It is essential to understand the linked homeostatic  mechanisms controlling water and sodium balance  when interpreting the plasma sodium concentration  and managing the clinically common disturbances of  water and sodium balance. This is of major importance  in deciding on the composition and amount, if any, of  intravenous fluid to give. It must also be remembered  that plasma results maybe affected by such intravenous  therapy, and can be dangerously misunderstood.   Water is an essential body constituent, and  homeostatic processes are important to ensure that  the total water balance is maintained within narrow  limits, and the distribution of water among the  vascular, interstitial and intracellular compartments is  maintained. This depends on hydrostatic and osmotic  forces acting across cell membranes.   Sodium is the most abundant extracellular cation  and, with its associated anions, accounts for most of  the osmotic activity of the extracellular fluid (ECF); it  is important in determining water distribution across  cell membranes.   Osmotic activity depends on concentration, and  therefore on the relative amounts of sodium and water  in the ECF compartment, rather than on the absolute  quantity of either constituent. An imbalance may cause  hyponatraemia (low plasma sodium concentration) or  hypernatraemia (high plasma sodium concentration),  and therefore changes in osmolality. If water and sodium  are lost or gained in equivalent amounts, the plasma  sodium, and therefore the osmolal concentration, is  unchanged; symptoms are then due to extracellular  volume depletion or overloading (Table 2.1). As the  metabolism of sodium is so inextricably related to that  of water, the two are discussed together in this chapter.   In a 70-kg man, the total body water (TBW) is about  42 L and contributes about 60 per cent of the total body  weight; there are approximately 3000 mmol of sodium,   mainly in the ECF (Table 2.2). Water and electrolyte  intake usually balance output in urine, faeces, sweat  and expired air.   The daily water and sodium intakes are variable, but in  an adult amount to about 1.5-2 L and 60-150 mmol,  respectively.   The kidneys and intestine deal with water and electrolytes  in a similar way. Net loss through both organs depends  on the balance between the volume filtered proximally    Table 2.2 The approximate volumes in different body  compartments through which water is distributed in a  70-kg adult   and that reabsorbed more distally. Any factor affecting  either passive filtration or epithelial cellular function  may disturb this balance.   Approximately 200 L of water and 30 000 mmol of  sodium are filtered by the kidneys each day; a further  10 L of water and 1500 mmol of sodium enter the  intestinal lumen. The whole of the extracellular water  and sodium could be lost by passive filtration in little  more than an hour, but under normal circumstances  about 99 per cent is reabsorbed. Consequently, the  net daily losses amount to about 1.5-2 L of water and  100 mmol of sodium in the urine, and 100 mL and  15 mmol, respectively, in the faeces.   Fine adjustment of the relative amounts of water  and sodium excretion occurs in the distal nephron and  the large intestine, often under hormonal control. The  effects of antidiuretic hormone (ADH) or vasopressin  and the mineralocorticoid hormone aldosterone on  the kidney are the most important physiologically,  although natriuretic peptides are also important.   About 1 L of water is lost daily in sweat and expired  air, and less than 30 mmol of sodium a day is lost in  sweat. The volume of sweat is primarily controlled  by skin temperature, although ADH and aldosterone  have some effect on its composition. Water loss in  expired air depends on the respiratory rate. Normally,  losses in sweat and expired air are rapidly corrected by  changes in renal and intestinal loss. However, neither  of these losses can be controlled to meet sodium and  water requirements, and thus they may contribute  considerably to abnormal balance when homeostatic  mechanisms fail.   Both the intake and loss of water are controlled by  osmotic gradients across cell membranes in the brain’s  hypothalamic osmoreceptor centres. These centres,  which are closely related anatomically, control thirst  and the secretion of ADH.   Antidiuretic hormone is a polypeptide with a half-life  of about 20 min that is synthesized in the supraoptic  and paraventricular nuclei of the hypothalamus and,  after transport down the pituitary stalk, is secreted  from the posterior pituitary gland (see Chapter 7).   The secretion of ADH is stimulated by the flow of  water out of cerebral cells caused by a relatively high  extracellular osmolality. If intracellular osmolality  is unchanged, an extracellular increase of only   2 per cent quadruples ADH output; an equivalent  fall almost completely inhibits it. This represents a  change in plasma sodium concentration of only about   3 mmol/L. In more chronic changes, when the osmotic  gradient has been minimized by solute redistribution,  there may be little or no effect. In addition, stretch  receptors in the left atrium and baroreceptors in the  aortic arch and carotid sinus influence ADH secretion  in response to the low intravascular pressure of severe  hypovolaemia, stimulating ADH release. The stress  due to, for example, nausea, vomiting and pain may  also increase ADH secretion. Inhibition of ADH  secretion occurs if the extracellular osmolality falls,  for whatever reason.   Antidiuretic hormone, by regulating aquaporin  2, enhances water reabsorption in excess of solute  from the collecting ducts of the kidney and so  dilutes the extracellular osmolality. Aquaporins are  cell membrane proteins acting as water channels  that regulate water flow. When ADH secretion is a  response to a high extracellular osmolality with the  danger of cell dehydration, this is an appropriate  response. However, if its secretion is in response to  a low circulating volume alone, it is inappropriate to  the osmolality. The retained water is then distributed  throughout the TBW space, entering cells along the  osmotic gradient; the correction of extracellular  depletion with water alone is thus relatively inefficient  in correcting hypovolaemia. Plasma osmolality  normally varies by less than 1-2 per cent, despite  great variation in water intake, which is largely due to  the action of ADH.   In some circumstances, the action of ADH is  opposed by other factors. For example, during an  osmotic diuresis the urine, although not hypo-osmolal,  contains more water than sodium. Patients with severe  hyperglycaemia, as in poorly controlled diabetes  mellitus, may show an osmotic diuresis.   Control of sodium balance  The major factors controlling sodium balance are renal  blood flow and aldosterone. This hormone controls  loss of sodium from the distal tubule and colon.   Aldosterone, a mineralocorticoid hormone, is secreted  by the zona glomerulosa of the adrenal cortex (see  Chapter 8). It affects sodium-potassium and sodium-  hydrogen ion exchange across all cell membranes. Its  main effect is on renal tubular cells, but it also affects  loss in faeces, sweat and saliva. Aldosterone stimulates  sodium reabsorption from the lumen of the distal  renal tubule in exchange for either potassium or  hydrogen ions (Fig. 2.1). The net result is the retention  of more sodium than water, and the loss of potassium  and hydrogen ions. If the circulating aldosterone  concentration is high and tubular function is normal,  the urinary sodium concentration is low.   Many factors are involved in the feedback control of  aldosterone secretion. These include local electrolyte  concentrations, such as that of potassium in the adrenal  gland, but they are probably of less physiological  and clinical importance than the effect of the renin-  angiotensin system.   Renin is an aspartyl protease secreted by the  juxtaglomerular apparatus, a cellular complex adjacent  to the renal glomeruli, lying between the afferent arteriole    Figure 2.1 The action of aldosterone on the  reabsorption of Na + in exchange for either K + or H +  from the distal renal tubules. See text for details. CD,  carbonate dehydratase; Eh, associated anion.   and the distal convoluted tubule. Renin is derived from  prorenin by proteolytic action, and secretion increases  in response to a reduction in renal artery blood flow,  possibly mediated by changes in the mean pressure in the  afferent arterioles, and (3-adrenergic stimulation. Renin  splits a decapeptide (angiotensin I) from a circulating oy-  globulin known as renin substrate. Another proteolytic  enzyme, angiotensin-converting enzyme (ACE), which  is located predominantly in the lungs but is also present  in other tissues such as the kidneys, splits off a further  two amino acid residues. This is the enzyme that ACE  inhibitors (used to treat hypertension and congestive  cardiac failure) act on. The remaining octapeptide,  angiotensin II, has a number of important actions:   • It acts directly on capillary walls, causing  vasoconstriction, and so probably helps to maintain  blood pressure and alter the glomerular filtration rate  (GFR) . Vasoconstriction may raise the blood pressure  before the circulating volume can be restored.   • It stimulates the cells of the zona glomerulosa to  synthesize and secrete aldosterone.   • It stimulates the thirst centre and so promotes oral  fluid intake.   Poor renal blood flow is often associated with an  inadequate systemic blood pressure. The release of  renin results in the production of angiotensin II, which  tends to correct this by causing aldosterone release,  which stimulates sodium and water retention and hence  restores the circulating volume. Thus, aldosterone  secretion responds, via renin, to a reduction in renal  blood flow. Sodium excretion is not directly related  to total body sodium content or to plasma sodium  concentration.   A peptide hormone (or hormones) secreted from  the right atrial or ventricular wall in response to the  stimulation of stretch receptors may cause high sodium  excretion (natriuresis) by increasing the GFR and by  inhibiting renin and aldosterone secretion. However,  the importance of this hormone (or hormones) in  the physiological control of sodium excretion and in  pathological states has not yet been fully elucidated,  although it is important in the pathophysiology of  congestive cardiac failure (see Chapter 22).   The most important factor in assessing changes in  day-to-day fluid balance is accurate records of fluid   intake and output; this is particularly pertinent for  unconscious patients. ‘Insensible loss’ is usually  assumed to be about 1 L/ day, but there is endogenous  water production of about 500mL/day as a result  of metabolic processes. Therefore the net daily  ‘insensible loss’ is about 500 mL. The required daily  intake may be calculated from the output during the  previous day plus 500 mL to allow for ‘insensible  loss’; this method is satisfactory if the patient is  normally hydrated before day-to-day monitoring  is started. Serial patient body weight determination  can also be useful in the assessment of changes in  fluid balance.   Pyrexial patients may lose 1 L or more of fluid in sweat  and, if they are also hyperventilating, respiratory water  loss may be considerable. In such cases an allowance  of about 500 mL for ‘insensible loss’ may be totally  inadequate. In addition, patients may be incontinent  of urine, and having abnormal gastrointestinal losses  makes the accurate assessment of fluid losses very  difficult.   Keeping a cumulative fluid balance record is a useful  way of detecting a trend, which may then be corrected  before serious abnormalities develop.   In the example shown in Table 2.3, 500 mL has been  allowed for as net ‘insensible daily loss’; calculated losses  are therefore more likely to be underestimated than  overestimated. This shows how insidiously a serious  deficit can develop over a few days.   The volume of fluid infused should be based on the  calculated cumulative balance and on clinical evidence  of the state of hydration, and its composition adjusted  to maintain normal plasma electrolyte concentrations.   Assessment of the state of hydration of a patient relies  on clinical examination and on laboratory evidence of  haemodilution or haemoconcentration.   • Haemodilution Increasing plasma volume with  protein-free fluid leads to a fall in the concentrations  of proteins and haemoglobin. However, these findings   may be affected by pre-existing abnormalities of  protein or red cell concentrations.   • Haemoconcentration ECF is usually lost from  the vascular compartment first and, unless the  fluid is whole blood, depletion of water and small  molecules results in a rise in the concentration of  large molecules, such as proteins and blood cells,  with a rise in blood haemoglobin concentration and  haematocrit, raised plasma urea concentration and  reduced urine sodium concentration.   Table 2.4 shows various intravenous fluid regimens  that can be used clinically. A summary of the British  Consensus Guidelines on Intravenous Fluid Therapy  for Adult Surgical Patients (GIFTASUP) can be found  at www.bapen.org.uk/pdfs/bapen_pubs/giftasup.pdf.   In mild disturbances of the balance of water and  electrolytes, their total amounts in the body may be of  less importance than their distribution between body  compartments (see Table 2.2).   Water is distributed between the main body  fluid compartments, in which different electrolytes  contribute to the osmolality. These compartments are:   • extracellular, in which sodium is the predominant  cation, and which can be subdivided into:   Sodium is the predominant extracellular cation, its  intracellular concentration being less than one-tenth  of that within the ECF. The intracellular potassium  concentration is about 30 times that of the ECF. About  95 per cent of the osmotically active sodium is outside   Table 2.3 Hypothetical cumulative fluid balance chart assuming an insensible daily loss of 500mL   cells, and about the same proportion of potassium is  intracellular. Cell-surface energy- dependent sodium/  potassium adenosine triphosphatase pumps maintain  these differential concentrations.   Other ions tend to move across cell membranes  in association with sodium and potassium. (The  movement of hydrogen ions is discussed in Chapter 4.)  Magnesium and phosphate ions are predominantly  intracellular, and chloride ions extracellular.   The cell membranes of the capillary endothelium are  more permeable to small ions than those of tissue cells.  The plasma protein concentration is relatively high,  but that of interstitial fluid is very low. The osmotic  effect of the intravascular proteins is balanced by very  slightly higher interstitial electrolyte concentrations  (Gibbs-Donnan effect); this difference is small and, for  practical purposes, plasma electrolyte concentrations  can be assumed to be representative of those in the ECF  as a whole.   Over half the body water is intracellular (see Table  2.2). About 15-20 per cent of the extracellular water is  intravascular; the remainder constitutes the interstitial  fluid. The distribution of water across biological  membranes depends on the balance between the  hydrostatic pressure and the in vivo effective osmotic  pressure differences on each side of the membrane.   The net movement of water across a membrane that is  permeable only to water depends on the concentration  gradient of particles - either ions or molecules - across  that membrane, and is known as the osmotic gradient.  For any weight-to-volume ratio, the larger the particles,  the fewer there are per unit volume, and therefore the  lower the osmotic effect they exert. If the membranes  were freely permeable to ions and smaller particles as  well as to water, these diffusible particles would exert  no osmotic effect across membranes, and therefore the  larger ones would become more important in affecting  water movement. This action gives rise to the effective  colloid osmotic (oncotic) gradient. Water distribution  in the body is thus dependent largely on three factors,  namely:   Units of measurement of osmotic pressure  Osmolar concentration can be expressed as:   If solute is dissolved in pure water at concentrations  such as those in body fluids, osmolarity and osmolality  will hardly differ. However, as plasma is a complex  solution containing large molecules such as proteins,  the total volume of solution (water + protein) is   greater than the volume of solvent only (water) in  which the small molecules are dissolved. At a protein  concentration of 70g/L, the volume of water is about  6 per cent less than the total volume of the solution (that  is, the molarity should theoretically be about 6 per cent  less than the molality). Most methods for measuring  individual ions assess them in molarity (mmol/L). If  the concentration of proteins in plasma is markedly  increased, the volume of solvent is significantly reduced  but the volume of solution remains unchanged.  Therefore the molarity (in mmol/L) of certain ions  such as sodium will be reduced but the molality will be  unaltered. This apparently low sodium concentration is  known as pseudohyponatraemia.   Osmometers measure changes in the properties of a  solution, such as freezing point depression or vapour  pressure, which depend on the total osmolality  of the solution - the osmotic effect that would be  exerted by the sum of all the dissolved molecules and  ions across a membrane permeable only to water.  These properties are known as colligative properties.  Sodium and its associated anions (mainly chloride)  contribute 90 per cent or more to this measured plasma  osmolality, the effect of protein being negligible. As the  only major difference in composition between plasma  and interstitial fluid is in protein content, the plasma  osmolality is almost identical to the osmolality of the  interstitial fluid surrounding cells.   It is the osmolam, rather than the osmolar, concentration  that exerts an effect across cell membranes and that  is controlled by homeostatic mechanisms. However,  as discussed below, the calculated plasma osmolarity  is usually as informative as the measured plasma  osmolality.   Although, because of the space-occupying effect of  protein, the measured osmolality of plasma should be  higher than the osmolarity, calculated from the sum of  the molar concentrations of all the ions, there is usually  little difference between the two figures. This is because  there is incomplete ionization of, for example, NaCl to  Na + and CL; this reduces the osmotic effect by almost  the same amount as the volume occupied by protein  raises it.   Consequently, the calculated plasma osmolarity is a  valid approximation to the true measured osmolality.  However, if there is gross hyperproteinaemia or  hyperlipidaemia such that either protein or lipid   contributes much more than 6 per cent to the measured  plasma volume, the calculated osmolarity may be  significantly lower than the true osmolality in the plasma  water. A hypothetical example is shown in Figure 2.2.   Many formulae of varying complexity have been  proposed to calculate plasma osmolarity. None of them  can predict the osmotic effect , but the following formula  (in which square brackets indicate concentration) gives  a close approximation to plasma osmolality (although  some equations omit the potassium, which may be  preferable):   Plasma osmolality = 2[Na + ] + 2[K + ] + [urea]   The factor of 2, which is applied to the sodium and  potassium concentrations, allows for the associated  anions and assumes complete ionization. This  calculation is not valid if gross hyperproteinaemia  or hyperlipidaemia is present or an unmeasured  osmotically active solute, such as mannitol, methanol,  ethanol or ethylene glycol, is circulating in plasma.   A significant difference between measured  and calculated osmolality in the absence of  hyperproteinaemia or hyperlipidaemia may suggest  alcohol or other poisoning. For example, a plasma  alcohol concentration of lOOmg/dL contributes about  20 mmol/kg to the osmolality. This osmotic difference is  known as the osmolar gap and can be used to assess the  presence in plasma of unmeasured osmotically active  particles. In such cases the plasma sodium concentration  may be misleading as a measure of the osmotic effect. It  is not possible to calculate urinary osmolarity because  of the considerable variation in the concentrations of  different, sometimes unmeasured, solutes; the osmotic  pressure of urine can be determined only by measuring  the osmolality.   Because the hydrostatic pressure difference across the  cell membrane is negligible, cell hydration depends on  the effective osmotic difference between intracellular  and extracellular fluids. The cell membranes are freely  permeable to water and to some solutes, but different  solutes diffuse (or are actively transported) across cell  membranes at different rates, although always more  slowly than water. In a stable state, the total intracellular  osmolality, due mostly to potassium and associated  anions, equals that of the interstitial fluid, due mostly  to sodium and associated anions; consequently, there is   Figure 2.2 The consequence of gross hyperproteinaemia or hyperlipidaemia on the plasma water volume and its  effect on the calculated plasma osmolarity and the true plasma osmolality.   no net movement of water into or out of cells. In some  pathological states, rapid changes of extracellular solute  concentration affect cell hydration; slower changes may  allow time for the redistribution of solute and have  little or no effect.   Sodium In normal subjects sodium and its associated  anions account for at least 90 per cent of extracellular  osmolality. Rapid changes in their concentration therefore  affect cellular hydration. If there is no significant change  in the other solutes, a rise causes cellular dehydration  and a fall causes cellular overhydration.   Urea Normal extracellular concentrations are so  low as to contribute very little to the measured plasma  osmolality. However, concentrations 15-fold or more  above normal can occur in severe uraemia and can  then make a significant contribution (see Chapter 3).  However, urea does diffuse into cells very much more  slowly than water. Consequently, in acute uraemia, the   increased osmotic gradient alters cell hydration, but  in chronic uraemia, although the measured plasma  osmolality is often increased, the osmotic effect of urea  is reduced as the concentrations gradually equalize on  the two sides of the membrane.   Glucose Like urea, the normally low extracellular  concentration of glucose does not contribute significantly  to the osmolality. However, unlike urea, glucose is actively  transported into many cells, but once there it is rapidly  metabolized, even at high extracellular concentrations,  and the intracellular concentration remains low. Severe  hyperglycaemia, whether acute or chronic, causes a  marked osmotic effect across cell membranes, with  movement of water from cells into the extracellular  compartment causing cellular dehydration.   Although hyperglycaemia and acute uraemia can  cause cellular dehydration, the contribution of normal  urea and glucose concentrations to plasma osmolality   is so small that reduced levels of these solutes, unlike  those of sodium, do not cause cellular overhydration.   Solutes such as potassium, calcium and magnesium  are present in the ECF at very low concentrations.  Significant changes in these are lethal at much lower  concentrations than those that would change osmolality.   Mannitol is an example of an exogenous substance  that remains in the extracellular compartment because it  is not transported into cells, and may be infused to reduce  cerebral oedema. Ethanol is only slowly metabolized,  and a high concentration in the ECF may lead to cerebral  cellular dehydration; this may account for some of the  symptoms of a hangover. High glucose concentrations  account for the polyuria of severe diabetes mellitus.   Large rises in the osmotic gradient across cell  membranes may result in the movement of enough  water from the intracellular compartment to dilute  extracellular constituents. Consequently, if the change  in osmolality has not been caused by sodium and its  associated anions, a fall in plasma sodium concentration  is appropriate to the state of osmolality. If, under such  circumstances, the plasma sodium concentration is not  low, this indicates hyperosmolality.   Generally, plasma osmolarity calculated from sodium,  potassium, urea and glucose concentrations is at least as  clinically valuable as measured plasma osmolality. It has  the advantage that the solute responsible, and therefore  its likely osmotic effect, is often identified.   The maintenance of blood pressure depends on  the retention of fluid within the intravascular  compartment at a higher hydrostatic pressure than that  of the interstitial space. Hydrostatic pressure in capillary  lumina tends to force fluid into the extravascular space.  In the absence of any effective opposing force, fluid  would be lost rapidly from the vascular compartment.  Unlike other cell membranes, those of the capillaries are  permeable to small ions. Therefore sodium alone exerts  almost no osmotic effect and the distribution of water  across capillary membranes is little affected by changes in  electrolyte concentration.   The very small osmotic effect of plasma protein  molecules produces an effective osmotic gradient  across capillary membranes; this is known as the  colloid osmotic, or oncotic, pressure. It is the most  important factor opposing the net outward hydrostatic  pressure (Fig. 2.3). Albumin (molecular weight 65kDa)   is the most important protein contributing to the  colloid osmotic pressure. It is present intravascularly  at significant concentration but extravascularly only at  a very low concentration because it cannot pass freely  across the capillary wall.   The osmotic gradient across vascular walls cannot be  estimated by simple means. The total plasma osmolality  gives no information about this. Moreover, the plasma  albumin concentration is a poor guide to the colloid  osmotic pressure. Although other proteins, such as  globulins, are present in the plasma at about the same  concentration as albumin, their estimation for this  purpose is even less useful: their higher molecular weights  mean that they have even less effect than albumin.   In normal subjects, the concentrations of sodium and  its associated anions are the most important osmotic  factors affecting ADH secretion. Plasma volume, by its  effect on renal blood flow, controls aldosterone secretion  and therefore sodium balance. The homeostatic  mechanisms controlling sodium and water excretion  are interdependent. (A simplified scheme is shown  in Fig. 2.4.) Thirst depends on a rise in extracellular  osmolality, whether due to water depletion or sodium  excess, and also on a very large increase in the activity  of the renin-angiotensin system.   A rise in extracellular osmolality reduces water  loss by stimulating ADH release and increases intake  by stimulating thirst; both these actions dilute the  extracellular osmolality. Osmotic balance (and therefore  cellular hydration) is rapidly corrected.   As already discussed, the plasma sodium concentration  is important because of its osmotic effect on fluid  distribution. Plasma sodium concentrations should be  monitored while volume is being corrected to ensure  that the distribution of fluid between the intracellular  and extracellular compartments is optimal. The  presence of other osmotically active solutes should be  taken into account.   Urinary sodium excretion is not related to body content  but to renal blood flow.   Estimation of the urinary sodium concentration in  a random specimen may be of value in the diagnosis of  the syndrome of inappropriate antidiuretic hormone    Figure 2.3 Osmotic factors that control the distribution of water between the fluid compartments of the body.   secretion (SIADH) and may help to differentiate renal  circulatory insufficiency (pre-renal) from intrinsic  renal damage (see Chapter 3).   The fractional excretion of sodium (FENa%) may  also be useful in helping to assess renal blood flow and  can be measured using a simultaneous blood sample  and spot urine sample:   A value of less than 1 per cent may be found in poor  renal perfusion, for example pre-renal failure, and of  more than 1 per cent in intrinsic renal failure.   The initial clinical consequences of primary sodium  disturbances depend on changes of extracellular  osmolality and hence of cellular hydration, and those  of primary water disturbances depend on changes in  extracellular volume.   Plasma sodium concentration is usually a substitute  for measuring plasma osmolality. Plasma sodium  concentrations per se are not important, but their effect  on the osmotic gradient across cell membranes is, and  it should be understood that the one does not always  reflect the other.   If the concentration of plasma sodium alters rapidly,  and the concentrations of other extracellular solutes  remain the same, most of the clinical features are due  to the consequence of the osmotic difference across cell   membranes, with redistribution of fluid between cells  and the ECF. However, gradual changes, which allow  time for redistribution of diffusible solute such as urea,  and therefore for equalization of osmolality without  major shifts of water, may produce little effect on fluid  distribution.   We now discuss in some detail conditions involving  water and sodium deficiency and excess. These are  discussed together, as the two are so closely inter-related  in vivo and can result in abnormal plasma sodium  concentrations.   Water and sodium deficiency (Figs 2. 5-2. 7)  Apart from the loss of solute-free water in expired  air, water and sodium are usually lost together from  the body. An imbalance between the degrees of their  deficiency is relatively common and may be due to   Figure 2.5 Homeostatic correction of isosmotic volume depletion. The reduced intravascular volume impairs renal blood  flow and stimulates renin and therefore aldosterone secretion. There is selective sodium reabsorption from the distal  tubules and a low urinary sodium concentration. (Shading indicates primary change.) ADH, antidiuretic hormone.   Figure 2.6 Infusion of hypotonic fluid as a cause of predominant sodium depletion. Increased circulating volume  with reduction in plasma osmolality inhibits aldosterone and antidiuretic hormone (ADH) secretion. (Shading  indicates primary change.)    Figure 2.7 Initial effect of aldosterone deficiency is impaired sodium retention and hypovolaemia; later, severe  hypovolaemia stimulates increased antidiuretic hormone (ADH) secretion with water retention, sometimes  causing a dilutional hyponatraemia. (Shading indicates primary change.)   the composition of the fluid lost or to that of the fluid  given to replace it. The initial effects depend on the  composition of the fluid lost compared with that of  plasma.   • Isosmolar volume depletion results if the sodium  concentration of the fluid lost is similar to that of  plasma; changes in plasma sodium concentration are  then unusual.   • Predominant sodium depletion is usually the result  of inappropriate treatment, as only bile, secreted in  small volumes, has a significantly higher sodium  concentration than that of plasma. Hyponatraemia  (a low plasma sodium concentration) usually results.   • Predominant water depletion results if the sodium  concentration of the lost fluid is much less than that  of plasma. Hypernatraemia (an abnormally high  concentration of sodium) may occur, and indicates  loss of relatively more water than sodium, even if  there is little evidence of volume depletion.   The term ‘dehydration’ can be misleading. It is often  used interchangeably to describe the conditions listed  above, although the clinical and biochemical findings  are very different. The consequent confusion may lead  to inappropriate and possibly dangerous treatment;  therefore, an attempt should be made to assess the  approximate composition of fluid lost by identifying its  origin.   The sodium concentrations of all small intestinal  secretions, and of urine when tubular function is grossly  impaired, are between 120mmol/T and 140mmol/L.  Clinical conditions causing approximately isosmolar  loss are as follows:   • small intestinal obstruction and paralytic ileus, in  which the fluid accumulating in the gut lumen has,  like urine in the bladder, been lost from the ECF,   • severe renal tubular damage with minimal glomerular  dysfunction, for example the recovery phase of acute  oliguric renal dysfunction, or polyuric chronic renal  dysfunction (see Chapter 3).   Hypovolaemia reduces renal blood flow and causes renal  circulatory insufficiency with oliguria with uraemia.  Sodium and water are lost in almost equivalent  amounts, and the plasma sodium concentration is  usually normal; for this reason the patient may not  complain of thirst despite some volume depletion.   Haemoconcentration confirms considerable loss of  fluid other than blood, although its absence does not  exclude such loss.   Postural hypotension (a fall in blood pressure on  standing) is a relatively early sign of volume depletion,  and tachycardia may also occur.   The ability to respond to hormonal homeostatic  changes depends mainly on renal tubular function  and cannot occur if the isosmolar volume depletion  is due to tubular damage. The reduced intravascular  volume impairs renal blood flow and stimulates renin,  and therefore aldosterone, secretion. There is selective  sodium reabsorption from the distal tubules and  therefore a low urinary sodium concentration.   The tendency of the retained sodium to increase  plasma osmolality stimulates ADH secretion, and  water is reabsorbed; this tends to correct the circulating  volume and keep the plasma sodium concentration  normal. Severe intravascular volume depletion may also  stimulate ADH secretion and therefore water retention,  causing mild hyponatraemia. This additional water is  distributed throughout the total body water and moves  from the depleted, and now slightly hypo-osmolar,  ECF into the relatively well-hydrated intracellular  compartment.   Even maximal renal water and sodium retention  cannot correct extrarenal losses that exceed those of  a normal urine output. Water and sodium must be  replaced in adequate amounts.   Patients unable to absorb adequate amounts of oral  fluid because of gastrointestinal loss usually need  intravenous replacement.   Fluid replacement in a patient who presents with  hypovolaemia can be monitored by clinical observation  and by measurement of urine output, plasma  electrolytes and urea.   The infusion of protein-free fluid increases the  hydrostatic gradient and reduces the opposing  colloid osmotic gradient by diluting plasma proteins.  The increase in glomerular filtration caused by the  overcorrection of hypovolaemia results in an increase  in urine output, and is a common cause of a low plasma  urea concentration.   Measurement of urinary sodium concentration may  sometimes help. If tubular function is adequate, a urinary  sodium concentration of less than about 20mmol/L  suggests that renal blood flow is still low enough to  stimulate maximal renin and aldosterone secretion,  and infusion should be increased. A urinary sodium   concentration greater than 20 mmol/L in a patient with  adequate tubular function suggests overcorrection and  the need to slow the rate of infusion. In cases in which  all losses, other than in sweat, faeces and expired air, can  be measured, further maintenance of normal balance  should be based on accurate fluid balance charts.   For crystalloid resuscitation or replacement,  ‘balanced’ solutions, such as Ringer’s lactate/acetate or  Hartmann’s solution, should replace 0.9 per cent saline,  except in cases of hypochloraemia, due for example  to vomiting or gastric drainage or if lactic acidosis is  present. Losses from diarrhoea/ileostomy/small bowel  fistula/ileus/ obstruction should be replaced volume for  volume with Hartmann’s or Ringer’s lactate/ acetate-type  solutions. ‘Saline depletion’, for example due to excessive  diuretic exposure, is best managed with a balanced  electrolyte solution such as Hartmann’s solution.   Intravenous solutions such as 4 per cent dextrose/  0.18 per cent saline and 5 per cent dextrose are  important sources of free water for maintenance, but  should be used with caution as excessive amounts may  cause dangerous hyponatraemia, especially in children  and the elderly and post-operatively. These solutions are  not suitable for resuscitation or replacement therapy,  except in conditions of significant free water deficit,  such as diabetes insipidus (see Actions of antidiuretic  hormone above).   To meet maintenance requirements, adult patients  should receive sodium 50-100 mmol/day, and  potassium 40-80 mmol/day in 1.5-2.5L water by the  oral, enteral or parenteral route (or a combination of  routes). Additional amounts should only be given to  correct deficit or continuing losses. It is essential to  carefully monitor patients by clinical examination, fluid  balance charts, and regular body weighing if possible.   An important and common cause of sodium depletion  is infusion of intravenous fluid of inappropriate  composition. No bodily secretion (other than bile,  which is secreted in very small amounts) has a sodium  concentration significantly higher than that of plasma.  The composition of the fluid is even more important  than the volume.   Patients with isosmolar fluid depletion, or those  recovering from major surgery, may be infused  with fluid such as ‘dextrose saline’, which contains  about 30 mmol/L of sodium. Glucose in the infused  fluid renders it isosmolar despite the low sodium   concentration, but the glucose is metabolized, and both  plasma sodium concentration and osmolality are diluted  by the remaining hypo-osmolar fluid. Homeostatic  mechanisms tend to correct this hypo-osmolality but  may be overwhelmed if the infusion rate is high. Severe,  and even life-threatening, hyponatraemia can result  from the imprudent administration of hypotonic fluid,  such as 5 per cent dextrose.   Excess hypo-osmolar infused fluid dilutes the  plasma sodium, causing a dilutional hyponatraemia  with hypo-osmolality. The homeostatic mechanisms  that tend to correct this hypo-osmolality involve the  inhibition of ADH secretion. The excess water is lost  in the urine until the restoration of normal plasma  osmolality again stimulates normal ADH secretion.  Correction of osmolality may occur at the expense of  intravascular volume. This would stimulate renin and  aldosterone secretion and sodium would be retained,  with the consequent restoration of osmolality and  normal ADH secretion.   However, if intravascular volume is maintained by  replacing the urinary volume with effectively hypotonic  fluid, hypo-osmolality with hyponatraemia persists and  sodium depletion is aggravated (Fig. 2.6). Restoration  of the plasma volume inhibits renin and aldosterone  secretion and sodium is lost in the urine despite hypo-  osmolality. The net effect of this procedure is the  restoration of circulating volume at the expense of  sodium depletion and cellular overhydration.   • a large volume of dilute urine due to the inhibition  of ADH secretion,   • a low plasma urea concentration due to the high GFR  (excessive intravenous infusion is one of the com-  monest causes of a low plasma urea concentration),   • high urinary sodium concentration due to the  inhibition of aldosterone secretion.   During the immediate post-operative period,  pain and stress also stimulate ADH secretion and  therefore water retention; this effect is short lived. Such  hyponatraemia is rarely a problem, but may become so  if hypo-osmolar fluid is being infused. In rare cases it  can then be lethal due to cerebral damage.   Aldosterone deficiency, such as occurs in Addison’s  disease, is a rare cause of sodium depletion. Initial  homeostatic reactions tend to maintain osmolality at  the expense of volume (see Chapter 8). Although less  than 1.5 per cent of the filtered sodium is reabsorbed  in the renal distal convoluted tubules, this is where the  fine adjustment is made in the ratio of sodium to water  and therefore to plasma osmolality, and thus normal  cell hydration is safeguarded. If aldosterone cannot  be secreted normally in response to appropriately  increased amounts of renin and angiotensin, this  adjustment cannot be made (Fig. 2.7). Under such  circumstances, although a greater proportion of  sodium may be reabsorbed from the proximal tubules,  there may still be relative sodium deficiency and  hypovolaemia. Initially plasma osmolality and therefore  plasma sodium concentration are maintained by water  loss; loss of relatively more sodium than water reduces  plasma osmolality and cuts off ADH secretion. Later,  hypovolaemia stimulates ADH secretion, with water  retention in excess of sodium; dilutional hyponatraemia  may occur despite intravascular volume depletion.   The clinical features include circulatory insufficiency  with postural hypotension and the following findings:   • renal circulatory insufficiency with mild uraemia  due to volume depletion,   • an inappropriately high urinary sodium  concentration in the presence of volume depletion,   Predominant water depletion is caused by loss of  water in excess of sodium. It is usually due to loss of  fluid that has a sodium concentration less than that  of plasma, deficient water intake, or both. The rise in  extracellular osmolality stimulates both ADH secretion  (which minimizes water loss) and thirst. Faboratory  abnormalities are most marked if the patient is unable  to respond to thirst.   The causes of predominant water depletion can be  divided into the following groups.   • Excessive loss of fluid that has a sodium concentration  less than that of plasma. The causes include:   - loss of excessive fluid stools of low sodium  concentration, usually in infantile gastroenteritis,   - loss of large amounts of sweat, such as in  pyrexial patients,   - loss of fluid from the body surface after  extensive burns.   • Deficiency of water intake as a result of inadequate  water supply, or mechanical obstruction to its  intake.   - osmotic diuresis, which can be caused by  hypertonic intravenous infusions, such as  parenteral nutrition tissue damage and hence  increased production of urea from protein,  and glycosuria in severe diabetes mellitus with  a very high plasma glucose concentration.   • Cranial diabetes insipidus, a syndrome associated  with impairment of ADH secretion. It may be  idiopathic in origin or due to either pituitary or  hypothalamic damage caused by head injury or by  invasion of the region by tumour or infiltration.    hypernatraemia, due to water depletion, stimulate aldosterone and antidiuretic hormone (ADH) secretion.  (Shading indicates primary change.)   Figure 2.9 Consequences of antidiuretic hormone (ADH) deficiency (diabetes insipidus). Impaired water retention  results in an increased plasma osmolality with stimulation of thirst and hypovolaemia with increased aldosterone  secretion. (Shading indicates primary change.)   Diabetes insipidus following a head injury may  present with polyuria, and then pass through a  temporary ‘recovery’ phase following transient  release of ADH from the remaining granules in the  pituitary stalk; this results in water retention and  occasionally causes a dilutional hyponatraemia.  Some patients with diabetes insipidus due to trauma  recover partially or completely as cerebral oedema  resolves. There is a hereditary autosomal dominant  form of cranial diabetes insipidus due to mutations  in the arginine vasopressin-neurophysin II gene.  In addition, there is the autosomal recessive form  DIDMOAD (diabetes insipidus, diabetes mellitus,  optic atrophy and deafness - see Chapter 12), due to  mutations in the wolframin gene.   • Nephrogenic diabetes insipidus is caused by the  reduced action of ADH on the renal collecting ducts.  The disorder may be either familial or acquired.  There is an X-linked recessive form due to mutations  in the vasopressin type 2 (V2) receptor gene and also  an autosomal recessive form due to mutations in the  aquaporin 2 gene (chromosome 12), which codes  for the vasopressin-dependent water channel in the  renal collecting ducts. Causes of secondary acquired  diabetes insipidus include:   - drugs, such as lithium carbonate, amphotericin  or demeclocycline, which interfere with the  action of ADH causing the clinical picture of  nephrogenic diabetes insipidus,   - hypercalcaemia or hypokalaemia,both ofwhich  impair the urine-concentrating mechanism  and may present with polyuria.   Water deficiency without thirst is called adipsic, or  hypodipsic, hypernatraemic syndrome or essential  hypernatraemia, and is thought to be due to a  hypothalamic disorder. In this syndrome there is a lack  of thirst without polyuria.   • loss of water in excess of sodium: the plasma  sodium concentration, and therefore osmolality,  increases,   • reduction of circulating volume, which reduces  renal blood flow and stimulates aldosterone  secretion; sodium is retained and hypernatraemia is  aggravated.   Compensatory effects occur because the increase in  the plasma osmolality stimulates:   • thirst, increasing water intake, if water is available  and if the patient can respond to it,   • ADH secretion: urinary volume falls and water loss  through the kidney is reduced.   If an adequate amount of water is available, depletion  is rapidly corrected. If there is an inadequate intake  to replace the loss, hypernatraemia may occur before  clinical signs of volume depletion are detectable. The  clinical features of predominant water depletion include:   • later - signs of volume depletion: the signs of  intravenous volume depletion are relatively less than  isotonic fluid loss as the deficit is distributed across  TBW space.   Conversely, the clinical features differ if there is ADH  deficiency, and include polyuria and dilute urine. The  laboratory findings are:   • mild uraemia due to volume depletion and therefore  low GFR,   • high urinary osmolality and urea concentration due  to the action of ADH,   • low urinary sodium concentration in response to  high aldosterone levels stimulated by the low renal  blood flow.   An excess of water or sodium is usually rapidly  corrected. Syndromes of excess are usually associated  with impaired homeostatic mechanisms. These can be  conveniently classified into those with:   • predominant sodium excess, which may be caused  by hyperaldosteronism or, in mentally disturbed  subjects, by excessive salt intake or by deliberate salt  poisoning as infants; there may be hypernatraemia,   • predominant water excess, which may result from  excessive ADH secretion or increased water intake  and usually results in hyponatraemia.   Volume excess not primarily due to an osmotic difference  across cell membranes may cause hypertension and  cardiac failure. Oedema occurs only if:   • the capillary hydrostatic pressure is increased, as in  congestive cardiac failure,   • the capillary colloid osmotic pressure is reduced due  to hypoalbuminaemia.   Laboratory findings are characteristic of  haemodilution and, unless there is glomerular  dysfunction, plasma urea concentrations tend to be low.   Some factors that increase fluid accumulation in the  interstitial space are summarized in Box 2.1.   By convention, the term secondary aldosteronism  usually refers to the clinical condition in which long-  standing aldosterone secretion is stimulated by the  renin-angiotensin system following a low renal blood  flow (Fig. 2.10). This maybe due to local abnormalities  in renal vessels or to a reduced circulating volume and  is therefore usually associated with the following:   Box 2.1 Some factors that increase the  accumulation of fluid within the interstitial  space (oedema)   • Redistribution of ECF, leading to a reduced plasma  volume despite a normal or even high total ECF  volume. The resulting conditions may be caused by  a reduction in plasma colloid osmotic pressure, and  are therefore associated with low plasma albumin  concentrations. Oedema is present. Persistent  hypoalbuminaemia may be due to liver disease,  nephrotic syndrome or protein undernutrition.   • Cardiac failure, in which two factors may reduce  renal blood flow and a third factor aggravates the  hyperaldosteronism:   - an increased capillary hydrostatic pressure in  cardiac failure may cause the redistribution of  fluid into the interstitial space, with oedema,   - both impaired catabolism of aldosterone  and elevated plasma atriuretic peptide  concentration may aggravate the condition.   • Damage to renal vessels, reducing renal blood flow.  These conditions are rarely associated with oedema  and include:   - renal hypertension, such as that due to renal  artery stenosis.   The reduced renal blood flow stimulates aldosterone  secretion, with enhanced sodium reabsorption  from the distal convoluted tubules and subsequent  water retention. These processes tend to restore the  intravascular volume. If there is hypoalbuminaemia    aldosterone secretion; if pronounced, antidiuretic hormone (ADH) secretion may be increased and thirst stimulated,  resulting in hyponatraemia despite an increase in total body sodium. (Shading indicates primary change.)   or heart failure, more fluid passes into the interstitial  fluid as the retained water dilutes the plasma albumin  concentration and therefore lowers the colloid  osmotic pressure and raises the capillary hydrostatic  pressure. The circulating volume can be maintained  only by further fluid retention. A vicious cycle leads  to the accumulation of excess fluid in the interstitial  compartment (oedema).   Initially, this cycle stimulates a par allel increase in water  and sodium retention, with normonatraemia. However,  stimulation of ADH secretion by hypovolaemia, if long  standing, may cause enough sodium-free water retention  to produce mild hyponatraemia despite the excess of  total body sodium. Angiotensin II also stimulates thirst  and therefore increases water intake.   Hypokalaemia is less common in secondary than  in primary hyperaldosteronism, perhaps because the  low GFR reduces the amount of sodium reaching the  distal tubules and therefore the amount available for  exchange with either hydrogen or potassium ions (see  Chapters 4 and 5). However, if reabsorption of sodium  without water is inhibited by loop diuretics, potassium  depletion occurs more readily than in subjects without  hyperaldosteronism.   The clinical features found in these patients are  those of the underlying primary condition. Laboratory  findings include:   Predominant sodium excess is rare. It is usually caused  by inappropriate secretion of aldosterone, such as in  primary hyperaldosteronism (Conn’s syndrome), or by  glucocorticoids, as in Cushing’s syndrome (see Chapter  8). In these syndromes, sodium retention stimulates  the retention of water, minimizing changes in plasma  sodium concentration. Sodium excess can also be  caused by excessive sodium intake.   About 50 per cent of cases of this syndrome are due to  a single benign adenoma of the glomerulosa cells of  the adrenal cortex; about 10 per cent of adenomas are  multiple, and most of the remaining cases are associated  with bilateral nodular hyperplasia of the adrenal  cortex. Rarer causes include aldosterone-producing  carcinoma of the adrenal gland or ectopic aldosterone-  secreting tumours. Another rare form is glucocorticoid-  suppressible hyperaldosteronism. In all these conditions,  aldosterone secretion is relatively autonomous (Fig. 2.11):   • The rise in plasma sodium concentration stimulates  ADH secretion and water retention.   • Water retention tends to restore plasma sodium  concentrations to normal.   • Aldosterone secretion is not subject to normal  feedback suppression. Its action causes sodium and  water retention at the expense of potassium loss;  hypokalaemic alkalosis is common (see Chapters 3,  4 and 5).    causing sodium retention and increasing the plasma osmolality. This stimulates antidiuretic hormone (ADH)  secretion. The increase in intravascular volume inhibits renin secretion. (Shading indicates primary change.)   The typical clinical features of primary hyper-  aldosteronism are those of hypervolaemia (patients  have mild to moderate hypertension but are rarely  oedematous) and those associated with hypokalaemia.  The laboratory findings include the following:   • A plasma sodium concentration that may be  within the higher reference range but may show  hypernatraemia.   • Hypokalaemic alkalosis (low plasma potassium and  raised plasma bicarbonate concentrations) due to  excess aldosterone secretion:   - a urinary potassium concentration greater than  about 20mmol/L, indicating kaliuria (urinary  potassium loss) in the face of hypokalaemia  (see Chapter 5),   - a low urinary sodium concentration in the early  stages; later, sodium excretion may rise, possibly  because of hypokalaemic tubular damage.   These findings in a patient without an obvious  reason for potassium loss and with mild to moderate  hypertension suggest the diagnosis of primary  hyperaldosteronism. The finding of high plasma  aldosterone with low renin activity confirms the  diagnosis; in secondary aldosteronism both the plasma  aldosterone concentration and renin activity are high,  although various antihypertrensive drugs may interfere  with these tests (see Chapter 8, Investigation of a patient  with suspected primary hyperaldosteronism).   This results from glucocorticoid excess, such as is  found with adrenal or pituitary adenomas, ectopic  adrenocortico trophic hormone (ACTH) secretion or   Sodium excess may be due to increased sodium intake,  such as excessive salt intake or overenthusiastic infusion  of saline or sodium- containing solutions, such as sodium  bicarbonate, or infusion of sodium salts of antibiotics.   Hyperosmolar saline is dangerous if used as an emetic  in cases of poisoning. The movement of water into the  gut along the osmotic gradient and absorption of some  of the sodium can cause marked hypernatraemia.  Death has occurred as a consequence of this practice,  particularly when patients could not respond to  hyperosmolality by drinking because of vomiting or  because they were unconscious.   Predominant water overload may occur in  circumstances in which normal homeostasis has failed,  for example in the following:   • If fluid of low sodium concentration, such as  5 per cent dextrose, has been infused, especially in  the post-operative period or if there is glomerular  dysfunction (see Fig. 2.12 and Chapter 3).   • If there is ‘inappropriate’ ADH secretion. Antidiuretic  hormone release may be stimulated by stress, such as  that due to pain, nausea, chest infection and cerebral  trauma (Box 2.2).   Hormone secretion is defined as inappropriate  if it continues when it should be cut off by negative  feedback control, in this case by a low plasma  osmolality. Tike many other peptide hormones, ADH   Figure 2.12 Homeostatic correction of water excess. Increased intravascular water volume, with decreased plasma  osmolality, inhibits aldosterone and antidiuretic hormone (ADH) secretion. (Shading indicates primary change.)   Box 2.2 Some causes of hyponatraemia  due to the syndrome of inappropriate  antidiuretic hormone secretion (SIADH)   Drugs, e.g. vincristine, chlorpropamide, carbamazepine,  non-steroidal anti-inflammatory drugs, certain anti-  depressants, oxytocin and opiates   can sometimes be synthesized by non-endocrine  malignant cells, such as small cell (oat cell) carcinoma  of the lung, and released ectopically. Inappropriate  secretion of ADH, from the pituitary or hypothalamus,  and therefore not ‘ectopic’, is very common in many  illnesses, such as disorders of the pulmonary and  central nervous systems.   The diagnosis of SIADH is usually made by finding  a urinary sodium concentration more than 20 mmol/L  in the presence of euvolaemic hyponatraemia or low  plasma osmolality and in the absence of hypovolaemia,  oedema, impaired renal function, diuretic usage,  adrenal insufficiency or hypothyroidism. According  to the Bartter and Schwartz criteria for SIADH, the  urinary osmolality is inappropriately concentrated in  relation to the plasma osmolality, that is, in the hypo-  osmolar state the urine is not maximally diluted. In  SIADH, the GFR is usually high, and plasma urea and  urate concentrations tend to be low:   • During the intravenous administration of the  posterior pituitary hormone oxytocin (Syntocinon)  to induce labour (see Chapter 7). Oxytocin has an  antidiuretic effect similar to that of the chemically  closely related ADH. If 5 per cent dextrose saline  is used as a carrier, the glucose is metabolized and  the net effect is retention of solute-free water. Death  from acute hypo-osmolality has resulted after  prolonged infusion; oxytocin should be given in the  smallest possible volume of isotonic saline, and the  fluid balance and plasma sodium concentrations  should be monitored carefully.   • Excess water intake can also occur if water intake  exceeds water clearance (about 20L/day), for   • TURP (transurethral resection of the prostate)  syndrome: water intoxication caused by an overload  of hypotonic irrigation fluid (such as glycine)  absorption from the open prostatic sinusoids during  the procedure.   • increases renal blood flow and cuts off aldosterone  secretion, increasing urinary sodium loss and  therefore further decreasing its plasma concentration,   • lowers plasma osmolality and therefore cuts off ADH  secretion; a large volume of dilute urine is passed.   When glomerular function is impaired, the excretion  of excess water may be limited. If there is appropriate  ADH secretion, or if oxytocin is being infused, for  example during labour, water retention continues  despite the low plasma osmolality, and compensation  cannot occur.   Hyponatraemia is usually caused by excessive water  relative to sodium within the extracellular compartment  or, more rarely, by sodium deficiency, and may reflect  extracellular hypo-osmolality (Box 2.3). If this is the  case, it may cause cellular overhydration. However,  hyponatraemia associated with a normal plasma  osmolality may occur in the following situations:   • Artefactual hyponatraemia may be due to blood  being taken from the limb into which fluid of low  sodium concentration, such as 5 per cent dextrose,  is being infused.   • Pseudohyponatraemia may be due to gross  hyperlipidaemia or to hyperproteinaemia. The  sodium concentration in plasma water, and therefore  the osmolality at cell membranes, may then be  normal (see Fig. 2.2).   • Appropriate hyponatraemia may be due, for example,  to hyperglycaemia, acute uraemia, the infusion of  amino acids or mannitol, a high plasma alcohol  or other solute concentrations (redistributive  hyponatraemia). All these may increase extracellular  osmolality; the consequent homeostatic dilution  of total extracellular solute concentration towards   A 74-year-old retired man presented to his general  practitioner with haemoptysis and weight loss. He  had smoked 20 cigarettes a day for 55 years. Chest  radiograph revealed a left upper lobe shadow, and  bronchoscopy confirmed a primary lung carcinoma.  His blood pressure was 132/80 mmHg and clinically  he was euvolaemic. There was no evidence of adrenal  insufficiency or thyroid disease and he was not taking  any medications. Some results were as follows:   The most likely explanation for the severe  hyponatraemia is syndrome of inappropriate  antidiuretic hormone secretion (SIADH). Some  lung carcinomas ectopically release ADH. Note that  the patient was clinically euvolaemic and there was  no other evidence of other causes of euvolaemic  hyponatraemia, such as adrenal insufficiency or  hypothyroidism. The urinary sodium concentration is  inappropriate for the hyponatraemia (> 20 mmol/L).   normal causes hyponatraemia, which reflects partial  or complete compensation of hyperosmolality rather  than hypo-osmolality.   The infusion of isosmolal sodium-containing fluids  with the aim of ‘correcting’ artefactual, appropriate  or pseudohyponatraemia is dangerous. When  hyponatraemia reflects true hypo-osmolality, cerebral  cellular overhydration may cause headache, confusion,  fits and even death. If the plasma sodium concentration  falls slowly over days or weeks, the brain can compensate  by the redistribution of water and solute between the  intracellular and extracellular fluid. However, if the  plasma sodium concentration declines rapidly over  24-48 h, these processes are inefficient and cerebral  oedema can occur. Cerebral pontine myelinolysis can  occur if the plasma sodium concentration is corrected  too rapidly, before redistribution can be reversed. The  rapid change in osmolality causes focal demyelination   Hypovolaemic (decreased extracellular volume)  Non-renal losses, e.g. vomiting, diarrhoea  Renal losses, e.g. diuretics or salt-losing nephropathy  Adrenal insufficiency (Addison’s disease)  Euvolaemic   Excess fluid replacement, e.g. hypotonic (sodium-  free) 5 per cent dextrose   Potomania or psychogenic polydipsia  Hypervolaemic (increased extracellular volume)  Congestive cardiac failure  Nephrotic syndrome  Cirrhosis and ascites   in the pons and extrapontine regions, which may lead  to hypotension, seizures, quadriparesis and death.   Some causes of hyponatraemia are shown in Box 2.3.  These can be conveniently grouped into hypovolaemic,  euvolaemic and hypervolaemic forms, based on the  clinical features:   • Hypovolaemic hyponatraemia The TBW decreases  although the total body sodium decreases to a larger  extent. Thus the ECF volume is decreased.   • Euvolaemic hyponatraemia Total body sodium is  normal, while TBW increases. The ECF volume is  slightly increased but there is no oedema. One cause  is SIADH (Box 2.2).   • Hypervolaemic hyponatraemia The total body  sodium increases but TBW increases to a greater  extent. The ECF is markedly increased and oedema  is present.   • Exclude artefactual hyponatraemia when blood is  taken from the vein into which fluid is being infused.   This occurs whether the blood is taken close to the  site of infusion, where the infused fluid is at a higher  concentration than in the rest of the circulation.   • Is there hyperlipidaemia or hyperproteinaemia?  Exclude pseudohyponatraemia when plasma  osmolality is normal.   • Assess the patient’s medication history, including  intravenous fluid therapy and also alcohol intake.   • Hyperglycaemia can result in excessive extracellular  solute and water shift out of cells:   • Abnormal electrolyte shifts may occur very rarely in  critically ill patients (the sick cell syndrome).   • It is important to exclude adrenal insufficiency  (Addison’s disease) and severe hypothyroidism (see  Chapters 8 and 11). These are rare causes.   • Clinically assess the state of the ECF volume;  is there obvious hypovolaemia, euvolaemia or  hypervolaemia?   - If there is obvious hypovolaemia, check the  sodium concentration in a random urine  specimen (assuming not on a diuretic): if it is  less than or equal to 20 mmol/L, it is suggestive  of extrarenal sodium loss; if it is more than  20 mmol/L, it suggests renal sodium loss.   - If the patient is clinically euvolaemic, check a  random urinary sodium concentration: if it  is less than or equal to 20 mmol/L, consider  acute water overload, for example due to  the administration of too much fluid post-  operatively; if it is more than 20 mmol/L,  consider SIADH (see Box 2.2).   - If the patient is oedematous and is not in  cardiac or renal tubular failure, consider  nephrotic syndrome.   See Box 2.3 for some of the other causes of  hyponatraemia.   It cannot be stressed too strongly that treatment  should not be based on plasma sodium concentrations  alone. Hyponatraemia per se only rarely needs to be  treated. Ignorance of the causes of hyponatraemia is,  unfortunately, still common. For example, iatrogenic  post-operative hyponatraemia due to the injudicious  use of intravenous isotonic dextrose is still encountered   and can result in neurological damage or death.  Menopausal women, children and the elderly are  especially susceptible to hyponatraemia, particularly  if they are taking diuretics or certain antidepressant  drugs. Furthermore, there is also misunderstanding  about the most appropriate treatment of severe  hyponatraemia: rapid correction can cause cerebral  pontine myelinolysis, and correction that is too slow  may allow cerebral oedema to develop; because of these  risks, treatment should be instigated by experts on a  high-dependency unit.   Severe hyponatraemia known to have been present  for less than 48 h should be treated as a medical  emergency; because of the danger of brainstem  herniation, the aim should be to abolish symptoms  or to raise the plasma sodium concentration to about  125 mmol/L. Some people recommend infusion of  sodium at the rate of 1 or 2mmol/h. Patients with  signs of acute brainstem herniation such as coma or   A 74-year-old woman underwent a left total hip  replacement and had received 5L of 5 per cent  dextrose intravenously over one day. She had the  following post-operative results:   The patient was being infused with large amounts of  5 per cent dextrose intravenously. This was the most  likely cause of her post-operative hyponatraemia.  The administration of hypotonic and sodium-free  fluid was causing a dilutional hyponatraemia. This  is a particularly dangerous practice post-operatively,  when antidiuretic hormone (ADH) secretion is  increased due to the stress of the operation and of  nausea and pain, thus increasing the likelihood of  water retention.   seizures may need twice-normal (hypertonic) saline  rapidly to correct plasma sodium concentration to  about 120mmol/L; only enough should be given to  stop neurological symptoms.   Conversely, similar severe degrees of hyponatraemia  that are known to have been present for more than 48 h  and without clinical evidence of cerebral oedema can  be treated by slow infusion of 0.5 mmol/L per hour until  the plasma concentration reaches 130 mmol/L. Normal  (isotonic) or hypertonic saline should be infused, and  fluid restriction or administration of loop diuretics  depends on the severity of the hyponatraemia. Close  monitoring of plasma (hourly) and urinary sodium is  required to ensure safe correction.   If the patient is not dehydrated and SIADH has been  diagnosed, fluid intake restriction to 800-1000 mL/ day  may be indicated. Administration of demeclocycline, by  antagonizing the action of ADH on renal tubules, may also  help but may have side effects such as photosensitivity.  Recently vasopressin 2 receptor antagonists such  as tolvaptan have been used to treat SIADH and  hyponatraemia of cardiac failure and cirrhosis.   • 0.9 per cent (normal) saline contains 154 mmol/L of  sodium,   • 1.8 per cent (twice-normal) saline contains  308 mmol/L of sodium.   children’s TBW = 0.6 x body weight  adults’ TBW = 0.55 x body weight  elderly people’s TBW = 0.5 x body weight.   The example below shows how to calculate saline  replacement in an elderly patient weighing 80 kg with  symptomatic severe hyponatraemia of 110 mmol/L.  The patient is euvolaemic and the aim is for plasma  sodium of 125 mmol/L.   If you aim to give treatment over 30 h = 20mmol/h  of sodium. That is 20 X 1000/1 54 mL of normal saline  an hour = approximately 130 mL, or 65mL/h twice-  normal saline.   Figure 2.14 Homeostatic mechanisms involved in the  correction of hypernatraemia. ADH, antidiuretic hormone.   Hypernatraemia always reflects hyperosmolality if  artefactual causes such as the use of sodium heparin  as an anticoagulant in the specimen container have  been excluded. Severe hypernatraemia can never  be appropriate to the osmolal state; the plasma  concentrations of solutes other than sodium are very  low and do not contribute significantly to the plasma  osmolality. For example, although hyperglycaemia  can increase the plasma osmolality by as much as  50 mmol/kg (about 17 per cent of the ‘normal’ plasma  osmolality), complete absence of glucose would reduce  it by only about 5 mmol/kg (about 2 per cent).   The symptoms associated with hypernatraemia may  include weakness, malaise, mental confusion, delirium  and coma.   Some causes of hypernatraemia are shown in  Box 2.4. These can also be grouped into euvolaemic,  hypovolaemic and hypervolaemic forms, based on the  clinical features.   • Remember that hypernatraemia is most commonly  due to predominant loss of water rather than to  excess of sodium.   • Is fluid of high sodium concentration being infused?  Much less commonly there may be a very high oral  sodium intake, but this is often deliberate. Some  antibiotics have a high sodium content.   • Clinically assess the patient’s state of hydration and  fluid balance.   Excessive sodium retention (sodium gains greater than  water gains - hypervolaemic)   • Is there polyuria? If so, look for evidence of diabetes  insipidus. Determine the ratio of urinary to plasma  osmolality (see later).   • If there is mild hypernatraemia with hypokalaemia  and hypertension, consider mineralocorticoid excess,  due, for example, to administration of steroids or to  Cushing’s syndrome or primary hyper aldosteronism.   Other causes of hypernatraemia are shown in Box  2.4, and may be clinically obvious.   A 5-year-old girl was admitted to hospital because  of a 4-day history of diarrhoea and vomiting.  On examination she was found to be clinically  ‘dehydrated’ with loss of skin turgor; her pulse was  120 beats/min and her blood pressure 74/50 mmHg.  Her admission results were as follows:   The severe hypernatraemia is in keeping with  hypotonic fluid loss due to the diarrhoea and  vomiting. The loss of skin turgor, tachycardia and  hypotension suggest hypovolaemia. The low urinary  sodium concentration indicates renal sodium  conservation. Children are particularly susceptible  to hypernatraemia induced by hypotonic fluid loss.   If the hypernatraemia is due to water depletion, oral  repletion is the treatment of choice. If this is not possible,  fluid of low sodium concentration, such as 5 per cent  dextrose saline, should be infused slowly. The aim  should be to lower the plasma sodium concentration at  no more than 1-2 mmol/L per hour or less, especially if  the hypernatraemia is long standing.   In hypervolaemic hypernatraemia, the  hypernatraemia may improve if patients reduce their  voluntary salt intake. The management of primary  hyperaldosteronism (Conn’s syndrome) and Cushing’s  syndrome is discussed in Chapter 8. Cranial diabetes   insipidus is usually treatedwith desmopressin. Hereditary  nephrogenic diabetes insipidus can be difficult to treat,  although thiazide diuretics or prostaglandin inhibitors  such as indometacin have been used.   The causes of polyuria are given in Box 2.5. Polyuria is  usually defined as more than 3 L of urine per day. The  causes and investigation of anuria (no urine) or oliguria  (less than 400 mL/ day) are discussed in Chapter 3.   - Distinguish between true polyuria (check 24-h  urinary volume) and frequency (a normal 24-h  volume but abnormally frequent micturition).  A midstream urine specimen may be useful to  exclude urinary tract infection as a cause of  urinary frequency.   - Is the patient taking diuretics? Consider other  drugs, for example lithium or demeclocycline.   Cerebral tumours and other infiltrations  Drugs and other agents, e.g. alcohol   Chronic kidney disease with predominant tubular  dysfunction, e.g. interstitial nephritis, medullary  cystic disease   - If there is a recent history of oliguric renal  failure, the patient has probably entered  the polyuric phase, and should be treated  accordingly (see Chapter 3).   • Exclude chronic kidney disease, hypokalaemia,  hypercalcaemia, adrenal insufficiency and thyrotoxi-  cosis.   - severe glycosuria due to diabetes mellitus or  infusion of dextrose,   - in infants, consider inborn errors of metabolism  (see Chapter 27),   • Accurately monitor fluid intake and output. The  patient should be carefully watched for secret fluid  ingestion (psychogenic polydipsia). Note also that  some patients may even add fluid to their urine or  drink ‘toilet’ water.   - Low or low-normal plasma urea or creatinine  concentrations, especially if there is mild  hyponatraemia, suggest that polydipsia is  the primary cause and that the polyuria is  appropriate. Question the patient about his  or her intake in relation to true thirst. Patients  with true psychogenic polydipsia may give  misleading answers.   • Measure the urinary and plasma osmolality. In an  osmotic diuresis the urine to plasma osmolality ratio  is usually more than 1.   • If urinary osmolality is more than 750 mmol/kg,  a water deprivation test is not usually indicated. A  useful screening test is an early morning urine test.   • If it is still not possible to distinguish between  psychogenic polydipsia and diabetes insipidus, contact  the laboratory to arrange a water deprivation test, if  necessary followed by administering l-desamino-8-  D-arginine vasopressin (DDAVP) (see below). This  may help distinguish cranial diabetes insipidus from  nephrogenic diabetes insipidus (see below).   • In difficult cases when diabetes insipidus is suspec-  ted and the water deprivation test has not been  helpful, a hypertonic saline test may be indicated  (see below).   • In cases of cranial diabetes insipidus, a neurological  opinion maybe necessary. Is there a possible obvious  cause for diabetes insipidus, such as a head injury?  (See Chapter 7.) Consider brain imaging such as  computerized tomography or magnetic resonance  imaging.   The causes of nephrogenic diabetes insipidus are  given in Box 2.5 (see also Chapter 3).   This section should be read in conj unction with Chapter  7, in which pituitary hormonal function is discussed.  The restriction of water intake for some hours should  stimulate posterior pituitary ADH secretion. Solute-  free water is reabsorbed from the collecting ducts  and concentrated urine is passed. Maximal water  reabsorption is impaired if:   Below are the pre-operative blood results of a  44-year-old man. The sample was reported by the  laboratory to be grossly ‘lipaemic’.   The plasma osmolality was not low despite  hyponatraemia, suggesting pseudohyponatraemia.  This is probably due to the gross lipaemia when  measured by indirect sodium ion-detecting electrodes  which some laboratories use to assay sodium (direct  electrodes do not usually detect pseudohypo-  natraemia). Note that calculated plasma osmolality  is 2 [ 1 18 + 4] + 6.0 + 4.0mmol/L = 254mmol/kg,  which differs markedly from the measured value of  290 mmol/kg.   If the feedback mechanism is intact, plasma ADH  levels will already be high and the administration  of exogenous hormone will not improve the renal  concentrating power. If diabetes insipidus is the  cause, tubular function will be normal and therefore  the administration of ADH will increase the renal  concentrating ability and increase the urinary  osmolality.   Warning The test is potentially dangerous, and  should not be performed if the patient is volume  depleted or has even mild hypernatraemia as fatality  could result. In such cases the finding of a low urinary  to plasma osmolality ratio (see below) should be  diagnostic, and further fluid restriction may be  dangerous. If the test is necessary, it must be stopped if:   • the plasma osmolality (or sodium concentration)  rises to high or high-normal concentrations - usually  taken as more than 300 mmol/kg; urine and blood  specimens should be collected at once,   • the patient loses more than 3 per cent of his or her  body weight.   Always contact the laboratory before starting the test,  both to ensure efficient and speedy analysis and to  check local variations in the protocol. The test should be  performed by a clinician experienced in the technique.  Patients are not allowed food or water after about   20.00 h on the night before the test. They must be in  hospital and closely observed during the period of fluid  restriction and weighed regularly during the test. The  duration of water deprivation depends on the clinical  presentation and the degree of polyuria.   08.00 h The bladder is emptied. Blood and urine are  collected and plasma and urinary osmolalities are  measured. If the plasma osmolality is low-normal or  low, water depletion is unlikely and polyuria is probably  due to an appropriate response to a high intake. If the  plasma osmolality is high-normal or high and the  urinary osmolality is more than 750 mmol/kg, the test  should be stopped.   09.00 h Blood and urine are again collected and the  plasma and urinary osmolalities are measured hourly.   The duration of the test depends on changes in the  urinary osmolalities.   • If the urinary osmolality is more than 750 mmol/  kg, neither significant tubular disease nor diabetes  insipidus is likely and the test may be stopped.   • If the urinary osmolality is less than 750 mmol/kg,  and plasma osmolality is normal, fluid restriction  should be continued and the estimations repeated at  hourly intervals. The test should be stopped as soon  as the urinary osmolality exceeds 750 mmol/kg.   • Failure of concentration of three consecutive urine  specimens indicates either tubular disease or diabetes  insipidus. If the diagnosis of diabetes insipidus is  considered, continue with the DDAVP test.   Give 2 pg DDAVP intramuscularly; continue to collect  urine and plasma samples.   If there is tubular dysfunction or nephrogenic diabetes  insipidus, there will be no change in the urinary  osmolality, but the plasma osmolality may increase  owing to water loss during the test. In cranial diabetes  insipidus the urinary osmolality increases, usually to a  value of more than 750 mmol/kg.   After prolonged overhydration, usually due to  psychogenic polydipsia, concentrating power may be  impaired, even after the administration of exogenous  hormone. This is due to ‘washing out’ of medullary  hyperosmolality. The test should be repeated after  several days of relative water restriction. The patient  should be kept under close observation, both for signs  of genuine distress associated with a rise in plasma  osmolality and for surreptitious drinking.   Table 2.5 Summary of the results of water deprivation/  DDAVP tests   In some cases in which there is diagnostic difficulty  with the water deprivation test, the hypertonic saline  test is used, but expert advice should be sought. This  test is sometimes used to make a diagnosis of cranial  diabetes insipidus in patients with polyuria but normal  plasma osmolality. It is potentially dangerous, as fluid  overload can occur, and therefore should not be used  in patients with cardiac or cerebral disease, or epilepsy.   The test is based on the principle that the infusion  of 5 per cent hypertonic saline, by raising plasma  osmolality, normally causes maximal ADH secretion. It  can be summarized as follows.   • 5 per cent saline is infused (0.04mL/kg per min for 2h).   • Plasma ADH is measured before and after the test  along with urine and plasma osmolality.   • The patient must be closely monitored during the  test, and the blood pressure and weight recorded.   Patients with cranial diabetes insipidus have little or  no rise in ADH. Conversely, in nephrogenic diabetes  insipidus there is usually ADH release during the test.   Figure 2.17 Fluid balance chart. It is essential to keep these accurate and regularly updated and reviewed. I  am grateful for the permission of Marian Davies and Dr Marthin Mostert, of University Hospital Lewisham, who  helped design the chart.   • Water and sodium homeostasis are intimately related,  and it is essential that doctors understand them  when investigating and managing hyponatraemia  and hypernatraemia. Summary algorithms for the  investigation of hyponatraemia and hypernatraemia  are depicted in Figures 2.13 and 2.15.   • Sodium is the main ECF cation and is controlled  by adrenal aldosterone (renal sodium retention if  ECF falls) and atrial natrial peptides.   • Body water excretion is controlled by ADH  (vasopressin) . Decreased ECF volume and increased   Polyuria (daily urine output more than 3 L) may be  due to failures of renal concentrating ability or lack  of ADH (Fig. 2.16).   Patient fluid balance must be monitored carefully,  with attention to the input and output, particularly  for those on ‘drip’ therapy (Fig. 2.17).   Polyuria (daily urine output more than 3 L) may be  due to failures of renal concentrating ability or lack  of ADH (Fig. 2.16).   In this chapter kidney function and how it can be  altered in disease states is discussed. It is best read in  conjunction with Chapters 2, 4 and 5. Interpretation  of renal function tests is also discussed.   The kidneys excrete metabolic waste products,  and have an essential homeostatic function in that  they control the body solute and water status and  the acid-base balance. There are about one million  nephrons per kidney, each of which is made up of  five main functional segments (Fig. 3.1).   The glomeruli, in the cortex of the kidney, are  invaginated and surround a capillary network of  blood vessels derived from the afferent, and draining  into the efferent, arterioles. Small molecules and  water are passively filtered during the passage of  blood through these capillaries, the ultrafiltrate  passing through the vessel walls and the glomerular   The proximal convoluted tubules, also in the cortex,  receive filtrate from the glomerular spaces. Convolution  increases the tubular length and therefore contact  between the luminal fluid and the proximal tubular  cells.   The loops of Henle extend down into the renal  medulla and ascend again after forming the loop.   The distal convoluted tubules, situated in the  cortex, are important for fine adjustment of luminal  fluid. They lie near the afferent arterioles, with the  juxtaglomerular apparatus between them. The enzyme  renin is produced by the latter and its release is  controlled by local blood flow.   The collecting ducts start as the distal tubules lead  down into the medulla and end by opening into the    Figure 3.1 The anatomical relation between the nephron and the juxtaglomerular apparatus.   renal pelvis. The modified fluid from the original filtrate  flows from the collecting ducts into the renal tract.   • an adequate blood supply, which under normal  circumstances is about 20 per cent of the cardiac  output, flowing through the kidneys,   • normal secretion and feedback control of hormones  acting on the kidney,   In addition to the excretory function and acid-  base control, the kidneys have important endocrine  functions, including:   • production of 1,25-dihydroxyvitamin D, the active  metabolite of vitamin D, which is produced following  hepatic hydroxylation of 25-hydroxyvitamin by the  renal enzyme 1 -hydroxylase,   About 200 L of plasma ultrafiltrate usually enter the  tubular lumina daily, mainly by glomerular filtration  into glomerular capsules but also through the spaces  between cells lining the tubules (tight junctions).  Production of ultrafiltrate depends on the blood  flow through normal glomeruli and on the difference  between the hydrostatic pressure gradient and the  plasma effective colloid osmotic (oncotic) pressure  gradient across the membranes (Fig. 3.2) and tight  junctions. The colloid osmotic effect is weak relative  to the hydrostatic gradient but does facilitate some  reabsorption of fluid from the proximal renal tubules.   Figure 3.2 The relationship between flow of blood through  the glomerulus and the factors that affect the rate of  filtration across the glomerular basement membrane.   The filtrate contains diffusible constituents at  almost the same concentrations as in plasma. About  30 000 mmol of sodium, 800 mmol of potassium,  800 mmol of urea, 300 mmol of free ionized calcium  and 1000 mmol of glucose are filtered daily. Proteins  (mainly low-molecular-weight proteins) and protein-  bound substances are filtered in only small amounts by  normal glomeruli and most are reabsorbed. The huge  volume of filtrate allows adequate elimination of waste  products such as urea; death from water and electrolyte  depletion would occur within a few hours were the bulk  of this water containing essential solutes not reclaimed.   Changes in filtration rate alter the total amount of water  and solute filtered, but not the composition of the filtrate.  From the 200 L of plasma filtered daily, only about 2L  of urine are formed. The composition of urine differs  markedly from that of plasma, and therefore of the  filtrate. The tubular cells use adenosine triphosphate-  dependent active transport, sometimes selectively, against  physicochemical gradients. Transport of charged ions  tends to produce an electrochemical gradient that inhibits  further transport. This is minimized by two processes.   Isosmotic transport This occurs mainly in the  proximal tubules and reclaims the bulk of filtered  essential constituents. Active transport of one ion leads  to passive movement of an ion of the opposite charge in  the same direction, along the electrochemical gradient.  The movement of sodium (Na + ) depends on the  availability of diffusible negatively charged ions, such  as chloride (Cl"). The process is ‘isosmotic’ because the  active transport of solute causes equivalent movement  of water reabsorption in the same direction. Isosmotic  transport also occurs to a lesser extent in the distal part  of the nephron.   Ion exchange This occurs mainly in the more  distal parts of the nephrons and is important for fine  adjustment after bulk reabsorption has taken place.  Ions of the same charge, usually cations, are exchanged  and neither electrochemical nor osmotic gradients are  created.   Therefore, during cation exchange there is  insignificant net movement of anions or water. For  example, Na + may be reabsorbed in exchange for  potassium (K + ) or hydrogen (H + ) ions. Na + and H +  exchange also occurs proximally, but at that site it is  more important for bicarbonate reclamation than for  fine adjustment of solute reabsorption (see Chapter 4).  In the cells lining the renal tubules, the intestine and   many secretory organs, the pumps are located on the  membrane on one side of the cell only and therefore  solute flows in one direction.   Other substances, such as phosphate and urate, are  secreted into, as well as reabsorbed from, the tubular  lumen. The tubular cells do not deal actively with waste  products such as urea and creatinine to any significant  degree. Most filtered urea is passed in urine (which  accounts for most of the urine’s osmolality), but some  diffuses back passively from the collecting ducts with  water; by contrast, some creatinine is secreted into the  tubular lumen.   Almost all the potassium is actively reabsorbed from  the proximal tubules, as is more than 70 per cent  of the filtered sodium, free ionized calcium and  magnesium. Some free ionized calcium is reabsorbed  at more distal sites, possibly from the loops of Henle.  This reabsorption may be stimulated by parathyroid  hormone (PTH) and inhibited by loop diuretics such  as furosemide. Only about 2 per cent of filtered calcium  appears in the urine.   Many inorganic anions follow an electrochemical  gradient; the reabsorption of sodium is limited by the  availability of chloride, the most abundant diffusible  anion in the filtrate. Bicarbonate is almost completely  recovered following exchange of sodium and hydrogen  ions (see Chapters 2 and 4). Specific active transport  mechanisms result in the almost complete reabsorption  of glucose, urate and amino acids. Some urate is secreted  into the lumina, mainly in the proximal tubules, but  most of this is reabsorbed.   Phosphate reabsorption is incomplete; phosphate  in tubular fluid is important for buffering hydrogen  ions. Inhibition of phosphate reabsorption by PTH  occurs in both the proximal and the distal convoluted  tubules, and accounts for the hypophosphataemia of  PTH excess. Thus almost all the reusable nutrients and  the bulk of electrolytes are reclaimed from the proximal  tubules, with fine homeostatic adjustment taking place  more distally. Almost all the filtered metabolic waste  products, such as urea and creatinine, which cannot be  reused by the body, remain in the luminal fluid.   Water is always reabsorbed passively along an osmotic  gradient. However, active solute transport is necessary   to produce this gradient (see also Chapter 2). Two main  processes are involved in water reabsorption:   • Isosmotic reabsorption of water from the proximal  tubules. The nephrons reabsorb 99 per cent of the  filtered water, about 70-80 per cent (140-160 T/day)  of which is returned to the body from the proximal  tubules. Active solute reabsorption from the filtrate  is accompanied by passive reabsorption of an  osmotically equivalent amount of water. Therefore,  fluid entering the lumina of the loops of Henle,  although much reduced in volume, is still almost  isosmotic.   • Dissociation of water reabsorption from that of  solute in the loops of Henle, distal tubules and  collecting ducts. Normally between 40 and 60 L of  water enter the loops of Henle daily. This volume  is reduced to about 2 L as varying amounts of  water are reabsorbed, helping to correct for  changes in extracellular osmolality. At extremes  of water intake, urinary osmolality can vary from  about 40 to 1400 mmol/kg. The proximal tubules  cannot dissociate water and solute reabsorption,  and the adjustment must occur between the  end of the proximal tubule and the end of the  collecting duct.   • Countercurrent multiplication is an active process  occurring in the loops of Henle, whereby a high  osmolality is created in the renal medulla and  urinary osmolality is reduced. This can occur in the  absence of antidiuretic hormone (ADH), also called  arginine vasopressin or vasopressin, and a dilute  hypo-osmolal urine is produced.   • Countercurrent exchange is a passive process,  occurring only in the presence of ADH. Water  without solute is reabsorbed from the collecting  ducts into the ascending vasa recta along the osmotic  gradient created by countercurrent multiplication  and by the high osmolality in the medulla, producing  a concentrated urine.   Countercurrent multiplication  This occurs in the loops of Henle. It depends on the  close apposition of the descending and ascending  limbs of the loops to the vasa recta. The vasa recta  make up a capillary network derived from the efferent  arterioles and, like the loops of Henle, pass deep into  the medulla.   The descending limbs are permeable to water but the  thick ascending limbs are impermeable to water and  solute. Chloride is actively pumped from the thick  ascending to the descending limbs as fluid flows through  the lumina of the loops; positively charged sodium ions  follow along the electrochemical gradient. Thus, the  osmolality progressively increases in the descending  limbs and renal medullary interstitium; it decreases in  the ascending limbs, but, as these are impermeable to  water, this change is not transmitted to the interstitium.   The almost isosmolal fluid enters the descending limbs  having the same osmolality as the plasma, just under  300 mmol/kg. If the fluid in the loops was stationary and  no pumping had taken place, the osmolality throughout   the loops and the adjacent medullary tissue would be  about 300 mmol/kg (Fig. 3.3a).   Suppose the fluid column remained stationary and  1 mmol of solute per kilogram were pumped from the  ascending into the descending limb, the result would be  as in Figure 3.3b. If this pumping continued and there  were no flow, the fluid in the descending limb would  become hyperosmolal and that in the ascending limb  correspondingly hypo-osmolal.   Suppose that the fluid flowed so that each figure ‘moved  two places’ (Fig. 3.3c). As this happened, more solute  would be pumped from the ascending to the descending  limbs (Fig. 3.3d). If the fluid again flowed ‘two places’, the  situation would be as shown in Figure 3.3e.    Figure 3.3 The renal counter-regulatory system. D, descending loop of Hen le; A, ascending loop of Henle.   If these steps occurred simultaneously and  continuously, the consequences would be as follows:   • Increasing osmolality in the tips of the loops of  Henle Because the walls of most of the loops are  permeable to water and solute, osmotic equilibrium  would be reached with the surrounding tissues in the  deeper layers of the medulla, including the plasma  within the vasa recta.   • Hypo-osmolal fluid leaving the ascending limbs  (Fig. 3.3f) In the absence of ADH, the walls of the  collecting ducts are impermeable to water, and  therefore no further change in osmolality occurs,  and hypo-osmolal urine would be passed.   Countercurrent exchange is essential, together  with multiplication, for regulating the osmolal  concentration of urine. It can only occur in the presence  of ADH and depends on the ‘random’ apposition  of the collecting ducts and the ascending vasa recta.  Antidiuretic hormone increases the permeability of  the cell membranes (via the aquaporins) lining the  distal parts of the collecting ducts to water, which   then moves passively along the osmotic gradient  created by multiplication. Consequently luminal fluid  is concentrated as the collecting ducts pass into the  increasingly hyperosmolal medulla.   The increasing concentration of the fluid would  reduce the osmotic gradient as it passes down the  ducts if it did not meet even more concentrated plasma  flowing in the opposite (countercurrent) direction.  The gradient is thus maintained, and water continues  to be reabsorbed until the fluid reaches the deepest  layers, where the osmolality is about four or five times  that of plasma (Fig. 3.3f). The low capillary hydrostatic  pressure at this site and the osmotic effect of plasma  proteins ensure that much of the reabsorbed water  within the interstitium enters the vascular lumina.  The diluted blood is carried towards the cortex and  ultimately enters the general circulation and helps to  dilute the extracellular fluid.   The osmotic action of urea in the medullary  interstitium may potentiate the countercurrent  multiplication. As water is reabsorbed from the  collecting ducts under the influence of ADH, the  luminal urea concentration increases. Because the distal  collecting ducts are permeable to urea, it enters the   Figure 3.4 The countercurrent mechanism, showing the relationship between the renal tubules and the vasa  recta. ADH, antidiuretic hormone.   deeper layers of the medullary interstitium, increasing  the osmolality and drawing water from the lower parts  of the descending limbs of the loops. The amount of  urea reabsorbed depends on:   • the rate of flow of tubular fluid: as much as  50 per cent of filtered urea may be reabsorbed when  flow is significantly reduced.   In summary, both concentration and dilution  of urine depend on active processes, which may be  impaired if tubules are damaged.   Renal homeostatic control of water excretion  In this section, the mechanisms involved in the normal  homeostatic control of urinary water excretion in  the extremes of water intake are discussed. It may be  helpful to read it in conjunction with Chapter 2, which  deals with sodium and water balance.   By increasing the plasma osmolality, water restriction  increases ADH secretion and allows countercurrent  exchange with enhanced water reabsorption. Reduced  circulatory volume results in a sluggish blood flow in the  vasa recta and increased urea reabsorption, allowing a  build-up of the medullary hyperosmolality produced by  multiplication. This potentiates water reabsorption in  the presence of ADH. The reduced capillary hydrostatic  pressure and increased colloid osmotic pressure, due to  the haemoconcentration following non-protein fluid  loss, ensure that much of the reabsorbed water enters  the vascular compartment.   A high water intake dilutes the extracellular fluid, and  the consequent fall in plasma osmolality reduces ADH  secretion. The walls of the collecting ducts therefore  remain impermeable to water and the countercurrent  multiplication produces a dilute urine and a high  osmolality within the medulla and medullary vessels.  Blood from the latter flows into the general circulation,  so helping to correct the fall in systemic osmolality.   During maximal water diuresis the osmolality at  the tips of the medullary loops may be 600 mmol/kg  or less, rather than the maximum of about 1400 mmol/  kg. Increasing the circulating volume increases renal  blood flow; the rapid flow in the vasa recta ‘washes  out’ medullary hyperosmolality, returning some of  the solute, without extra water, to the circulation.   Thus, not only is more water than usual lost in the  urine, more solute is ‘reclaimed’. Because medullary  hyperosmolality, and therefore the ability to concentrate  the urine maximally, is dependent on medullary blood  flow, under normal circumstances urinary osmolality  will be fully restored only several days after a prolonged  water load has stopped (see Chapter 2).   An excess of filtered solute in the proximal tubular  lumina impairs the bulk water reabsorption from  this site by its osmotic effect. Unabsorbed solute  concentration rises progressively as water is reabsorbed  with other solute during passage through the proximal  tubules, and this opposes further water reabsorption.  Thus a larger volume than usual reaches the loops of  Henle. Moreover, fluid leaving the proximal tubules,  although still isosmotic with plasma, has a lower  sodium concentration than plasma. The relative lack  of the major cation (sodium) to accompany the anion  chloride along the electrochemical gradient inhibits  the pump in the loops. The resulting impairment of  build-up of medullary osmolality inhibits distal water  reabsorption, under the influence of ADH from the  collecting ducts, resulting in a diuresis (see Chapter 2).   Normally most filtered water leaves the proximal  tubular lumina with reabsorbed solute. For example,  glucose (with an active transport system) and urea  (which diffuses back passively) are sometimes filtered  at high enough concentration to exceed the proximal  tubular reabsorptive capacity. They can then act as  osmotic diuretics and cause water depletion. This  is important, for example, in diabetes mellitus or in  uraemia.   The most effective osmotic diuretics are substances  that cannot cross cell membranes to any significant  degree; therefore, they must be infused, as they cannot  be absorbed from the gut. One example is mannitol, a  sugar alcohol, which is sometimes used therapeutically  as a diuretic.   Sodium reabsorption in exchange for hydrogen ions  occurs throughout the nephrons. In the proximal  tubules the main effect of this exchange is on  reclamation of filtered bicarbonate. In the distal  tubules and collecting ducts, the exchange process is  usually associated with net generation of bicarbonate  to replace that lost in extracellular buffering. Potassium   and hydrogen ions compete for secretion in exchange  for sodium ions. The possible mechanism stimulated  by aldosterone is discussed in Chapter 2. The most  important stimulus to aldosterone secretion is mediated  by the effect of renal blood flow on the release of renin  from the juxtaglomerular apparatus; this method of  reabsorption is part of the homeostatic mechanism  controlling sodium and water balance.   Different parts of the nephrons are in close anatomical  association and are dependent on a common blood  supply. Renal dysfunction of any kind affects all parts of  the nephrons to some extent, although sometimes either  glomerular or tubular dysfunction is predominant. The  net effect of renal disease on plasma and urine depends  on the proportion of glomeruli to tubules affected and  on the number of nephrons involved.   To understand the consequences of renal disease it  may be useful to consider the hypothetical individual  nephrons, first with a low glomerular filtration rate  (GFR) and normal tubular function, and then with  tubular damage but a normal GFR. It should be  emphasized that these are hypothetical examples, as  in clinical reality a combination of varying degree may  exist.   Uraemia is the term used to describe a raised plasma  urea concentration and is almost always accompanied  by an elevated creatinine concentration: in North  America this is usually referred to as azotaemia (a raised  nitrogen concentration).   The total amounts of urea and creatinine excreted  are affected by the GFR. If the rate of filtration fails  to balance that of production, plasma concentrations  will rise.   Phosphate and urate are released during cell  breakdown. Plasma concentrations rise because less  than normal is filtered. Most of the reduced amount  reaching the proximal tubule can be reabsorbed, and  the capacity for secretion is impaired if the filtered  volume is too low to accept the ions; these factors  further contribute to high plasma concentrations.   A large proportion of the reduced amount of filtered  sodium is reabsorbed by isosmotic mechanisms; less  than usual is then available for exchange with hydrogen  and potassium ions distally. This has two main outcomes:   • reduced hydrogen ion secretion throughout the  nephron: bicarbonate can be reclaimed only if  hydrogen ions are secreted; plasma bicarbonate  concentrations will fall,   • reduced potassium secretion in the distal tubule,  with potassium retention (potassium can still be  reabsorbed proximally).   If there is a low GFR accompanied by a low renal  blood flow:   • Systemic aldosterone secretion will be maximal: in  such cases, any sodium reaching the distal tubule will  be almost completely reabsorbed in exchange for H +  and K + , and the urinary sodium concentration will  be low.   • ADH secretion will be increased: ADH acting on  the collecting ducts allows water to be reabsorbed  in excess of solute, further reducing urinary volume  and increasing urinary osmolality well above that of  plasma and reducing plasma sodium concentration.  This high urinary osmolality is mainly due to  substances not actively dealt with by the tubules. For  example, the urinary urea concentration will be well  above that of plasma. This distal response will occur  only in the presence of ADH; in its absence, normal  nephrons will form a dilute urine.   If the capacity of the proximal tubular cells to  reabsorb solute, and therefore water, is normal, a larger  proportion than usual of the reduced filtered volume  will be reclaimed by isosmotic processes, thus further  reducing urinary volume.   In summary, the findings in venous plasma and  urine from the affected nephrons will be as follows.   • Low (appropriate) sodium concentration - only  if renal blood flow is low, stimulating aldosterone  secretion.   • High (appropriate) urea concentration and  therefore a high osmolality - only if ADH secretion  is stimulated.   Damage to tubular cells impairs adjustment of the  composition and volume of the urine. Impaired solute  reabsorption from proximal tubules reduces isosmotic  water reabsorption. Countercurrent multiplication  may also be affected, and therefore the ability of the  collecting ducts to respond to ADH is reduced. A large  volume of inappropriately dilute urine is produced.   The tubules cannot secrete hydrogen ions and  therefore cannot reabsorb bicarbonate normally  or acidify the urine. The response to aldosterone  and therefore the exchange mechanisms involving  reabsorption of sodium are impaired; the urine contains  an inappropriately high concentration of sodium for  the renal blood flow. Potassium reabsorption from the  proximal tubule is impaired and plasma potassium  concentrations may be low. Reabsorption of glucose,  phosphate, magnesium, urate and amino acids is  impaired. Plasma phosphate, magnesium and urate  concentrations may be low.   Thus, the findings in venous plasma and urine from  the affected nephrons will be as follows.   - even if renal blood flow is low, an  inappropriately high sodium concentration  (inability to respond to aldosterone),   - even if ADH secretion is stimulated, an  inappropriately low urea concentration and  therefore osmolality (inability of the collecting  ducts to respond to ADH).   There may also be tubular proteinuria, which  usually refers to low-molecular-weight proteins that  are normally produced in the body, filtered across the  glomerular membrane and reabsorbed in the proximal  tubule, but appear in the urine as a result of proximal  tubular damage, for example a l -microglob ulin and  retinol binding protein. However, tubular proteinuria  also occurs when proximal tubular enzymes and  proteins, such as j\ r - a c e t \ i - |I - 1 ) - g I u c o s a m i n i d a s e   (NAG), are released into the urine due to tubular cell  injury. See Chapter 19.   The biochemical findings and urine output in renal  disease depend on the relative contributions of  glomerular and tubular dysfunction. When the GFR  falls, substances that are little affected by tubular action  (such as urea and creatinine) are retained. Although  their plasma concentrations start rising above the  baseline for that individual soon after the GFR falls,  they seldom rise above the reference range for the  population until the GFR is below about 60 per cent  of normal, although in individual patients they do rise  above baseline.   Plasma concentrations of urea and creatinine  depend largely on glomerular function (Fig. 3.5). By  contrast, urinary concentrations depend almost entirely  on tubular function. However little is filtered at the  glomeruli, the concentrations of substances in the initial  filtrate are those of a plasma ultrafiltrate. Any difference  between these concentrations and those in the urine is  due to tubular activity. The more the tubular function is  impaired, the nearer the plasma concentrations will be  to those of urine. Urinary concentrations inappropriate  to the state of hydration suggest tubular damage,  whatever the degree of glomerular dysfunction.   The plasma sodium concentration is not primarily  affected by renal disease. The urinary volume depends  on the balance between the volume filtered and the  proportion reabsorbed by the tubules. As 99 per cent  of filtered water is normally reabsorbed, a very small  impairment of reabsorption causes a large increase in  urine volume. Consequently, if tubular dysfunction  predominates, impairment of water reabsorption  causes polyuria, even though glomerular filtration is  reduced (see Chapter 2).   The degree of potassium, phosphate and urate  retention depends on the balance between the degree of  glomerular retention and the loss as a result of a reduced   Figure 3.5 The effects of glomerular and tubular dysfunction on urinary output and on plasma concentrations of  retained ‘waste’ products of metabolism, the volume depending on the proportion of nephrons involved.   proximal tubular reabsorptive capacity. If glomerular  dysfunction predominates, so little is filtered that plasma  concentrations rise, despite the failure of reabsorption.  Conversely, if tubular dysfunction predominates,  glomerular retention is more than balanced by impaired  reabsorption of filtered potassium, urate and phosphate,  and therefore plasma concentrations may be normal  or even low. A low plasma bicarbonate concentration  is found in association with metabolic acidosis, which  may worsen the hyperkalaemia.   This was previously known as acute renal failure. In  adults, oliguria is defined as a urine output of less than  400 mL/ day, or less than 15mL/h; it usually indicates  a low GFR and a rapid decline in renal function over  hours to weeks, with retention of creatinine and  nitrogenous waste products. Oliguria may be caused by  the factors discussed below.   This is caused by factors that reduce the hydrostatic  pressure gradient between the renal capillaries and  the tubular lumen. A low intracapillary pressure is the   A 17-year-old man was involved in a road traffic  accident. Both femurs were fractured and his spleen  was ruptured. Two days after surgery and transfusion  of 1 6 units of blood, the following results were found:   The patient was producing only lOmL of urine per  hour and a spot urinary sodium was 8 mmol/L.   The results are compatible with pre-renal acute  kidney injury (AKI), secondary to massive blood loss.  Note the oliguria, low urinary sodium concentration,  hyperkalaemia, hyperphosphataemia and also low  plasma bicarbonate concentration, suggestive of a  metabolic acidosis.   • intravascular depletion of whole blood  (haemorrhage) or plasma volume (usually due to  gastrointestinal loss), or reduced intake,   • reduced pressure as a result of the vascular dilatation  caused by ‘shock’, causes of which include myocardial  infarction, cardiac failure and intravascular haemolysis,  including that due to mismatched blood transfusion.   The patient is usually hypotensive and clinically  volume depleted. If renal blood flow is restored within  a few hours, the condition is reversible, but, the longer it  persists, the greater the danger of intrinsic renal damage.   As most glomeruli are involved and tubular function is  relatively normal, the biochemical findings in plasma and  urine are those described earlier. Uraemia due to renal  dysfunction may be aggravated if there is increased protein  breakdown as a result of tissue damage, a large haematoma  or the presence of blood in the gastrointestinal lumen.  Intravenous amino acid infusion may have the same  effect because the urea is derived, by hepatic metabolism,  from the amino groups of amino acids. Increased  tissue breakdown may also aggravate hyperkalaemia,  hyperuricaemia and hyperphosphataemia.   Acute oliguria due to intrinsic renal damage  This may be due to:   • acute glomerulonephritis, usually in children - the  history of a sore throat and the finding of red cells in  the urine usually make the diagnosis obvious,   • septicaemia, which should be considered when the  cause of oliguria is obscure,   One problem in the differential diagnosis of acute  oliguria is distinguishing between renal circulatory  insufficiency and intrinsic renal damage that may have  followed it. Acute oliguric renal dysfunction often  follows a period of reduced GFR and renal circulatory  insufficiency.   The oliguria is due to reduced cortical blood flow  with glomerular damage, aggravated by back-pressure  on the glomeruli due to obstruction to tubular flow  by oedema. At this stage, the concentrations of many  constituents in plasma, such as urea and creatinine,  are raised with hyperkalaemia; tubular damage results   in an inappropriately dilute urine for the degree of  hypovolaemia. Fluid must be given with caution, and  only until volume depletion has been corrected; there is  a danger of overloading the circulation.   During recovery, oliguria is followed by polyuria.  When cortical blood flow increases, and as tubular  oedema resolves, glomerular function recovers before  that of the tubules. The biochemical findings gradually  progress to those of tubular dysfunction until they  approximate those for ‘pure’ tubular lesions. Urinary  output is further increased by the osmotic diuretic effect  of the high load of urea. The polyuria may cause water  and electrolyte depletion. The initial hyperkalaemia may  be followed by hypokalaemia. Mild acidosis (common to  both glomerular and tubular disorders) persists until late.  Recovery of the tubules may restore full renal function.   Oliguria or anuria (absence of urine) may occur in  post-renal failure. The cause is usually, but not always,  clinically obvious and may be due to the following:   • Intrarenal obstruction, with blockage of the tubular  lumina by haemoglobin, myoglobin and, very rarely,  urate or calcium. Obstruction caused by casts and  oedema of tubular cells is usually the result of true  renal damage.   • Extrarenal obstruction, due to calculi, neoplasms,  for example prostate or cervix, urethral strictures  or prostatic hypertrophy, any of which may cause  sudden obstruction. The finding of a palpable  bladder indicates urethral obstruction, and in males  is most likely to be due to prostatic hypertrophy,  although there are other, rarer, causes.   Early correction of outflow obstruction may rapidly  increase the urine output. The longer it remains  untreated, the greater the danger of ischaemic or  pressure damage to renal tissue. Imaging studies such  as renal tract ultrasound may be useful to confirm post-  renal obstruction (Box 3.1).   • A careful clinical history, especially of taking  nephrotoxic drugs, and examination may give  clues to the cause of acute kidney injury (AKI). It  is essential to exclude reversible causes of pre-renal  failure, including hypovolaemia or hypotension,  and also post-renal urinary tract obstruction (renal  tract imaging may be useful, such as abdominal   Acute tubular necrosis, e.g. hypotension, toxins,  contrast media, myoglobinuria, sepsis, drugs,  sustained pre-renal oliguria  Vasculitis   radiograph if calculi are suspected, and renal tract  ultrasound); see Box 3.1).   • Monitor urine output, plasma urea and creatinine  and electrolytes, as well as acid-base status.   • Hyperkalaemia, hypermagnesaemia, hyperphos-  phataemia, hyperuricaemia and metabolic acidosis  may occur in the oliguric phase of AKI.   • Urine microscopy may show granular casts  supportive of the diagnosis of acute tubular necrosis.   • The urinary to plasma urea ratio can be useful,  and when more than 10:1 is suggestive of pre-renal  problems. The urinary to plasma creatinine or  osmolality ratio may also be useful (Table 3.1).   • The fractional excretion of sodium (FENa%) is also  useful diagnostically and can be measured using  a simultaneous blood sample and spot urine (see  above and Fig. 3.6).   A 56-year-old man attended the renal out-patient  clinic because of polycystic kidneys, which had been  diagnosed 20 years previously. He was hypertensive  and the following blood results were returned:   Estimated glomerular filtration rate (eGFR) 14 mL/  min per 1.73 m 2   These results are typical of a patient with chronic  kidney disease (CKD) with raised plasma urea  and creatinine concentrations. The patient has  hyperkalaemia and a low plasma bicarbonate  concentration, suggestive of a metabolic acidosis. The  hypocalcaemia and hyperphosphataemia are also in  keeping with CKD stage 5.   Table 3.1 Some laboratory tests used to investigate  acute kidney injury   Note that in post-renal failure there is usually anuria.  FENa%, fractional excretion of sodium.   • An FENa% of less than 1 per cent is typical of pre-  renal failure, as is a urinary sodium concentration  more than 20 mmol/L.    Figure 3.6 Algorithm for the investigation of acute kidney injury (AKI). FENa%, fractional excretion of sodium.   • Blood may be necessary for full blood count,  coagulation screen and blood cultures. Also exclude  myeloma, and look for Bence Jones proteinuria  and cryoglobulins. Autoantibody screen, including  antineutrophil cytoplasmic antibody (ANCA),  antinuclear antibody (ANA), extractable nuclear  antigen (ENA) antibody, complement, antiglomerular  basement membrane antibodies and double-  stranded deoxyribonucleic acid (DNA), myoglobin,  plasma creatine kinase and plasma calcium, may also  be indicated, depending on the clinical situation.   • In obscure cases, renal biopsy may be necessary to  establish a diagnosis.   • Recently, urine neutrophil gelatinase-associated  lipocalin (NGAL) has been suggested as a marker of  renal injury and predictor of AKI.   Chro nicrenal dysfunction [definedasbeingreducedeGFR  (estimated GFR), proteinuria, haematuria and/or renal  structural abnormalities of more than 90 days’ duration]  is usually the end result of conditions such as diabetes  mellitus, hypertension, primary glomerulonephritis,  autoimmune disease, obstructive uropathy, polycystic  disease, renal artery stenosis, infections and tubular  dysfunction and the use of nephrotoxic drugs (Box  3.2). It is common, perhaps affecting about 13 per cent  of the population. Acute or chronic renal dysfunction  can occur when angiotensin-converting enzyme (ACE)  inhibitors or angiotensin II receptor blockers (ARBs) are  given to patients with renal artery stenosis; a clue to this   is an increase in plasma creatinine of about 20 per cent  and/or a decrease in eGFR of about 15 per cent soon  after initiation of the drug.   In most cases of acute oliguric renal disease there  is diffuse damage involving the majority of nephrons.  A patient who survives long enough to develop  chronic renal disease must have some functioning  nephrons.   Histological examination shows that not all  nephrons are equally affected: some may be  completely destroyed and others almost normal. Also,  some segments of the nephrons may be more affected  than others. The effects of chronic renal disease can  be explained by this patchy distribution of damage;  acute renal disease may sometimes show the same  picture.   In chronic kidney disease (CKD) the functional  adaptive effects can be divided into three main  categories: diminished renal reserve, renal insufficiency,  and end-stage uraemia. The loss of 75 per cent of renal  tissue produces a fall in GFR of 50 per cent. Although  there is a loss of renal function, homeostasis is initially  preserved at the expense of various adaptations  such as glomerulotubular changes and secondary  hyp erp ar athyroidism.   At first, glomerular function may be adequate to  maintain plasma urea and creatinine concentrations  within the reference range. As more glomeruli are  involved, the rate of urea excretion falls and the plasma  concentration rises. This causes an osmotic diuresis in  functioning nephrons; in other nephrons the tubules  may be damaged out of proportion to the glomeruli.  Both tubular dysfunction in nephrons with functioning  glomeruli and the osmotic diuresis through intact  nephrons contribute to the polyuria, other causes of  which should be excluded (see Chapter 2).   During the polyuric phase, the plasma concentration  of many substances, other than urea and creatinine,  may be anywhere between the glomerular and tubular  ends of the spectrum, although metabolic acidosis is  usually present.   If nephron destruction continues, the findings become  more like those of pure glomerular dysfunction.  Glomerular filtration decreases significantly and urine  output falls; oliguria precipitates a steep rise in plasma  urea, creatinine and potassium concentrations; and the  metabolic acidosis becomes more severe.   The diagnosis of CKD is usually obvious. In  the early phase, before plasma urea and creatinine  concentrations have risen significantly, there may  be microscopic haematuria or proteinuria. However,  haematuria may originate from either the kidney  or the urinary tract, and may therefore indicate  the presence of other conditions, such as urinary  tract infections, renal calculi or tumours (see Box  3.2).   • Plasma phosphate concentrations rise and  plasma total calcium concentrations fall. The  increased hydrogen ion concentration increases  the proportion of free ionized calcium, the plasma  concentration of which does not fall in parallel with  the fall in total calcium concentration. Impaired  renal tubular function and the raised phosphate  concentration inhibit the conversion of vitamin D  to the active metabolite and this contributes to  the fall in plasma calcium concentration. Usually,  hypocalcaemia should be treated only after correction  of hyperphosphataemia. After several years of CKD,  secondary hyperparathyroidism (see Chapter 6)  may cause decalcification of bone, with a rise in  the plasma alkaline phosphatase activity. Some  of these features of CKD can also evoke renal  osteodystrophy, associated with painful bones. The  increase in plasma PTH occurs early when the GFR  falls below 60 mh/min per 1.73 m 2 .   • Plasma urate concentrations rise in parallel with  plasma urea. A high plasma concentration does not  necessarily indicate primary hyperuricaemia; clinical  gout is rare unless hyperuricaemia is the cause of the  renal damage (see Chapter 20).   • Normochromic, normocytic anaemia due to  erythropoietin deficiency is common and, because  haemopoiesis is impaired, does not respond to  iron therapy; this can be treated with recombinant  erythropoietin.   • One of the commonest causes of death in patients  with CKD is cardiovascular disease, in part  explained by hypertension and a dyslipidaemia  of hypertriglyceridaemia and low high-density  lipoprotein cholesterol. Some of these effects maybe  due to reduced lipoprotein lipase activity.   • Abnormal endocrine function, such as  hyperprolactinaemia, insulin resistance, low plasma  testosterone and abnormal thyroid function, may  also be seen in chronic renal dysfunction.   • Some of the features of CKD may be explained by the  presence of ‘middle molecules’ - compounds that the  kidneys would normally excrete. These compounds,   of relatively small molecular weights, can exert toxic  effects upon body tissues.   • The presence of increasing proteinuria may be the  best single predictor of disease progression.   Irreversible but potentially modifiable complications  such as anaemia, metabolic bone disease, under-  nutrition and cardiovascular disease occur early in the  course of CKD. A summary of the clinical features of  chronic kidney disease is shown in Table 3.2.   There is a group of conditions that primarily affect  tubular function more than the function of the  glomeruli. However, scarring involving whole nephrons  may eventually cause chronic renal dysfunction.  Impaired function may involve a single transport  system, particularly disorders associated with amino  acid or phosphate transport, or may affect multiple  transport systems. Conditions associated with multiple  transport defects may cause renal tubular acidoses  - renal tubular disorders associated with a systemic  metabolic acidosis because of impaired reclamation of  bicarbonate or excretion of H + (see Chapter 4).   Disorders affecting the urine-concentrating  mechanism and causing nephrogenic diabetes insipidus  but which rarely in themselves cause a metabolic  acidosis are discussed elsewhere (see Chapter 2).   The nephrotic syndrome is caused by increased  glomerular basement membrane permeability, resulting  in protein loss, usually more than 3 g a day (or a urine  protein to creatinine ratio of >300 mg/mmol), with  consequent hypoproteinaemia, hypoalbuminaemia  and peripheral oedema. All but the highest molecular  weight plasma proteins can pass through the glomerular  basement membrane. The main effects are on plasma  proteins and are associated with hyperlipidaemia and  hyperfibrinoginaemia. (This is discussed more fully in  Chapter 19.) Uraemia occurs only in late stages of the  disorder, when many glomeruli have ceased to function.   This comprises reduced eGFR, oedema, hypertension  and proteinuria with significant haematuria. It is  usually associated with systemic disease such as post-  infectious glomerulonephritis, e.g. post-streptococcal  or immunoglobulin A (IgA) nephropathy, ANCA-  associated vasculitis, e.g. Wegener’s granulomatosis or  microscopic polyarteritis, or antiglomerular basement  membrane disease (Goodpasture’s disease).   As glomerular function deteriorates, substances that  are normally cleared by the kidneys, such as urea and  creatinine, accumulate in plasma.   Urea is derived in the liver from amino acids and  therefore from protein, whether originating from the  diet or from tissues. The normal kidney can excrete  large amounts of urea. If the rate of production exceeds  the rate of clearance, plasma concentrations rise. The  rate of production is accelerated by:   • absorption of amino acids and peptides from digested  blood after haemorrhage into the gastrointestinal  lumen or soft tissues,   • increased catabolism due to starvation, tissue  damage, sepsis or steroid treatment.   In catabolic states, glomerular function is often  impaired due to circulatory factors and this contributes  more to the uraemia than does increased production.  Conversely, the plasma urea concentration may be  lower than l.Ommol/L, the causes of which include the  following:   - use of amino acids for protein anabolism  during growth, especially in children,   - inborn errors of the urea cycle are rare and  usually only occur in infants.   Creatinine is largely derived from endogenous sources  by muscle creatine breakdown. Plasma creatinine usually  correlates with muscle mass, with 95 per cent of creatine  occurring in skeletal muscle. The plasma creatinine  concentration varies more than that of urea during the  day owing to creatine intake in meals. However, sustained  high-protein diets and catabolic states probably affect the  plasma concentration of creatinine less than that of urea.  Creatinine concentration is used to assess renal function;  however, its assay may be less precise than that of urea,  and can be prone to analytical interference by substances  such as bilirubin, ketone bodies and certain drugs.   If the plasma concentration of either urea or  creatinine is significantly raised, and especially if it is  rising, impaired glomerular function is likely. Serial  changes may be used to monitor changes in the GFR  and changes greater than 10-15 per cent are likely to be  clinically significant.   With a reduced GFR, plasma urea concentrations  tend to rise faster than those of creatinine and tend to  be disproportionately higher with respect to the upper  reference limit. The rate at which urea is reabsorbed  from the collecting ducts is dependent on the amount  filtered by the glomerulus and by the rate of luminal  fluid flow (see Table 3.2).   For a substance (S) that is filtered by the glomerulus,  but not reabsorbed from or secreted into the tubules,  the amount filtered (GFR X plasma[S]) must equal the  amount excreted in the urine (urinary[S] X volume per  unit time):   Figure 3.7 The inverse relationship between plasma  creatinine and creatinine clearance. (Shaded area is  approximate 95 per cent confidence intervals.)   The GFR thus measured is referred to as the clearance  - the volume of plasma that could theoretically be  completely cleared of a substance in lmin. Only  substances freely filtered by glomeruli and not acted on  by the tubules can be used to give true measurement  of GFR. There is no such endogenous substance, but  inulin, a polysaccharide, fulfils the criteria closely.  Inulin is not produced by the body; it must be given  either by constant infusion in order to maintain steady  plasma concentrations during the period of the test, or  by a single injection followed by serial blood sampling  to enable the concentration at the midpoint of the  collection to be calculated.   Radiochromium-labelled ethylenediamine tetra-  acetic acid (EDTA) is another exogenous compound  that some consider the ‘gold standard’ for calculating  patient GFR, although this requires the use of nuclear  medicine tests and is rarely used.   For endogenously produced substances such as  creatinine, with its relatively constant production, the  following equation can be used to calculate a clearance  that acts as an approximation for GFR:   urinary [creatinine] X urine volume (mL)  plasma [creatinine] X urine collection period (min)   The modification of diet in renal disease (MDRD)  formula can be used to estimate GFR (eGFR) and  has generally superseded the need to use creatinine  clearances in clinical practice and is also used to titrate  drug dosing in patients with renal impairment. This is  calculated by the isotope dilution mass spectrometry  (IDMS) traceable MDRD equation recommended  in the UK as: 175 x ([plasma creatinine] /88.4) -1154 x  (age) -0 - 203 x (0.742 if female) x (1.210 if black).   The equation has not been validated in the following  groups: those under 18 years old, those acutely ill,  patients with limb amputations, pregnant women,  the very elderly and the obese and malnourished. In  some of these situations there may be differences in  muscle mass and hence in creatine concentrations and  ultimately creatinine. Those with muscle breakdown  may show higher plasma creatinine concentration  and the converse may be seen in those with reduced  muscle bulk. There may be increased muscle bulk in  black compared with white people. Individuals taking  creatine supplements for body building may show  increased plasma creatinine and also plasma creatine  kinase (CK).   Creatinine clearance is higher than inulin clearance  because some creatinine is secreted by the tubules.  Urea clearance is lower than inulin clearance as some  urea is reabsorbed into the tubules (urea and inulin  clearance are now essentially obsolete in clinical  practice).   However, there are various factors that make the  measurement of creatinine clearance inaccurate:   • All laboratory assays have an inherent imprecision.  The combined imprecision of two assays is greater  than that of one. Urine as well as plasma is assayed  for clearance measurements.   • The most significant error of any method depending  on a timed urine collection is in the measurement  of urine volume. Inaccurate urine collection  may yield misleading results. The difficulties are  increased in infants and young children, and in  patients who have difficulty in bladder emptying  or are incontinent.   • Both creatinine and urea may be partly destroyed by  bacterial action in infected or old urine.   For an individual patient, plasma creatinine  concentrations may rise above the baseline level but  remain within the population reference range despite a  deterioration in glomerular function. The reciprocal of  the plasma creatinine concentration is called the renal  index.   The plasma creatinine concentration may not  exceed the upper limit of the reference range until the  GFR, and therefore the creatinine clearance, has been  reduced by approximately 60 per cent (see Fig. 3.7).  Thus the measurement of creatinine clearance should  be a more sensitive but less accurate indicator of early  glomerular dysfunction than of plasma creatinine  concentration.   Clearance values will be low whether the reduced  GFR is due to renal circulatory insufficiency, intrinsic  renal damage or ‘post-renal’ causes, and it cannot  distinguish among them. Creatinine clearance has  been said to be useful in deciding the dose of a renally  excreted drug.   Another endogenous substance that can be used as a  marker of GFR is plasma cystatin C (Cys C), and its  use may alleviate some of the problems associated  with creatinine clearance determinations. This is a  13-kDa protein that is a member of the family of  cystine proteinase inhibitors. Unlike other endogenous   compounds such as creatinine, Cys C is not secreted  by the renal tubules and does not return to the  bloodstream after glomerular filtration. It has been  suggested that plasma Cys C may approximate to  the ‘ideal’ endogenous marker for GFR, as blood  concentrations are independent of patient age and sex,  although currently this test is not routinely available in  most laboratories.   Renal tubular function tests  Reduced tubular function, with normal glomerular  function, impairs the adjustment of the composition and  volume of the urine with minimal effect on the plasma  urea or creatinine concentration. The investigations  used to diagnose tubular disorders can be divided into  those that predominantly identify proximal tubular  dysfunction and those that predominantly identify  distal tubular dysfunction.   Impaired solute reabsorption from the proximal tubules  reduces isosmotic water reabsorption. Countercurrent  multiplication may also be affected, and hence the  ability to respond to ADH is reduced. A large volume of  inappropriately dilute urine is produced.   The tubules cannot secrete hydrogen ions and so  cannot reabsorb bicarbonate normally and therefore  the urine is inappropriately alkaline for the degree of  acidosis in the blood.   The reabsorption of potassium, phosphate,  magnesium, urate, glucose and amino acids is impaired.  The following findings maybe present, and measurement  may occasionally be useful.   Tubular proteinuria can be diagnosed by measuring  specific low-molecular-weight proteins such as retinol-  binding protein, NAG or o^-microglobulin, which   are increased in the urine because of reduced tubular  reabsorption and increased renal tubular secretion. If  there is detectable glycosuria, phosphaturia and non-  selective amino aciduria, the condition is known as  Fanconi’s syndrome.   Impaired distal tubular function primarily affects  urine acidification, with a failure to excrete hydrogen  ions; the urinary pH rarely falls below 5.5. There  is an impaired response to aldosterone involving  reabsorption of sodium, and the urine contains an  inappropriately high concentration of sodium for the  renal blood flow. The associated findings may include  the following.   • Low bicarbonate and high chloride concentration with  low pH (hyperchloraemic acidosis), hypokalaemia.   • An inappropriately high sodium concentration, even  if renal blood flow is low (inability to respond to  aldosterone).   • An inappropriately low urea concentration, and  therefore osmolality, even if ADH secretion is  stimulated.   The ability to form concentrated urine in response  to fluid deprivation depends on normal tubular  function (countercurrent multiplication) and on  the presence of ADH. Failure of this ability is usually  due to renal disease, or cranial diabetes insipidus (see  Chapter 2 for discussion of the fluid deprivation test).  The investigation of renal tubular acidosis is covered in  Chapter 4.   Urinary sodium estimation may be used to differentiate  acute oliguria due to renal damage from that due to  renal circulatory insufficiency. Aldosterone secretion  will be maximal only if renal blood flow is reduced;  in such circumstances, functioning tubules respond  appropriately by selectively reabsorbing sodium by  distal tubular exchange mechanisms. A urinary sodium  concentration of less than about 20 mmol/L is usually  taken to indicate that tubular function is not significantly  impaired.   Measurement of urinary osmolality or other indicators  of selective water reabsorption, such as urinary urea  or creatinine concentrations, is less valuable than  assaying urinary sodium concentration, as ADH  secretion is stimulated for many other causes (see  Table 3.1).   Pre-renal AKI can sometimes be reversed by careful  control of fluid balance and prompt treatment  of hypovolaemia. Sometimes furosemide with  mannitol or dopamine infusion may re-establish  normal urine flow. If oliguria is due to parenchymal/  intrinsic damage, some clinicians may restrict fluid  and sodium intake, giving only enough fluid to  replace losses and provide an adequate low-protein  energy intake to minimize aggravation of uraemia.  If possible, the cause of the intrinsic renal failure  should be treated. Careful attention should be given  to nephrotoxic drugs in AKI. In post-renal failure,  prompt relief of the obstruction may reverse the  situation.   A polyuric phase may occur, particularly on relief  of urinary obstruction with excretion of potassium  and magnesium, and this can result in hypovolaemia,  hypokalaemia and hypomagnesaemia, which may need  correcting.   Renal replacement therapy (RRT) such as dialysis  or haemofiltration may improve fluid and electrolyte  imbalances (see below). RRT is important to prevent  dangerous hyperkalaemia or if resistant pulmonary  oedema is present. Other indications for RRT include  severe acidosis of pH less than 7.1, encephalopathy and  uraemic pericarditis.   Careful control of fluid and electrolyte balance is  important; water intake is usually only restricted if  the plasma sodium concentration is not maintained.  Similarly, sodium intake should be unrestricted unless  contraindications such as hypertension or oedema  exist. Plasma potassium monitoring is essential and  potassium restriction may become necessary (and  avoidance of potassium-retaining medication) if there  is hyperkalaemia, which may need specific therapy and  can be life threatening (see Chapter 5).   Control of blood pressure, lipids and, if present,  diabetes mellitus (optimization of glycaemic control) is  important and may help slow decline of eGFR and reduce  cardiovascular risk, which is common in these patients.  Angiotensin-converting enzyme inhibitors may slow  the decline in renal function, although patients should  be monitored for hyperkalaemia. Dietary modification  with increased caloric intake along with reduced dietary  protein intake may slow the decline in GFR by reducing  protein catabolism. Nevertheless, it is also important to  ensure that the patient is well nourished.   Tissue precipitation of calcium to phosphate  may occur early in renal disease and is related to  hyperphosphataemia and the calcium phosphate  product (calcium concentration X phosphate  concentration). This precipitation can be reduced by  adequate fluid intake. Dietary phosphate restriction is  used in the early stages of chronic renal dysfunction.  If the plasma phosphate concentration is raised,  phosphate-binding agents such as calcium acetate  or carbonate may be indicated. When GFR is below  60 mL/minper 1.73 m 2 , secondaryhyperparathyroidism  with elevated PTH concentration occurs. Giving  small doses of active vitamin D, such as calcitriol or  alfacalcidol, reduces the serum PTH, and improves  bone histology, and leads to increased bone mineral  density and helps avoid renal osteodystrophy,  hypocalcaemia and tertiary hyperparathyroidism (see  Chapter 6).   Recombinant erythropoietin and iron therapy may  be indicated to treat anaemia when haemoglobin is  less than 1 1 g/dL; this may slow progression of chronic  renal disease.   Dialysis removes urea and other toxic substances  from the plasma and corrects electrolyte balance by  dialysing the patient’s blood against fluid containing  no urea and appropriate concentrations of electrolytes,  free ionized calcium and other plasma constituents.  The following are the principal forms of dialysis:   • Haemofiltration is a form of haemodialysis in which  large volumes of fluid and solute can be removed  through a highly permeable membrane; dialysis  is dependent primarily on the blood pressure.  Haemofiltration is mainly used for AKI whereas the  following forms of dialysis are mainly for CKD.   • In haemodialysis, blood is passed through an  extracorporeal circulation and dialysed across an  artificial membrane with a solution of low solute  concentration before being returned to the body.   Negative pressure on the dialysate side of the membrane  can be varied to adjust the amount of water removed.   • In intermittent and continuous ambulatory  peritoneal dialysis, the folds of the peritoneum are  used as the dialysing membrane with capillaries  on one side, and an appropriate fluid of higher  osmolality is infused into the peritoneal cavity on the  other. After a suitable time to allow for equilibration  of diffusible solutes, depending on the type of  peritoneal dialysis, the peritoneal cavity is drained  and the cycle is repeated.   Dialysis is used in some cases of acute kidney injury  until renal function improves, or as a regularly repeated  procedure in suitable cases of end-stage kidney disease.  It may also be used to prepare patients for renal  transplantation.   Renal calculi are usually composed of products of  metabolism present in normal glomerular filtrate, often at  concentrations near their maximum solubility (Fig. 3.8).   • A high urinary concentration of one or more  constituents of the glomerular filtrate, due to:   - a low urinary volume with normal renal  function, because of restricted fluid intake or  excessive fluid loss over a long period of time  (particularly common in hot climates) - this  favours formation of most types of calculi,  especially if one of the other conditions listed  below is also present,    Figure 3.8 A renal calculus. Reproduced with  permission from Nyhan WL and Barshop BA. Atlas of  Inherited Metabolic Diseases, 3rd edition. London:  Hodder Arnold, 2012.   - a high rate of excretion of the metabolic  product forming the stone, due either to  high plasma and therefore filtrate levels or to  impairment of normal tubular reabsorption  from the filtrate.   • Changes in pH of the urine, often due to bacterial  infection, which favour precipitation of different  salts at different hydrogen ion concentrations.   • Urinary stagnation due to obstruction to urinary  outflow or renal tract structural abnormality.   • Lack of normal inhibitors: urine normally contains  inhibitors, such as citrate, pyrophosphate and  glycoproteins, which inhibit the growth of calcium  phosphate and calcium oxalate crystals respectively.  Hypocitraturia may partly explain the renal calculi  found in distal or type 1 renal tubular acidosis (see  Chapter 4).   About 80 per cent of all renal stones contain calcium.  Precipitation is favoured by hypercalciuria, and the type  of salt depends on urinary pH and on the availability  of oxalate. Any patient presenting with calcium-  containing calculi should have plasma calcium and  phosphate estimations performed, and, if the results are  normal, they should be repeated at regular intervals to  exclude primary hyperparathyroidism.   Hypercalcaemia causes hypercalciuria if glomerular  function is normal. The causes and differential   diagnosis of hypercalcaemia are discussed in Chapter  6. In many subjects with calcium-containing renal  calculi the plasma calcium concentration is normal.  Any increased release of calcium from bone (as  in actively progressing osteoporosis, in which loss  of matrix causes secondary decalcification, or in  prolonged acidosis, in which ionization of calcium  is increased) causes hypercalciuria; hypercalcaemia  is unusual in such cases. In distal renal tubular  acidosis there is an increased calcium load and,  because of the relative alkalinity of the urine, calcium  precipitation in the kidney and renal tract may occur  - nephrocalcinosis.   Hypercalciuria has been defined as a daily urinary  calcium excretion of more than 6.2 mmol in adult  females and 7.5 mmol in adult males.   A significant proportion of cases remain in which  there is no apparent cause for calcium precipitation.  A common cause is hypercalciuria despite   Hyperoxaluria favours the formation of the very  poorly soluble calcium oxalate, even if calcium  excretion is normal. The source of the oxalate may be  derived exogenously from the diet. Oxalate absorption  is increased by fat malabsorption: calcium in the bowel  is bound to fat instead of precipitating with oxalate,  which is then free to be absorbed. Foods rich in oxalate  include rhubarb, chocolate, beetroot, spinach, nuts  and tea.   Primary hyperoxaluria, a rare inborn error, should  be considered if renal calculi occur in childhood.  There are two main types, 1 and 2, the former being  more common. Type 1 is due to deficiency of alanine  glyoxylate aminotransferase, and type 2 is due to  deficient D-glycerate dehydrogenase. Hyperoxaluria  (urinary oxalate greater than 400pmol/24h) is a more  important risk factor for formation of renal stones than  is hypercalciuria.   Calcium- containing calculi are usually hard, white  and radio-opaque. Calcium phosphate may form  ‘staghorn’ calculi in the renal pelvis, while calcium  oxalate stones tend to be smaller and to lodge in the  ureters, where they are compressed into a fusiform  shape. Alkaline conditions favouring calcium phosphate  precipitation and stone formation are particularly  common in patients with chronic renal infection.   The treatment of calcium-containing calculi depends  on the cause. Urinary calcium concentration should be  reduced:   • if this is not possible, by reducing dietary calcium  (although this alone may exacerbate hyperoxaluria)  and oxalate intake,   • by maintaining a high fluid intake, unless there is  glomerular failure.   Thiazide diuretics reduce urinary calcium excretion,  and treatment of urinary tract infection may reduce the  risk of calculi formation.   These stones (about 10 per cent of all renal calculi)  are associated with chronic urinary tract infections by  organisms such as Proteus species capable of splitting  ammonium. The urinary pH is usually greater than 7.  These urease-containing bacteria convert urea to  ammonia and bicarbonate.   About 8 per cent of renal calculi contain uric acid; these  are sometimes associated with hyperuricaemia, with or  without clinical gout. In most cases, no predisposing  cause can be found. Precipitation is favoured in an acid  urine. Uric acid stones are usually small, friable and  yellowish brown, but can occasionally be large enough  to form ‘staghorn’ calculi. They are radiolucent but  may be visualized by ultrasound or by an intravenous  pyelogram.   The treatment of hyperuricaemia is discussed  in Chapter 20. If the plasma urate concentration is  normal, fluid intake should be kept high and the urine  alkalinized. A low-purine diet may help to reduce urate  production and excretion.   Cystine stones are rare. In normal subjects the  concentration of cystine in urine is soluble, but in  homozygous cystinuria this may be exceeded and the  patient may present with radio-opaque renal calculi.  Like urate, cystine is more soluble in alkaline than in  acidic urine; the principles of treatment are the same as  for uric acid stones. Penicillamine can also be used to  treat the condition (see Chapter 27).   Xanthine stones are very uncommon and may be the  result of the rare inborn error xanthinuria.   These are seen in patients with human immuno-  deficiency virus (HIV) infection who have been treated  with the protease inhibitor indinavir. The stones are  composed of pure protease inhibitor.   Other rare stones may consist of dihydroxyadenine (due  to adenine phosphoribosyltransferase deficiency) or  poorly calcified mucoproteinaceous material associated  with chronically infected kidneys (matrix stone). Some  stones may be factitious, as sometimes found in patients  with Munchausen’s syndrome, who may add ‘stones’ to  their urine.   Investigation of a patient with renal calculi  • If the stone is available, send it to the laboratory for  analysis (Fig. 3.9).   • Exclude hypercalcaemia (see Chapter 6) and  hyperuricaemia (see Chapter 20).   • Collect a 24-h specimen of urine for urinary volume,  calcium and oxalate estimations. These tests will help  to detect hypercalciuria or hyperoxaluria.   • If all these tests are negative, and especially if there  is a family history of calculi, screen the urine for  cystine. If the qualitative test is positive, the 24-h  excretion of cystine and basic amino acids should be  estimated.   • If fresh uninfected urine is alkaline despite a  systemic metabolic acidosis, the diagnosis of renal  tubular acidosis is likely (see Chapter 4). A pH more  than 7 is suggestive of a urinary infection with a  urea- splitting organism such as Proteus vulgaris, in  which case consider struvite calculi. A midstream  urine specimen is useful to exclude infection before  diagnosing renal tubular acidosis.   A 21 -year-old man presented to the urology out-  patient clinic because of renal calculi. There was also  a family history of renal calculi.   Estimated glomerular filtration rate (eGFR) > 90 mL/  min per 1.73m 2   Urinary excretion of both calcium and oxalate fell  within the laboratory reference ranges. However,  cystine was detected in the urine.   In conjunction with the family history and relatively  young age of presentation, the results are suggestive  of cystinuria manifesting cystine stones. This is one  of the most common amino acidurias, although a  rare cause of renal calculi, and is treated by increasing  fluid intake and alkalinizing the urine.   • Low plasma urate and high urinary xanthine  concentrations suggest xanthinuria, particularly in  a child.   • Determination of urinary citrate (an inhibitor of  some renal calculi) concentrations may sometimes  be useful, as low concentrations may be found.   • If the cause is still unclear, consider the rare causes of  renal calculi shown in Box 3.3.   • Renal tract imaging techniques to clarify the anatomy,  such as ultrasound or intravenous pyelogram, may  also be necessary.   Apart from specific treatments (see above), patients  with a tendency to form calculi are generally advised  to drink more water. The aim is usually to increase  the urinary volume to about 2-3 L in 24 h. Reducing  calcium intake may not be advisable, as it may increase  oxalate absorption and excretion. Calculi removal  by fragmentation using extracorporeal shock wave  lithotripsy has in some cases reduced the need for  surgical intervention.   • The kidneys are vital organs for the excretion  of various waste products as well as for acid-  base balance, fluid volume control, hormone  production and metabolic function, such as  calcium homeostasis.   • Plasma creatinine determination is a useful test of  renal function, but plasma creatinine concentration  can still remain within the reference range in the  presence of a significant decline in renal function.   • Acute kidney injury (AKI) can be due to pre-renal,  renal or post-renal causes. Raised plasma urea  and creatinine concentrations occur along with  fluid retention, anuria or oliguria, hyperkalaemia,  hyperphosphataemia and metabolic acidosis.   • End-stage chronic kidney disease (CKD5) implies  slow, irreversible renal disease. Raised plasma  urea and creatinine concentrations occur initially   and, as renal reserve declines, there is further  hyperkalaemia, hyperphosphataemia, metabolic  acidosis, hypocalcaemia and anaemia. This may  necessitate renal support such as dialysis.   • Renal calculi can be the result of urinary stasis or  infection associated with urinary supersaturation.  The commonest calculi are calcium containing.   • Nephrotic syndrome is defined as gross proteinuria  associated with oedema and hypoproteinaemia  (discussed further in Chapter 19). This is a disorder  of the renal glomerular membrane.   • Renal tubular disease can result in Fanconi’s  syndrome associated with acid-base and  potassium disturbance, glycosuria, amino aciduria,  hypouricaemia and hypophosphataemia.   • Renal replacement therapy, such as dialysis  (Fig. 3.10), maybe indicated in AKI and CKD5.    Figure 3.10 A renal dialysis machine used to give renal replacement therapy in some patients with end-stage  chronic kidney disease (CKD5). Reproduced with kind permission of Pemed.   The importance of acid-base homeostasis cannot  be overstated because of its importance in keeping  hydrogen ion (H + ) balance under control. Acid-base  abnormalities are relatively common medically, and it  is therefore essential for clinicians to be fully conversant  with their interpretation.   Our cells release between 50 and 100 mmol of H +  into the extracellular fluid (ECF) daily. Despite this, the  extracellular H + concentration ( [H + ] ) is maintained at  about 40nmol/L (pH 7.4).   • Anaerobic carbohydrate metabolism produces  lactate, and anaerobic metabolism of fatty acids  by (3-hydroxylation and of ketogenic amino acids  produces acetoacetate, which releases equimolar  amounts of H + . Lactic acidosis or ketoacidosis can  occur if the release of H + by these reactions exceeds  the compensatory capacity.   • Release of H + can occur during conversion of amino  nitrogen to urea in the liver, or of the sulphydryl  groups of some amino acids to sulphate.   Hydrogen ion balance is largely dependent upon  the secretion of H + from the body into the urine due  to renal tubular action. Aerobic metabolism of the  carbon skeletons of organic compounds converts  carbon, hydrogen and oxygen into carbon dioxide  and water. Carbon dioxide is an essential component  of the extracellular buffering system. Thus the body is  dependent upon healthy function of the kidneys and  the lungs for normal acid-base homeostasis.   Acids can dissociate to produce H + (protons), which  can be accepted by a base. An alkali dissociates to  produce hydroxyl ions (OH - ). Acidosis is commoner  than alkalosis because metabolism tends to produce H +  rather than OH - .   A strong acid is almost completely dissociated  in aqueous solution, and so produces many H + . For  example, hydrochloric acid is a strong acid and is  almost entirely dissociated in water to form H + and  chloride (Cl - ). Weak acids dissociate less, although  very small changes in [H + ] may have important  consequences.   Buffering is a process by which a strong acid (or  base) is replaced by a weaker one, with a consequent  reduction in the number of free H + , and therefore the  change in pH, after addition of acid, is less than it would  be in the absence of the buffer. For example:   The pH is a measure of H + activity. It is log 10 of the  reciprocal of [H + ] in mol/L. The log 10 of a number is  the power to which 10 must be raised to produce that  number:   log 100 = log 10 2 = 2, and log 10 7 = 7 (4.2)   If [H + ] is 10 -7 (0.0000001) mol/L, then -log [H + ] = 7.  But:   P H = log = - log [H + ] (4.3)   A change of 1 pH unit represents a 10-fold change  in [H + ]. Changes of this magnitude do not normally  occur in tissues. However, in pathological conditions  the blood pH can change by more than 0.3 of a unit;  a decrease of pH by 0.3, from 7.4 to 7.1, represents a  doubling of the [H + ] from 40 to 80nmol/L. Thus, the  use of the pH notation makes a very significant change  in [H + ] appear deceptively small.   A blood pH of 7.0 indicates a severe acidosis. A blood  pH of 7.7 similarly indicates a severe alkalosis. Urinary   pH is much more variable than that of blood, and [H + ]  can vary 1000-fold (a change of 3 pH units).   The Henderson-Hasselbalch equation expresses the  relation between pH and a buffer pair - that is, a weak  acid and its conjugate base. The equation is valid for  any buffer pair, the pH being dependent on the ratio  of the concentration of base to acid. Note that pK a is  the negative logarithm of the acid dissociation constant  (K ), and, the larger the value of pK a , the smaller the  extent of acid dissociation:   In this equation the base is bicarbonate (HC0 3 “) and  the acid is carbonic acid (H 2 C0 3 ). It is not possible to  measure the latter directly; however, it is in equilibrium  with dissolved CO,, of which the partial pressure  (Tco 2 ) can be estimated. The concentration of H 2 C0 3  is derived by multiplying this measured value by the  solubility constant (S) for CO,. Thus:   If the Pco 2 is expressed in kPa, S = 0.23, or in mmHg,  S = 0.03.   Plasma [HC0 3 “] is controlled largely by the kidneys  and Pco, by the lungs. In acid-base disturbances due  to respiratory problems the kidneys are essential for  compensation and, conversely, in metabolic (non-  respiratory) causes of acid-base imbalance the  compensation is due mainly to changes in pulmonary  function.   Despite considerable fluctuations in the rate of  release of H + into the ECF, the [H + ], and therefore pH,  is relatively tightly controlled in blood by the following  mechanisms.   • The reaction is reversible, and H + combines with  HCO " only if the reaction is driven to the right  by the removal of CO,. By itself, this would cause  HC0 3 ” depletion.   • Buffering of H + is a temporary measure, as the H +  has not been excreted from the body. The production   of the weak acid of the buffer pair causes only a  small change in pH (see Henderson-Hasselbalch  equation). If H + are not completely neutralized  or eliminated from the body, and if production  continues, buffering power will eventually be so  depleted that the pH will change significantly.   • There are two main ways by which H + can be lost  from the body: through the kidneys or some of the  intestine, mainly the stomach. This mechanism is  coupled with the generation of HC0 3 “. In the kidney  this is the method by which secretion of excess H +  ensures regeneration of buffering capacity.   Carbon dioxide and H + are potentially toxic products  of aerobic and anaerobic metabolism respectively. Most  CO, is lost through the lungs, but some is converted  to HC0 3 “, thus contributing important extracellular  buffering capacity; inactivating one toxic product  provides a means of minimizing the effects of the other.   A buffer pair is most effective at maintaining a pH  near its pK a , that is, when the ratio of the concentrations  of base to acid is close to 1. However, the optimum  pH of the ECF is about 7.4, and the pK a of the  bicarbonate system is 6.1. Although this may seem to  be disadvantageous, the bicarbonate system is the most  important buffer in the body because:   • it accounts for more than 60 per cent of the blood  buffering capacity,   • it is necessary for efficient buffering by haemoglobin  (Hb), which provides most of the rest of the blood  buffering capacity.   The partial pressure of C0 2 ( Pco,) in plasma is normally  about 5.3 kPa (40 mmHg) and depends on the balance  between the rate of production by metabolism and the  loss through the pulmonary alveoli. The sequence of  events is as follows:   1. Inspired oxygen (0 2 ) is carried from the lungs to the  tissues by Hb.   2. The tissue cells use the 0 2 for aerobic metabolism;  some of the carbon in organic compounds is  oxidized to C0 2 .   3. C0 2 diffuses along a concentration gradient from  the cells into the ECF and is returned by the blood  to the lungs, where it is eliminated in expired air.   4. The rate of respiration, and therefore the rate of CO,  elimination, is controlled by chemoreceptors in the  respiratory centre in the medulla of the brainstem  and by those in the carotid and aortic bodies. The  receptors respond to changes in the [CO,] or [H + ]  of plasma or of the cerebrospinal fluid. If the Pco 2  rises much above 5.3 kPa, or if the pH falls, the rate  of respiration increases.   Normal lungs have a very large reserve capacity for  elimination of C0 2 . Not only is there a plentiful supply  of CO,, the denominator in the Henderson-Hasselbalch  equation, but the normal respiratory centre and lungs  can control its concentration within narrow limits  by responding to changes in the [H + ] and therefore  compensate for changes in acid-base disturbances.   The erythrocytes and renal tubular cells generate  HCO, - , the buffer base in the bicarbonate system, from  C0 2 . Under physiological conditions:   • the erythrocyte mechanism makes fine adjustments  to the plasma [HCOy] in response to changes in  Pco 2 in the lungs and tissues,   • the kidneys play the major role in maintaining the  circulating [HC0 3 - ] and in eliminating H + from the  body.   The carbonate dehydratase system  Bicarbonate is produced following the dissociation  of carbonic acid formed from CO and H 2 0. This is  catalysed by carbonate dehydratase (CD; carbonic  anhydrase), which is present in high concentrations in  erythrocytes and renal tubular cells:   C0 2 + H,0 H 2 C0 3 H + + HC0 3 - (4.8)   Erythrocytes and renal tubular cells have high  concentrations of CD, but also have means of removing  one of the products, H + ; thus both reactions continue  to the right, and HC0 3 _ is formed. One of the reactants  (water) is freely available and one of the products (H + )  is removed. Bicarbonate ion generation is therefore  accelerated if the concentration of:   • H + falls because it is either buffered by erythrocytes  or excreted from the body by renal tubular cells.   Therefore, an increase in intracellular Pco 2 or a  decrease in intracellular [HC0 3 “] in the erythrocytes  and renal tubular cells maintains the extracellular  [HC0 3 “] by accelerating the production of HCO This  minimizes changes in the ratio of [HCO, - ] to Pco 2 and  therefore changes in pH.   In the normal subject, at a plasma Pco, of 5.3 kPa  (a [CO ] of about 1.2mmol/L), erythrocytes and  renal tubular cells maintain the extracellular HC0 3 -  at about 25mmol/T. The extracellular ratio of  [HC0 3 - ] to [CO,] (both in mmol/L) is just over 20:1.  It can be calculated from the Henderson-Hasselbalch  equation, and with a pK a of 6.1 this represents a pH  very near 7.4. An increase of intracellular Pco,, or a  decrease in intracellular [HCO "], accelerates HC0 3 -  production and minimizes changes in the ratio and  therefore in pH.   Bicarbonate generation by the erythrocytes (Fig. 4.1)  Haemoglobin is an important blood buffer. However,  it only works effectively in cooperation with the  bicarbonate system:   Erythrocytes produce little CO, as they lack aerobic  pathways. Plasma CO, diffuses along a concentration  gradient into erythrocytes, where CD catalyses its  reaction with water to form carbonic acid (H 2 C0 3 ),  which then dissociates. Much of the H + is buffered by  Hb, and the HC0 3 - diffuses out into the extracellular  fluid along a concentration gradient.   Electrochemical neutrality is maintained by  diffusion of Cl - in the opposite direction into cells. This  movement of ions is known as the ‘chloride shift’.    Figure 4.1 Generation of bicarbonate by erythrocytes,  showing the chloride shift. CD, carbonate dehydratase;  Hb, haemoglobin.   Under normal circumstances the higher Pco 2 in the  blood leaving tissues stimulates erythrocyte HCO ~  production; consequently the arteriovenous difference  in the ratio of [HC0 3 _ ] to [CO,], and therefore the pH,  is kept relatively constant.   Extracellular and intracellular buffers other than  HC0 3 “ and Hb do not contribute significantly to blood  buffering. They include:   • phosphate, which has a plasma concentration of  about 1 mmol/L, but a higher concentration in bone  and inside cells where buffering capacity is of more  importance,   • proteins, which, because of their low concentrations  in plasma, also have little blood-buffering capacity.   Carbonate dehydratase is also of central importance  in the mechanisms involved in H + secretion and in  maintaining the HC0 3 “ buffering capacity in the blood.  Hydrogen ions are secreted from renal tubular cells into  the lumina, where they are buffered by constituents of the  glomerular filtrate. Unlike Hb in erythrocytes, urinary  buffers are constantly being replenished by continuing  glomerular filtration. For this reason, and because most  of the excess H + can only be eliminated from the body  by the renal route, the kidneys are of major importance  in compensating for chronic acidosis. Without them, the  Hb buffering capacity would soon become saturated.  Two renal mechanisms control [HCO, - ] in the ECF:   • Bicarbonate reclamation (‘reabsorption’), the  predominant mechanism in maintaining the steady  state. The CO, driving the CD mechanism in renal  tubular cells is derived from filtered HCO s “. There is  no net loss of H + (Fig. 4.2).   • Bicarbonate generation, a very important mechanism  for correcting acidosis, in which the levels of C0 2 or  [HC0 3 “] affecting the CD reaction in renal tubular  cells reflect those in the extracellular fluid. There is a  net loss of H + (Fig 4.3).   Normal urine is nearly HC0 3 “ free. An amount  equivalent to that filtered by the glomeruli is returned  to the body by the tubular cells. The luminal surfaces  of renal tubular cells are impermeable to HC0 3 “.  Thus, HC0 3 “ can only be returned to the body if  first converted to C0 2 in the tubular lumina, and an  equivalent amount of C0 2 is converted to HC0 3 “  within tubular cells. The mechanism depends on the    Figure 4.2 Normal reabsorption of filtered bicarbonate  from the renal tubules. CD, carbonate dehydratase.    Figure 4.3 Net generation of bicarbonate by renal  tubular cells with excretion of hydrogen ions. By non-  bicarbonate base; CD, carbonate dehydratase.   action of CD, both in the brush border on the luminal  surfaces and within tubular cells, and on H + secreted  into the lumina in exchange for sodium (Na + ). This  occurs predominantly in the proximal tubules but also  in the first part of the distal tubules:   • Bicarbonate is filtered through the glomeruli at a  plasma concentration of about 25mmol/L.   • Filtered HC0 3 “ combines with H + , secreted by  tubular cells, to form H 2 C0 3 .   • The H 2 CO s dissociates to form C0 2 and water. In the  proximal tubules this reaction is catalysed by CD in  the brush border. In the distal tubules, where the pH is  usually lower, H 2 C0 3 probably dissociates spontaneously.   • As the luminal Pco 2 rises, C0 2 diffuses into tubular  cells along a concentration gradient, and as the  intracellular concentration of CO, rises, CD catalyses  its combination with water to form H,C0 3 , which  dissociates into H + and HC0 3 “.   • Hydrogen ions are secreted in the tubular lumina  in exchange for Na + and so the HC0 3 “-generating  reactions start again from the second stage. As the  intracellular concentration of HC0 3 “ rises, HC0 3 "  diffuses into the ECF accompanied by Na + , which  has been reabsorbed in exchange for H + .   This self-perpetuating cycle reclaims buffering  capacity that would otherwise have been lost from the  body by glomerular filtration. The secreted H + is derived  from cellular water, and is incorporated into water in the  lumina. Because there is no net change in H + balance and  no net gain of HC0 3 “, this mechanism cannot correct an  acidosis but can maintain a steady state.   The mechanism in renal tubular cells for generating  HC0 3 “ is identical to that of HC0 3 ~ reabsorption, but  there is net loss of H + from the body as well as a net gain  of HCO Therefore this mechanism is well suited to  correcting any type of acidosis.   • A rise in Pco 2 : in this case, the rise in [COJ is the  indirect result of a rise in the extracellular Pco 2 . Renal  tubular cells, unlike erythrocytes with anaerobic  pathways, constantly produce CO, aerobically. This  diffuses out of cells into the extracellular fluid along  a concentration gradient. An increase in extracellular  Pco 2 , by reducing the gradient, slows this diffusion  and the intracellular Pco 2 rises.   • A fall of [ HCOy] : reduction of extracellular [ HC0 3 _ ] ,  by increasing the concentration gradient across renal  tubular cell membranes, increases the loss of HC0 3 “  from cells.   Normally almost all the filtered HC0 3 " is reabsorbed.  Once the luminal fluid is HC0 3 “ free, continued  secretion of H + and the intracellular generation of  HC0 3 " depend on the presence of other filtered buffer  bases (IT). In their absence, the luminal acidity would  increase so much that further H + secretion would be  inhibited. These buffers, unlike HC0 3 “, do not form  compounds capable of diffusing back into tubular cells,  nor is H + incorporated into water.   There is net loss of H + in urine as HB. The HC0 3 “  formed in the cell is derived from cellular CO and  therefore represents a net gain in HC0 3 “. Whenever  lmmol of H + is secreted into the tubular lumen,  1 mmol of HC0 3 “ passes into the ECF with Na + . The  mechanism of HC0 3 " generation is very similar to that  in erythrocytes. However, unlike red cells, renal tubular  cells are exposed to a relatively constantPco,. Bicarbonate  generation, coupled with H + secretion, becomes very  important in acidosis, when it is stimulated by either a  fall in extracellular [HC0 3 “] (metabolic acidosis) or a  rise in extracellular Pco 2 (respiratory acidosis).   Apart from HC0 3 “, the two other major urinary buffers  are phosphate and ammonia (NH 3 ); they are also  involved in HC0 3 “ generation.   At pH 7.4, most of the phosphate in plasma, and also  in the glomerular filtrate, is monohydrogen phosphate  (HP0 4 2 “), which can accept H + to become dihydrogen  phosphate (H 2 P0 4 “). Bicarbonate can continue to  be generated within tubular cells, with H + , and to be  returned to the body after all that in the filtrate has  been reabsorbed. Therefore it can help to replace that  used in extracellular buffering. The pK of this buffer  pair is about 6.8:   Phosphate is normally the most important buffer in  the urine because its pK a is relatively close to the pH of  the glomerular filtrate and because the concentration  of phosphate increases 20-fold, to nearly 25 mmol/L, as  water is reabsorbed from the tubular lumen.   Even in a mild acidosis, more phosphate ions are  released from bone than at normal pH; the need for  increased urinary H + secretion is linked with increased  buffering capacity in the glomerular filtrate owing to the  increase of phosphate. At a urinary pH below 5.5, most  of the filtered phosphate is converted to dihydrogen  phosphate. Therefore, at low pH, urinary phosphate  cannot maintain the essential buffering of continued H +  secretion. The predominant urinary anion is Cl", but,  because hydrochloric acid is almost completely ionized  in aqueous solution, it cannot act as a buffer.   As the urine becomes more acid, it can be shown to  contain increasing amounts of ammonium ion (NH 4 + ).  Urinary ammonia probably allows H + secretion, and  therefore HC0 3 “ formation, to continue after other  buffers have been depleted.   Ammonia, produced by hepatic deamination of  amino acids, is rapidly incorporated into urea, with a net  production of H + . However, as the systemic [H + ] increases,  there is some shift from urea to glutamine (GluCONHJ  synthesis, with a slight fall in hepatic H + production.   Glutamine is taken up by renal tubular cells, where it  is hydrolysed by glutaminase to glutamate (GluCOO")  and NH + :   Ammonia and NH 4 + form a buffer pair with a pK of  about 9.8:   Because of the very high pK , at pH 7.4 and below  the equilibrium is overwhelmingly in favour of NH 4 +  production. Ammonia can diffuse out of the cell into  the tubular lumen much more rapidly than NH 4 + . If the  luminal fluid is acidic, NH 3 will be retained within the  lumen by avid combination with H + derived from the  CD mechanism. This allows H + , produced in the kidneys,  to be excreted as ammonium chloride (NH 4 + Ch); thus,  in severe acidosis, HC0 3 " formation can continue even  when phosphate buffering power has been exhausted.  There is a net gain of HC0 3 ". However, H + as well as NH 3  is released in renal tubular cells when NH , + dissociates;  this is maintained by passive diffusion of NH 3 into the  luminal fluid (Fig. 4.4).   On the face of it there seems no advantage in  buffering one H + secreted into the lumen if, at the same  time, another is produced within the cell.   Explanations for the role of NH 3 in the correction of  acidosis include the following:   • The fate of the GluCOO" produced at the same  time as the NH, + . After further deamination to  2-oxoglutarate, it can be converted to glucose;  gluconeogenesis uses an amount of H + equivalent to  that used in the generation of NH 4 + produced from  glutamine. Therefore, the H + liberated into the cell  may be incorporated into glucose.   • A shift from urea synthesis to glutamine production  in the liver with a fall in systemic H + production in  the presence of an acidosis. This is a minor factor.   The rate of gluconeogenesis and glutamine synthesis  and glutaminase activity all increase in an acidosis.    Figure 4.4 The role of ammonia in the generation  of bicarbonate by renal tubular cells. CD, carbonate  dehydratase. Modified with kind permission from  Williams DL, Marks V (eds), Biochemistry in Clinical  Practice. London: Heinemann Medical Books, 1983.  © Elsevier.   Carbonate dehydratase also catalyses the formation  of HCO “ in intestinal mucosal cells. The HC0 3 " may  pass either into the ECF or into the intestinal lumen -  a mechanism that can continue only if H + is pumped  in the opposite direction. Electrochemical neutrality is  maintained by one of two mechanisms:   • Na + exchange for H + , by a mechanism that is the  opposite of that in renal tubular cells,   The parietal cells of the stomach secrete H + into the  lumen together with CT. As H + CT enters the gastric  lumen, HC0 3 " diffuses into the ECF, thus accounting for  the post-prandial ‘alkaline tide’. In the healthy subject  this is rapidly corrected by HC0 3 “ secretion, mainly by  the pancreas, as food passes down the intestinal tract.  This mechanism explains the metabolic alkalosis that  occurs in pyloric stenosis.   Sodium bicarbonate secretion by the pancreatic and  biliary cells in response to stimulation by secretin  accounts for the alkalinization of the duodenal  fluid and occurs by the reverse process of NaHCO,  reabsorption in renal tubular cells. The pancreatic  and biliary mechanisms are accelerated by the local  rise in Pco 2 that results when H + is pumped into the  ECF and reacts with the HC0 3 “ generated by gastric  parietal cells. This is analogous to the stimulation of  renal HC0 3 “ formation by the rise in luminal Pco r  Loss of large amounts of duodenal fluid may cause  HC0 3 “ depletion.   As fluid passes down the intestinal tract, HC0 3 “ enters  and Cl" leaves the lumen by a reversal of the gastric  mucosal mechanism. Therefore the gastric loss of Cl-  an d the gain of HCO _ are finally corrected. Preferential  reabsorption of urinary CT by this mechanism after  ureteric transplantation into the ileum, ileal loops  or the colon explains the hyperchloraemic acidosis  (normal anion-gap acidosis) associated with this  operation (Fig. 4.5).    Net effect on plasma - gain of bicarbonate and loss of chloride    Net effect on plasma - loss of bicarbonate and gain of chloride  Figure 4.5 Acid-base balance in intestinal cells.   Disturbances of H + homeostasis always involve the  bicarbonate buffer pair. In respiratory disturbances  abnormalities of CO z are primary, whereas in so-called  metabolic or non-respiratory disturbances [HCO, - ] is  affected early and changes in C0 2 are secondary.   Acid blood pH is known as acidaemia, and alkaline  blood pH is called alkaemia. Abnormal processes, either  respiratory or metabolic, generate abnormal amounts  of acid or base - acidosis or alkalosis respectively. The  blood pH may or may not be abnormal because of the  compensatory mechanisms.   Acidosis or alkalosis can cause a variety of clinical  symptoms including arrhythmias, weakness, confusion   A 7-year-old boy was admitted unconscious to  a casualty department. On examination he was  found to be hyperventilating. He had inadvertently  consumed ethylene glycol antifreeze, which he had  found in his parents’ garage stored in a lemonade  bottle. Blood results were as follows:   The results show a high anion gap, normally  about 15-20 mmol/L, but in this case 37mmol/L,  with metabolic acidosis, i.e. (134 + 6)-(10 + 93).  Hyperkalaemia is present due to the movement of  intracellular K + out of cells because of the acidosis.  The compensatory mechanism of hyperventilation  ‘blows off’ volatile acid in the form of C0 2 , hence the  low Pco 2 .   and gastrointestinal problems. It is noteworthy that  various enzymes have optimal pH values and acid-base  disturbance can perturb their function.   Acidosis occurs if there is a fall in the ratio of [HC0 3 _ ]  to Pco 2 in the ECF. It may be due to:   • metabolic (non-respiratory) acidosis, in which the  primary abnormality in the bicarbonate buffer  system is a reduction in [HC0 3 “],   • respiratory acidosis, in which the primary abnormality  in the bicarbonate buffer system is a rise in Pco 2 .   The primary disorder in the bicarbonate buffer system  in a metabolic acidosis is a reduction in [HC0 3 “],  resulting in a fall in blood pH. The reduction in the  HCO, - may be due to:   • its use in buffering H + more rapidly than it can be  generated by normal homeostatic mechanisms,   • loss in the urine or gastrointestinal tract more  rapidly than it can be generated by normal  homeostatic mechanisms,   The causes of a metabolic acidosis are shown in Box  4. 1 and can be divided into those with a high and those  with a normal plasma anion gap.   One negative charge balances one positive charge, and  some substances are multivalent, having more than one  charge per mole. If the molecular weight is divided by  the valency, the charges on each resulting equivalent  will be the same as those on an equivalent of any other  chemical. Most of the ions in the following discussion  are monovalent, and hence the number of millimoles is  numerically the same as that of milliequivalents (mEq).  However, when calculating ion balance, the latter  notation should strictly be used, that is, concentration  of charges.   Sodium and potassium (K + ) provide more than  90 per cent of plasma cation concentration in the  healthy subject; the balance includes low concentrations  of calcium (Ca 2+ ) and magnesium (Mg 2+ ), which vary  only by small amounts.   Acute kidney injury and chronic kidney disease  Ketosis [diabetes mellitus (most common)]; also  ethanol excess or prolonged starvation  Lactic acidosis (see Box 4.2)   More than 80 per cent of plasma anions are accounted  for by Cl” and HC0 3 ”; the remaining 20 per cent or so  (sometimes referred to as unmeasured anion) is made  up of protein, and the normally low concentrations of  urate, phosphate, sulphate, lactate and other organic  anions.   The protein concentration remains relatively  constant, but the concentrations of other unmeasured  anions can vary considerably in disease.   The anion gap, represented as A” in the following  equations, is the difference between the total  concentration of measured cations (Na + and K + ) and  measured anions (Cl” and HCO ”); it is normally about  15-20 mEq/L. Therefore:   [Na + ] + [K + ] = [HCCr] + [Cl”] + [A”]   As we will see later, the urinary anion gap can also be  measured and is useful in the diagnosis of renal tubular  acidosis (Fig. 4.6).   A useful mnemonic to help remember some of the causes  of a high anion gap metabolic acidosis is DR MAPLES:  D = diabetic ketoacidosis, R = renal, M = methanol,  A = alcoholic ketoacidosis, P = paracetamol, L = lactic  acidosis, E = ethylene glycol, S = salicylates.   If the number of negatively charged ions (in this  case HC0 3 ”) is reduced, electrochemical neutrality  is maintained by replacing them with an equivalent  number of other anion(s) or with ketone bodies  (acetoacetate and 3-hydroxybutyrate) in ketoacidosis  or lactate in lactic acidosis. Other causes of unmeasured  anion (A”) are salicylate in aspirin overdose and  metabolites from methanol, paracetamol or ethylene  glycol poisoning.    Lungs C0 2 + H 2 0 H 2 0 Metabolic C0 2   In renal glomerular dysfunction (see Chapter 3),  even if tubular function is normal, HC0 3 ” generation  is impaired because the amount of Na + available  for exchange with H + and the amount of filtered  buffer A” available to accept H + are both reduced.  These buffer anions contribute to the unmeasured  A”. For each milliequivalent of buffer A” retained,  1 mEq fewer H + can be secreted, and therefore 1 mEq  fewer HC0 3 ” is generated. The retained A” therefore  replaces HC0 3 ”.   In the following example, the plasma [HC0 3 ”] will  be assumed to have fallen by 10mmol(mEq)/L:   [Na + ] + [K + ] = [HCO”] + [Ck] + [A”]   The [HC0 3 ”] has fallen from 25 to 15 mEq/L and the  anion gap, entirely due to [A - ] , has risen by the same  amount, from 19 to 29 mEq/L, that is, a high anion gap.  If renal HC0 3 ” generation is so impaired that it cannot  keep pace with its peripheral utilization, the pH will fall.   It is worth noting that the plasma anion gap can be  decreased by an increase in unmeasured cations, e.g.  hyperkalaemia, hypercalcaemia, hypermagnesaemia,  high immunoglobulin G (IgG), bromide or lithium  intoxication, or by a decrease in unmeasured anions,  for example hypoalbuminaemia.   Lactic acidosis is a common form of high anion gap  metabolic acidosis; some of the causes are shown in  Box 4.2. One definition used is an arterial pH of less  than 7.2 with a plasma lactate concentration greater  than 5.0mmol/L. The normal fasting blood lactate  is between 0.4 and l.Ommol/L, which can rise to  1.5mmol/L on prolonged exercise (hyperlactataemia).   Most forms of lactic acidosis are due to increased  L-lactate, although in some malabsorption states, with  bacterial overgrowth, D-lactic acidosis may occur.   The Cohen and Woods classification of lactic acidosis  is as follows. The commonest type of lactic acidosis is  type A, due to poor tissue perfusion or hypoxia. Type  B is lactic acidosis not due to tissue underperfusion.  Type B1 is due to organ or tissue dysfunction including  severe hepatic disturbances as the liver is involved in  lactate metabolism. Metastatic malignant disease  can also evoke a lactic acidosis, probably because the  carcinoma tissue produces lactic acid. Type B2 lactic  acidosis is due to drugs and toxins. Type B3 is due to  inborn errors of metabolism.   Glucose-6-phosphatase deficiency  Pyruvate dehydrogenase/carboxylase deficiency  Fructose 1,6-diphosphatase deficiency  Mitochondrial defects  Beri beri (thiamine deficiency)   The combination of a low plasma [HC0 3 “] and a high  [Cl - ], known as hyperchloraemic acidosis, is rare. The  anion gap in such cases is normal:   [Na + ] + [K + ] = [HCO - ] + [Cl“] + [A - ]   The causes, which can usually be predicted on clinical  grounds, include HC0 3 - loss in a one-to-one exchange  for Cl - . This occurs if the ureters are transplanted  into the ileum or colon, usually after cystectomy for  carcinoma of the bladder. If Ch-containing fluid such  as urine enters the ileum, ileal loops or colon, the cells  exchange some of the Cl - for HC0 3 - .   Duodenal fluid, with a [HC0 3 - ] about twice that  of plasma, is alkaline. If the rate of loss (for example  through small intestinal fistulae) exceeds that of the   renal ability to regenerate HC0 3 - , the plasma [HC0 3 - ]  may fall enough to cause acidosis.   Overenthusiastic infusion of saline can also be  associated with a hyperchloraemic acidosis (see  Chapter 2 for discussion on intravenous fluid therapy  and the GIFTASUP guidelines). Acetazolamide  therapy is used to treat glaucoma. By inhibiting CD  activity in the eye, it reduces the formation of aqueous  humour. Inhibition of the enzyme in renal tubular  cells and erythrocytes impairs H + secretion and HCO -  formation. Hyperchloraemic acidosis sometimes  complicates treatment with acetazolamide and can be  associated with hypokalaemia.   Another cause of hyperchloraemic acidosis is  impaired H + secretion, and therefore HCO " produc-  tion, due to renal tubular acidosis.   In normal tubules, most filtered Na + is reabsorbed  with Cl - ; the rest is exchanged for secreted H + or K + . If  H + secretion is impaired, and yet the same amount of  Na + is reabsorbed, Na + must be accompanied by Cl" or  exchanged for K + . This type of hyperchloraemic acidosis  is therefore often accompanied by hypokalaemia - like  acetazolamide treatment. This is an unusual finding in  acidosis, which is usually associated with hyperkalaemia  (Box 4.3).   Box 4.3 Some causes of a hyperchloraemic  acidosis (normal anion gap acidosis)   Gastrointestinal disease, e.g. fistula or diarrhoea  Ureteric diversion, e.g. ileal bladder or  ureterosigmoidostomy  Intravenous saline excess   There are various forms of renal tubular acidosis, which  can present as a hyperchloraemic metabolic acidosis.  (See Box 4.4 for some causes.)   The more common, sometimes called classic, renal  tubular acidosis (type I) is due to a distal tubular  defect causing a failure of acid secretion by the cortical  collecting duct system of distal nephrons. The urinary  pH cannot fall much below that of plasma, even in  severe acidosis. The distal luminal cells are abnormally   permeable to H + , and this impairs the ability of distal  tubules to build up a [H + ] gradient between the tubular  lumina and cells. The re-entry of H + into distal tubular  cells inhibits CD activity at that site; proximal HCO, -  reabsorption is normal. The inability to acidify the  urine normally can be demonstrated by using the  furosemide test or an NH ,C1 load.   A 52-year-old woman with Sjogren’s syndrome and  distal or type I renal tubular acidosis attended a renal  out-patient clinic. Her blood results were as follows:   The results are suggestive of a normal anion gap  metabolic acidosis or hyperchloraemic acidosis.  Hypokalaemia is unusual in the face of an acidosis,  one of the exceptions being renal tubular acidosis  type I or II. Note that the anion gap here is  (144 + 3)-(13 + 118) = 16mmol/L, which is a normal  gap although with high plasma [Cl - ] and low [HC0 3 - ] .   Box 4.4 Some causes of type I and type II  renal tubular acidosis   Autoimmune disorders: Sjogren's syndrome, chronic  active hepatitis  Hyperparathyroidism  Drugs/toxins: lithium, amphotericin   In renal tubular acidosis type II there is impairment  of HC0 3 - reabsorption in the proximal tubule. Loss  of HCO, - may cause systemic acidosis, but the ability  to form acid urine when acidosis becomes severe is  retained; the response to NH 4 C1 loading may therefore  be normal. Type II renal tubular acidosis is also  associated with amino aciduria, phosphaturia and  glycosuria, as in Fanconi’s syndrome.   Renal tubular acidosis type IV is associated with  hyporeninism hypoaldosteronism (see Chapter 8)  and with a hyperkalaemic hyperchloraemic acidosis.  Sometimes plasma renin and aldosterone measurement  may be useful to confirm this.   • Measure the urinary anion gap and pH in a fresh  spot urine sample along with the plasma anion gap.  In practice, the urinary anion gap is the difference  between the sum of the urinary [Na + ] and [K + ]  minus the urinary [Cl - ].   • A urinary anion gap of less than 100 mmol/L implies  low urinary [NH 4 + ] .   • In such cases a urinary pH of more than 5.5 in the  presence of a hyperchloraemic metabolic acidosis  and hypokalaemia is suggestive of type I, or distal,  renal tubular acidosis. Conversely, a urinary pH of  less than 5.5 in the presence of hyperchloraemic  metabolic acidosis and hyperkalaemia is suggestive  of renal tubular acidosis type IV. Plasma aldosterone  and renin concentrations may show hyporeninism  hypoaldosteronism in renal tubular acidosis type IV.   • A urinary anion gap of more than 100 mmol/L  implies high urinary [NH 4 + ].   • Measure fractional excretion of HCO, - (FE% HC0 3 - ),  which equals:   urine[HCO, ] x plasma [creatinine]  plasma [HCO, - ] x urine [creatinine]   • If the fractional excretion of HCO, - is increased,  this is suggestive of renal tubular acidosis type II,  particularly if glycosuria, hypophosphaturia and  hypophosphataemia are also present.   • Sometimes a diagnosis of acidosis is difficult to  establish and additional tests are indicated. See  Table 4.1.   The test relies on the fact that increased Na + delivery  to the distal tubules results in exchange for H + and K + ,   dRTA, distal renal tubular acidosis; FE% HC0 3 _ , fractional excretion  of bicarbonate.   resulting in a decrease in urinary pH to less than 5.5  under normal conditions.   Furosemide 40 mg is given orally and urine is  collected for pH every half-hour. However, like the  ammonium chloride test, a urinary pH of less than 5.5  usually precludes its need. Generally this is simpler and  safer than the ammonium chloride test and may be  preferable, assuming that there are no contraindications  to the use of furosemide.   This test is not necessary if the pH of a urinary specimen,  collected overnight, is already less than 5.5.   The NH 4 + is potentially acid because it can dissociate  to NH 3 and H + . After ingestion of ammonium chloride,  the kidneys usually secrete the H + , and the urinary pH  falls.   • 08.00 h: gelatine-encapsulated ammonium chloride  is given orally in a dose of 0. 1 g/kg body weight. This  can act as a gastric irritant.   • Urine specimens are collected hourly and the pH of  each is measured immediately in the laboratory.   In normal subjects, the urinary pH falls to 5.5 or below  between 2 and 8h after the dose. In generalized tubular  disease there may be a sufficient number of functioning  nephrons to achieve a normal level of acidification. In  distal renal tubular acidosis this degree of acidification   does not occur. Urinary acidification is normal in  proximal tubular acidosis.   This test can cause nausea and vomiting and is  contraindicated in liver disease.   • the pH is low (uncompensated or partly  compensated) or near normal (fully compensated),   • plasma [Cl - ] is unaffected in most cases unless  there is hyperchloraemic acidosis, for example it is  raised after ureteric transplantation, saline excess,  diarrhoea/ fistulae, in renal tubular acidosis or during  acetazolamide treatment.   In many cases the diagnosis may be obvious from  the clinical history (particularly drug history) and  examination (Fig. 4.7).   Tests that may help to elucidate the cause of the  metabolic acidosis are:   • tests for drugs or poisons, for example ethanol,  paracetamol, salicylate (if indicated),   Treatment of a metabolic acidosis is usually that of  the underlying condition. Dialysis may be necessary in  the face of uraemia. Overdoses may also require dialysis,  for example ethylene glycol, methanol and salicylate.  Infusion of sodium bicarbonate (usually small boluses  containing about 50-100 mmol) is only rarely indicated  in severe metabolic acidosis of pH less than 7.1 and can  cause complications such as hypernatraemia, volume  overload, hypokalaemia, ‘overshoot’ alkalosis and  paradoxical central nervous system acidosis probably  due to the rapid diffusion of CO, across cell membranes  in comparison with slower HC0 3 - movement.   In distal type I and type II renal tubular acidosis,  oral HCO therapy may be necessary. In type IV, if due  to hyporeninism hypoaldosteronism, fludrocortisone  (sometimes in conjunction with furosemide) may be  useful.   The findings in respiratory acidosis differ significantly  from those in non-respiratory disturbances. Some of  the causes of respiratory acidosis are given in Box 4.5.   The primary abnormality in the bicarbonate buffer  system is CO, retention, usually due to impaired  alveolar ventilation with a consequent rise in Pco r As in  the metabolic disturbance, the acidosis is accompanied  by a fall in the ratio of [ HCOy | to Pay.   In acute respiratory failure, for example due to  bronchopneumonia or status asthmaticus, both the  erythrocyte and renal tubular mechanisms increase the  rate of generation as soon as the Pco, rises. In the short  term, renal contribution to HCO ~ production is limited  by time. A relatively large proportion of the slight rise in   plasma [HCOy] is derived from erythrocytes because,  in normal circumstances, the Hb buffering mechanism  is not saturated. This degree of compensation is rarely  adequate to prevent a fall in pH.   In chronic respiratory failure, for example due  to chronic obstructive pulmonary disease (COPD),  the renal tubular mechanism is of great importance.  Haemoglobin buffering power is of limited capacity,  but, as long as the glomerular filtrate provides an  adequate supply of Na + for exchange with H + and  buffers to accept H + , tubular cells continue to generate  HCOy until the ratio of [HCOy] to Pco 2 is normal.  In stable chronic respiratory failure the pH may be  normal despite a very high Pco 2 , because of an equally  high plasma [HCOy].    Figure 4.7 Algorithm for the investigation of a metabolic acidosis. RTA, renal tubular acidosis.   Opiates, benzodiazepines, anaesthetic agents  Head injury, cerebral tumours, central nervous  system infection   - pH is near normal or low, depending upon  chronicity (allowing time for compensation to  occur),   A 67-year-old retired printer presented to casualty  because of increasing breathlessness. He had smoked  20 cigarettes a day for 50 years. On examination he  was found to be centrally cyanosed and coughing  copious green phlegm. His arterial blood results  were as follows:   The patient had chronic obstructive pulmonary  disease, and the blood gases show a respiratory  acidosis with hypercapnia and hypoxia. The latter  has resulted in central cyanosis. Compensation is via  the kidneys, with increased acid excretion and HC0 3 “  reclamation. Chronic cases of respiratory acidosis are  usually almost totally compensated as there is time  for the kidneys and buffer systems to adapt. This is  unlike an acute respiratory acidosis due to bilateral  pneumothorax, in which the rapid acute changes  do not give sufficient time for the compensatory  mechanisms to take place. This patient had an acute  exacerbation of his lung disease and the C0 2 retention  exceeded the compensatory mechanisms.   In many cases the cause of the respiratory acidosis can  be deduced from the clinical history and examination in  conjunction with a chest radiograph and lung function  tests, if indicated (Fig. 4.8).   Treatment of a respiratory acidosis is usually of the  underlying disorder. Sodium bicarbonate infusion (see  above) is rarely indicated, except perhaps if the pH is  less than 7.1, such as in cardiopulmonary arrest. In  cases of bronchospasm or COPD, bronchodilators such  as (3-agonists (for example salbutamol), anticholinergic  agents (for example ipratropium) or methylxanthines  (for example theophylline) may be useful. Sedative  drugs should be avoided, and naloxone may reverse  the actions of opiates and flumazenil the effects of  benzodiazepines.   Mechanical ventilation may be needed. Respiratory  stimulants such as doxapram or nikethamide are not  without side effects, for example they may lower the  epilepsy threshold. Medroxyprogesterone can increase  respiratory drive and has been used in pickwickian   syndrome (obesity-hypoventilation). Oxygen therapy  maybe indicated if the patient is hypoxic and, in patients  with COPD who fulfil the criteria for 0 2 therapy,  this may decrease mortality and reduce pulmonary  hypertension. However, as discussed later, 0 2 therapy  should be used with caution if there is hypercapnia  (raised Pco 2 ), which may aggravate the acidosis.   Alkalosis occurs if there is a rise in the ratio of [HC0 3 “]  to Pco 2 in the ECF.   In metabolic alkalosis the primary abnormality  in the bicarbonate buffer system is a rise in [HCO ~].  There is little compensatory change in Pco r   In respiratory alkalosis the primary abnormality is  a fall in the Pco r The compensatory change is a fall in  [HC0 3 -].   As the primary products of metabolism are H + and  CO,, not OH" and HC0 3 “, alkalosis is less common than  acidosis. The presenting clinical symptom of alkalosis  may be tetany despite a normal plasma total calcium  concentration; this is due to a fall in the free ionized  calcium fraction.   A primary rise in plasma [HC0 3 “] may occur in the  following situations:   • Bicarbonate administration, such as the ingestion  of large amounts of HC0 3 ~ to treat indigestion  (milk-alkali syndrome, which is rare) or during  intravenous HCO - infusion. Usually both the cause  and the treatment are obvious.   • Severe K + depletion with the generation of HC0 3 “  by the kidney. This is one of the most common  causes (discussed in Chapter 5). Here, H + shifts  into the intracellular space. As the extracellular [K + ]  decreases, K + flow out of the cells and, to maintain  electrical neutrality, H + move into the intracellular  space.   • Loss of H + : if a H + is excreted, a HC0 3 “ is gained in  the extracellular space. The most likely loss of H + is  through either the kidneys or the gastrointestinal  tract. In the case of the former, renal losses occur  when the distal tubule [Na + ] absorption increases  in the presence of excessive aldosterone, which  stimulates the electrogenic epithelial Na + channel in  the collecting duct. As this channel reabsorbs Na + ,  the tubular lumen becomes more negative, resulting  in H + and K + secretion into the lumen. In the case  of the gastrointestinal tract, nasogastric suction or  vomiting causes loss of gastric secretions, which are  rich in hydrochloric acid.   • Contraction alkalosis: thiazide or loop diuretic use  or Ch-losing diarrhoea results in the loss of HC0 3 “-  poor, Cl“-rich ECF. This results in a contraction of  ECF volume and, because the original HCO " is  now dissolved in a smaller fluid volume, a slight  (2-4mmol/L) increase in [HC0 3 “] occurs. This  is sometimes called ‘saline-responsive’ metabolic  alkalosis.   Compensation for metabolic alkalosis is relatively  ineffective. Although acidosis stimulates the respiratory  centre, alkalosis cannot usually depress it sufficiently  to bring the pH back to normal; respiratory inhibition  not only leads to C0 2 retention but also causes hypoxia,   Endogenous mineralocorticoid excess, e.g. Cushing’s  syndrome, primary hyperaldosteronism (Conn’s  syndrome), Liddle’s syndrome, 11 -hydroxylase and  17-hydroxylase deficiencies, Bartter’s or Gitelman’s  syndrome   which can over-ride the inhibitory effect of alkalosis on  the respiratory centre. Consequently, CO, may not be  retained in adequate amounts to compensate for the  rise in plasma [HC0 3 “].   Two factors may impair the ability to lose HC0 3 “ in  the urine and so may aggravate a metabolic alkalosis:   • A reduced glomerular filtration rate due to volume  depletion limits the total amount of HCO " that can  be lost.   • The [Cl - ] in the glomerular filtrate may be reduced  if there is severe hypochloraemia due to gastric Cl“  loss. Isosmotic Na + reabsorption in proximal tubules  depends on passive reabsorption of Cl" along the  electrochemical gradient. A reduction in available  Cl" limits isosmotic reabsorption, and more Na +  becomes available for exchange with H + and K + . The  urine becomes inappropriately acid, and H + secretion  stimulates inappropriate HC0 3 “ reabsorption. The  increased K + loss aggravates the hypokalaemia due to  alkalosis. Thus prolonged vomiting such as pyloric  stenosis vomiting or gastric aspiration may cause:   The hypokalaemia may become apparent only when  the plasma volume has been restored, but it should  be anticipated. Pyloric stenosis is usually treated  before severe hypochloraemic alkalosis develops.  Nevertheless, the typical changes in plasma HC0 3 “ and  Cl" may indicate the diagnosis. Chronic vomiting due  to pyloric stenosis is one of the most common causes  of hypochloraemia. If the pyloris is patent, the fluid  vomited is mixed with alkaline intestinal fluid and does  not cause this syndrome.   A clinical and drug history and physical examination  may reveal the cause of the metabolic alkalosis (Fig.  4.9). Useful laboratory investigations in a metabolic  alkalosis may include:   • plasma Na + , K + , Cl“, Mg 2+ urea and creatinine,   • spot urine [Cl - ] less than 20mmol/L suggests  the saline-responsive form (volume depletion or  contraction) of metabolic alkalosis, and more than  20 mmol/L the saline-non- responsive form.   Other tests are indicated according to the clinical  situation, for example if primary hyperaldosteronism  (Conn’s syndrome) or Cushing’s syndrome is suspected  (mineralocorticoid excess syndromes; see Chapter 8).   Hypokalaemia should be investigated and treated  (see Chapter 5). If the patient is on a thiazide or loop  diuretic, this may need to be reduced or stopped.  Antiemetics may help stop vomiting, and proton pump  inhibitors may reduce gastric acid secretion.   If a saline-responsive alkalosis occurs with volume  depletion, intravenous infusion of isotonic saline may  help. The saline non-responsive form is often due to  mineralocorticoid excess.   The primary abnormality in the bicarbonate buffer  system in respiratory alkalosis is a fall in Pco 2 . This is due  to abnormally rapid or deep respiration when the CO,  transport capacity of the pulmonary alveoli is relatively  normal. The fall in Pco 2 reduces the CD activity in renal  tubular cells and erythrocytes. The compensatory fall in  plasma [HC0 3 “] tends to correct the pH.   It may be difficult to distinguish clinically between  the overbreathing due to metabolic acidosis, in which  the fall in plasma [HC0 3 “] is the primary biochemical  abnormality, and that due to respiratory alkalosis, in  which it is compensatory. In doubtful cases, estimation  of arterial pH and Pco 2 is indicated.   • pH is raised (uncompensated or partly compensated)  or near normal (fully compensated).   Acute hypocapnia (low Pco 2 ) can cause hypokalaemia  due to increased intracellular uptake of K + . A reduction in  free or ionized calcium can lead to tetany and paraesthesiae.   The causes of respiratory alkalosis are shown in Box  4.7. One of the most common causes is anxiety-induced  hyperventilation. Salicylates stimulate the respiratory   centre directly, and overdosage initially causes respiratory  alkalosis. They also uncouple oxidative phosphorylation,  and may evoke a lactic acidosis. Both these effects lower  plasma [HC0 3 “], but the pH may be high if respiratory  alkalosis is predominant, normal if the two cancel each  other out, or low if metabolic acidosis is predominant.   The cause of the hyperventilation maybe obvious from  the clinical history and examination (Fig. 4.10). A chest  radiograph and lung function tests may be indicated.  Laboratory tests could include:   • plasma [K + ] and ionized [Ca 2+ ] may be low,   • elevated white cell count and relevant cultures may  point to sepsis as a cause of hyperventilation,   A baby girl a few days old had had projectile vomiting  since birth due to pyloric stenosis. Her blood results  were as follows:   The results are suggestive of a metabolic alkalosis  due to the severe vomiting. Note also the low plasma  [Cl - ] due to loss of hydrochloric acid in vomit,  and hypokalaemia resulting from K + movement  into cells due to the alkalosis. Compensation is by  hypoventilation and retention of CO,.   A 20-year-old woman presented to casualty with a  panic attack. She had noticed peri-oral paraesthesia  and was found on examination to be hyperventilating.  Her arterial blood results were as follows:   The patient is showing a respiratory alkalosis. The  panic attack had resulted in hyperventilation. The  peri-oral paraesthesia is due to a lowering of plasma  ionized calcium concentration as a result of the  alkalosis. Compensation is by the kidneys, which  increase HC0 3 “ excretion and reduce acid excretion.   Treatment is usually of the underlying condition,  which is rarely life threatening unless arterial pH is  more than 7.5. During hyperventilation syndrome,  patients may benefit from reassurance, the treatment of  underlying psychological stress and rebreathing into a  paper bag during acute episodes.   Exogenous agents that increase respiratory drive,  e.g. salicylates, theophylline, catecholamines,  nikethamide, doxapram, thyroxine and progestagens  Hypoxaemia due to early and non-severe pulmonary  disease, e.g. asthma, pulmonary embolus,  pneumonia, or high altitude  Increased cerebral respiratory centre drive, e.g. head  injury, cerebral tumour, meningitis, cerebrovascular  accidents   Increased non-cerebral causes of respiratory centre  drive, e.g. pregnancy, heat exposure, hepatic failure,  septicaemia   Compensatory changes in acid-base disturbances  In either metabolic or respiratory acidosis the ratio of  [HCOy] to Pco 2 , and therefore the pH, can be corrected  by a change in concentration of the other member of  the buffer pair in the same direction as the primary  abnormality. This compensation may be either partial  or complete, but never over compensates, that is, there  is no pH overshoot.   The compensatory change in a metabolic acidosis is  a reduction in Pco 2 by hyperventilation, whereas in a  respiratory acidosis it is a rise in plasma [HCO ~] by the  action of the renal tubules.   In a metabolic alkalosis, compensation by  hypoventilation results in an elevation of Pco,, whereas  in a respiratory alkalosis the kidneys increase HC0 3 "  excretion, thereby lowering plasma [HC0 3 “].   The pH in a fully compensated acidosis is  normal. However, the concentrations of the other  components of the Henderson-Hasselbalch equation  are abnormal. All parameters can return to normal  only if the primary abnormality is corrected: if  normalization occurs, this removes the ‘drive’ of the  compensation.   See Table 4.2 for a summary of acid-base disorders  and their respective compensations. These are  simplifications, as, in practice, mixed acid-base  disturbances may occur (e.g. in metabolic acidosis with  respiratory acidosis).   Table 4.2 Summary of findings in arterial blood in  disturbances of hydrogen ion homeostasis   Arrows, primary change; *arrows, compensatory change; N, normal;  RTA, renal tubular acidosis.   Potassium depletion can cause alkalosis, or alkalosis can cause  hypokalaemia. The clinical history may differentiate the cause of the  combination of hypokalaemia and alkalosis.   Metabolic acidosis, with a low [HC0 3 i, causes overbreathing.  Measurement of blood pH and Ft o 2 can differentiate these two.   In an uncomplicated metabolic acidosis with a  high anion gap the change in [HC0 3 “] is equimolar  with the change in the [A - ]. Deviations from this  relationship indicate a mixed acid-base disturbance,  for example a metabolic acidosis and respiratory  acidosis (Fig. 4.11).   If a disease is chronic, then there is more time for  compensatory mechanisms to come into action to try  and restore pH.   The amount of O z in blood is determined by the amount  dissolved, the Hb concentration and the affinity of  Hb for O,. Haemoglobin consists of four subunits,  each made up of a haem, a porphyrin ring containing  iron, and a polypeptide. As a haem takes up an 0 2  molecule, there is a rearrangement of the subunits that  facilitates the uptake of additional ,. This accounts for  the shape of the oxyhaemoglobin dissociation curve  (see Fig. 4.12). Factors that affect the affinity of haem  for oxygen include the following.    A - Acute respiratory acidosis  B - Chronic respiratory acidosis  C - Chronic metabolic alkalosis  D - Acute respiratory alkalosis  E - Chronic respiratory alkalosis  F - Chronic metabolic acidosis  G- Acute metabolic acidosis  Central shaded area = Normal   Figure 4.1 1 Siggaard-Andersen acid-base chart for  arterial blood. Copyright Radiometer Medical Aps.  Adapted with permission.   • pH of blood: as the pH falls, the affinity for  O, decreases. This is known as the Bohr effect.  Deoxygenated Hb binds H + more avidly and  accounts for the increased buffering capacity of Hb  in venous blood.   • 2,3-diphosphoglycerate (2,3-DPG) is formed during  glycolysis and is plentiful within the erythrocyte.  It binds to Hb, liberating more O,, so that the  dissociation curve shifts to the right. A fall in pH  decreases 2,3-DPG levels, thus potentially reducing  tissue oxygenation. Erythrocyte levels of 2,3-  DPG increase in anaemia and in some conditions  associated with chronic hypoxia.   • Fetal Hb has a greater affinity for O, than adult Hb,  thus facilitating the transfer of O across the placenta.   Factors affecting blood gas results  In respiratory acidosis it is often important to know the  partial pressure of oxygen (Po 2 ) as well as the pH, Pco 2  and [HC0 3 “].   Normal gaseous exchange across the pulmonary  alveoli involves loss of C0 2 and gain of 0 2 . However,  in disease a fall in Po 2 and a rise in Pco 2 do not always  coexist. The reasons for this are as follows:   • Carbon dioxide is much more soluble in water than  0 2 and its rate of diffusion is about 20 times as high.  For example, in pulmonary oedema, diffusion of 0 2  across alveolar walls is hindered by oedema fluid and  arterial Po 2 falls. The hypoxia and alveolar distension  stimulate respiration and C0 2 is excreted. However,   Figure 4.12 The oxyhaemoglobin dissociation curve  showing the effect of pH (Bohr effect) on oxygen  saturation.   the rate of O transport through the fluid cannot be  increased enough to restore normal arterial Po r This  results in a low or normal Pco 2 and a low Po 2 . Only  in very severe cases is the Pco 2 raised.   • Haemoglobin in arterial blood is normally 95 per cent  saturated with O,, and the small amount of O, in  simple solution in the plasma is in equilibrium with  oxyhaemoglobin. Increased air entry at atmospheric  Po, cannot significantly increase O, carriage in  blood leaving normal alveoli, but can reduce the  Pco . Breathing pure O, increases arterial Po 2 , but  not Hb saturation.   • In conditions such as lobar pneumonia, pulmonary  collapse and pulmonary fibrosis or infiltration, not  all alveoli are affected equally. At first the gaseous  composition of blood leaving them is normal.  Increasing the rate or depth of respiration may later  lower the Pco 2 considerably, but does not alter either  the Po 2 or the Hb saturation.   • Obstruction of small airways means that air cannot  reach those alveoli supplied by them; the composition  of blood leaving them will be near that of venous  blood and there is a ventilation/perfusion mismatch  with a right-to-left shunt. Only if gaseous flow due  to increased respiratory exertion can overcome the  obstruction will it help to correct the low Po 2 and  high Pco 2 .   • Some alveoli may have normal air entry, but much  reduced blood supply. This is ‘dead space’. Increased  ventilation will have no effect, because there is little  blood to interact with the increased flow of gases.   Blood from unaffected alveoli and from those with  obstructed airways mixes in the pulmonary vein before  entering the left atrium. The high Pco, and low Po,  stimulate respiration and, if there are enough unaffected  alveoli, the very low Pco 2 in blood leaving them may  compensate for the high Pco 2 in blood from poorly  aerated alveoli. By contrast, neither the Po 2 nor the Hb  saturation will be significantly altered by mixing. The  systemic arterial blood will then have a low or normal  Pco 2 with a low Po,.   If the proportion of affected to normal alveoli is  very high, Pco 2 cannot adequately be corrected by  hyperventilation, and there will be a high arterial Pco,  with a low Po,.   If there are mechanical or neurological lesions  impairing respiratory movement or obstruction  of large, or most of the small, airways as in an acute  asthmatic attack, almost all alveoli will have a normal   blood supply; the poor aeration will cause a high Pco 2  and low Po 2 in the blood draining them, and therefore  in systemic arterial blood. Occasionally, stimulation  of respiration by alveolar stretching can maintain a  normal, or even low, Pco, early in an acute asthmatic  attack. A raised Pco 2 in an asthmatic indicates a severe  attack (Box 4.8).   • Hypoxaemic respiratory failure (type I) is defined  as hypoxaemia with a normal or low Paco 2 . This is  the most common form of respiratory failure and is  associated with most forms of pulmonary disease,  particularly when there is collapse of or fluid in  the alveoli. Examples are pulmonary oedema or  pneumonia. It may be difficult to determine from  blood gases whether hypoxaemic failure is acute or  chronic, although in chronic failure polycythaemia  or cor pulmonale may occur.   • Hypercapnic respiratory failure (type II) is  characterized by both hypoxaemia and hypercapnia.  Hypoxaemia may occur, particularly if the patient   In the list of examples given below the conditions  marked * may fall into either group, depending on  the severity of the disease.   Low arterial Po 2 with low or normal Pfco 2 (hypoxia)   Low arterial Po 2 with a high Pc o 2 (hypoxia and  hypercapnia)   is breathing room air. Common causes include  chest wall abnormalities, neuromuscular disease,  drug overdose or severe airway disease, for example  asthma or COPD. This may be acute (within a few  hours), when pH may be less than 7.3, because there  is not time for renal compensation to take place, or  chronic, when arterial pH is only slightly low.   As well as treatment for the cause of the condition,  some patients may need mechanical ventilation. It is  important to remember that CO, narcosis can occur in  some patients with hypercapnia who are given O . This  is because they may lose their hypoxic respiratory drive,  which can lead to rapid increases in Pco 2 .   Pulse oximeters give non-invasive estimation of the  arterial Hb O saturation. They are useful in anaesthesia,  recovery or intensive care (including neonatal units)  and during patient transport. There are two main  principles involved:   Oximetry gives a good estimation of adequate  oxygenation, but no direct information about  ventilation, including CO, status.   Bright overhead lighting may give pulsatile waveforms  and saturation values when there is no pulse. Dyes  and pigments, including nail varnish, may give  artificially low values. Abnormal haemoglobins, such  as methaemoglobinaemia, cause readings to tend  towards 85 per cent (see Fig. 4.12). Furthermore,  carboxyhaemoglobin, caused by carbon monoxide (CO)  poisoning, causes saturation values to tend towards  100 per cent. A pulse oximeter is thus misleading in  cases of CO poisoning for this reason and should not  be used. Oxygen saturation values less than 70 per cent  can be inaccurate.   Cardiac arrhythmias may interfere with the oximeter  picking up the pulsatile signal properly and with  calculation of the pulse rate. Vasoconstriction and  hypothermia cause reduced tissue perfusion and failure  to register a signal. Cardiac valve defects may cause  venous pulsation, and therefore venous 0 2 saturation is  recorded by the oximeter.   • Arterial specimens are usually preferable to capillary  specimens. Venous samples are avoided as the carbon  dioxide is raised compared with arterial samples as a  result of tissue metabolism.   • The heparin and specimen in the syringe must be  well mixed. Warning Excess heparin may dilute the  specimen and cause haemolysis.   • If sodium heparin is used, do not estimate [Na + ]  using the same specimen as a resultant factitious  hypernatraemia can occur.   • Expel any air bubbles at once. The nozzle should then  be stoppered. Leaving the specimen in the syringe  should minimize gas exchange with the atmosphere.   • Performing the assay as soon as possible should  minimize the effect on pH of anaerobic erythrocyte  metabolism. The specimen should be kept cool and  not sent through a vacuum tube system because of  potential shaking.   In newborn infants, arterial puncture may be used or  capillary samples taken as an alternative. However, the  following precautions are essential:   • In order for the composition of the blood to be as  near arterial as possible, the area from which the  specimen is taken should be warm and pink. If there  is peripheral cyanosis, results may be dangerously  misleading.   • The blood should flow freely. Squeezing the skin  while sampling may dilute the specimen with  interstitial fluid.   • The capillary tubes must be heparinized, and mixing  with the blood must be complete (see above).   • The tubes must be completely filled with blood. Air  bubbles invalidate the results.   Arterial blood gas estimations may be indicated in the  following situations:   • There is doubt about the cause of the abnormal  plasma HC0 3 “ (for example to differentiate metabolic  acidosis from respiratory alkalosis or metabolic  alkalosis from respiratory acidosis).   • An acid-base disturbance is suspected, for example  after cardiopulmonary arrest, in renal failure  complicated by lung disease or in salicylate overdose.   In such conditions, the estimations are required only  if the results will influence treatment.   • There is an acute exacerbation of COPD or acute,  potentially reversible, lung disease. In such cases,  vigorous therapy or artificial respiration may tide  the patient over until lung function improves; more  precise information than the plasma [HC0 3 “] is  needed to monitor and control treatment.   • Blood is being taken for estimation of Po and to  guide O, therapy or artificial ventilation.   • To complement oximetry, particularly if low  saturations are found or hypercapnia is suspected.   The following may be used to assess acid-base  disturbances in patients (see Fig. 4.11.)   Measurement of blood pH determines whether there  is an acidosis or alkalosis. If the pH is abnormal, the  primary abnormality may be in the control of CO, by  the lungs or respiratory centre, or in the balance between  HCO y utilization in buffering and its reabsorption and  regeneration by renal tubular cells and erythrocytes.  However, a normal pH does not exclude a disturbance  of these pathways: compensatory mechanisms may be  maintaining it.   Assessment of these factors can be made only by  measuring components of the bicarbonate buffer  system. There is reasonable correlation between arterial  and venous pH (the latter being about 0.04 units less)  but of course there are usually considerable Po 2 and  Pco 2 differences.   There are two methods that may be used to estimate  the circulating [HC0 3 “], although it can of course be  measured directly:   • Plasma total CO, (TCO z ) or sometimes termed  by some laboratories as bicarbonate on their  reports. The plasma [HC0 3 “] is probably the most  commonly measured index of H + homeostasis. If pH  and Pco 2 estimations are not needed, the assay has  certain advantages: venous blood can be used and it  can be performed together with assays for urea and  electrolytes. It is an estimate of the sum of plasma  HC0 3 ", carbonic acid and dissolved CO,. At pH 7.4   the ratio of | HCO, - j to the other two components is  about 20:1, and at pH 7.1 it is still 10:1. Thus, if the  TCO, were 21mmol/L, [HC0 3 - ] would contribute  20mmol/L at pH 7.4 and just over 19mmol/L at  pH 7.1. Only lmmol/L and just under 2mmol/L,  respectively, would be due to H 2 C0 3 and C0 2  respectively. Thus, TCO, is effectively a measure of  the plasma [HC0 3 - ].   • The [HC0 3 “] is calculated from the Henderson-  Hasselbalch equation, using the measured values  of pH and Pco 2 (in kPa) in whole arterial blood. It  is a measure of the whole blood [HC0 3 - ] and, for  the reasons discussed above, usually agrees well with  plasma TCO,. It is the estimate of choice if the other  two parameters are being measured.   • Base excess is defined as the amount of acid that  must be added to 1L of fully oxygenated blood  to return the pH to 7.40 at 37°C and Pco 2 at  40mmHg (5.3 kPa). The usual reference range is  -2 to +2mEq/T. It is a measure of the metabolic  component that is independent of the respiratory  component. In a metabolic alkalosis base excess is  more than + 2mEq/L, and in a metabolic acidosis  base excess is less than - 2 mEq/E.   • Standard bicarbonate is provided by some blood gas  machines and defined as the bicarbonate concentration  under standard conditions: Pco 2 = 40mmHg  (5.3 kPa), at 37°C, and saturated with oxygen and is a  good measure of the metabolic component.   The concentration of dissolved CO, is calculated by  multiplying the measured Pco, by the solubility constant  of the gas: 0.23 if Pco 2 is in kPa or 0.03 if it is in mmHg.   Although this chapter focuses on the Siggaard-  Andersen ‘classic’ theory of acid-base disturbance, there  is another approach. Stewart takes into consideration  the buffering capacity of albumin. Here:   _ log [SID] - xb [Atotal]/(Kb + 10 - pH)  " " a aPco   where [SID] = [Na + ] + [K + ] - [Ch] - [lactate],  [Atotal] = all plasma weak acids mainly albumin, and  a, b, and k are all constants.   This theory may help to explain the metabolic  alkalosis seen in severely ill hypoalbuminaemic patients  (see Chapter 19) and the hyperchloraemic acidosis seen  with excess saline administration.   Hyperchloraemia occurs in a normal anion gap  metabolic acidosis. Conversely, hypochloraemia is seen  in the metabolic alkalosis of pyloric stenosis or chronic  vomiting.   Plasma [Cl - ] estimation may help in two main  situations:   • If a patient who is vomiting has a high plasma  [HC0 3 “], the finding of a low [Cl - ] favours the  diagnosis of pyloric stenosis or chronic vomiting.   • If there is a low plasma [HC0 3 - ], the finding of a  high plasma [Cl - ] (and therefore a normal anion  gap) strengthens the suspicion of hyperchloraemic  acidosis. In metabolic acidosis due to most other  causes the plasma [Cl - ] is normal and the anion gap  is increased.   A spot urinary [Cl - ] can be useful in determining  the cause of a metabolic alkalosis. A [Cl - ] less than  20mmol/L is indicative of a saline-responsive  metabolic alkalosis, for example chronic vomiting, and  a urinary [Cl - ] more than 20mmol/T of a saline-non-  responsive form of metabolic alkalosis, for example as  in mineralocorticoid excess syndromes such as Bartter’s  syndrome. Diuretics can cause variable urine chloride  concentrations, which are usually raised initially and  then reduce with chronic use.   • Tight homeostatic control of acid-base balance is  essential, otherwise cell malfunction and death can  occur. The kidneys excrete non-volatile acid via the  renal tubules into the urine, while the lungs excrete  volatile acid as CO,.   • Blood pH is inversely proportional to the Pco 2 and  directly proportional to the [HCO, - ]. This can be  determined on a blood gas analyser (Fig. 4.13).   • Respiratory acidosis results from disorders of the  respiratory system and is caused by CO, retention.  Conversely, respiratory alkalosis is due to excess  CO, loss, as in hyperventilation. In the case of the  former, compensation is by the renal excretion of  non-volatile acid and reclamation of HC0 3 “. In the  latter, the kidneys compensate by losing HC0 3 “.   • Metabolic (non-respiratory) acidosis results from  increased non-volatile acid such as lactic acid or  certain ketones. Compensation is by the lungs, which  increase C0 2 excretion by hyperventilation. Metabolic  (non-respiratory) alkalosis is caused by HC0 3 “ excess  and acid loss, such as in prolonged vomiting, and its  compensation is via the lungs, which hypoventilate,  thereby retaining volatile acid as C0 2 .   Figure 4.13 Analyser used to determine patient arterial  blood gases. Reproduced with kind permission of  Medica Corporation.   There is an important inter-relationship between  the processes of potassium metabolism and those of  water and sodium balance, renal function and acid-  base disorders (discussed in the preceding chapters).  Abnormalities of potassium homeostasis such as  plasma concentrations that are too low (hypokalaemia)  or too high (hyperkalaemia) are also relatively common  in clinical practice and important to know about, as  they may become life threatening.   The total amount of potassium in the body is about  3000 mmol, of which about 98 per cent is intracellular.  For this reason the plasma potassium concentration is  a poor indicator of the total body content.   The intracellular potassium ion concentration ([K + ])  is large and provides a reservoir for the extracellular  compartment. Consequently changes in water balance  have little direct effect on the plasma [K + ], unlike  plasma [Na + ].   The normal potassium intake is about 60-100 mmol/  day. Potassium enters and leaves the extracellular  compartment by three main routes:   Potassium is principally absorbed in the small intestine.  Dietary intake replaces net urinary and faecal loss.  Prolonged starvation can cause or aggravate potassium  depletion leading to hypokalaemia. Meat, vegetables  and fruit including bananas have about 6.2 mmol/ 100 g  of potassium. Foods with a high content of potassium  (more than 12.5 mmol/ 1 00 g) include dried fruits (dates  and prunes), nuts, avocados and bran/wheat grain.  Dried figs, molasses and seaweed are rich in potassium  (more than 25 mmol/ 1 00 g).   Potassium leaves the extracellular compartment  in all intestinal secretions, usually at concentrations  near to or a little above that in plasma. A total of about  60 mmol/day is lost into the intestinal lumen, most of  which is reabsorbed. Less than 10 mmol/day is present  in formed faeces. Excessive intestinal potassium loss can  occur in diarrhoea, ileostomy fluid or through fistulae.   Potassium is filtered by the glomeruli and, owing to the  huge volume of the filtrate, about 800 mmol (about a  quarter of the total body content) would be lost daily if  there were no tubular regulation. The net loss is about  10 per cent of that filtered.   Potassium is normally almost completely reabsorbed  in the proximal tubules. Damage to these may cause  potassium depletion. Potassium is secreted in the  distal tubules and collecting ducts in exchange for Na + ;  hydrogen ions (H + ) compete with potassium ions (K + ).  Aldosterone stimulates both exchange mechanisms.  If the proximal tubules are functioning normally,  potassium loss in the urine depends on three factors:   1. The amount of sodium available for exchange: this  depends on the glomerular filtration rate, filtered  sodium load, and sodium reabsorption from the  proximal tubules and loops of Henle. As discussed  later, reabsorption in the loops is inhibited by many  diuretics.   2. The circulating aldosterone concentration: this is  increased following fluid loss, with volume con-  traction, which usually accompanies intestinal loss  of potassium, and in most conditions for which  patients are receiving diuretic therapy. Hyperkal-  aemia stimulates aldosterone release in synergy  with angiotensin II, while hypokalaemia inhibits it.   3. The relative amounts of H + and K + in the cells of the  distal tubules and collecting ducts, and the ability to   secrete H + in exchange for Na + : this may be impaired  during treatment with carbonate dehydratase  (CD) inhibitors and in some types of renal tubular  acidosis (Chapter 4).   The Na + /K + adenosine triphosphatase (ATPase) ‘pump’  on cell surfaces maintains a high intracellular [K + ] .  This exchanges three Na + ions from cells in exchange  for two K + ions in the extracellular fluid (ECF), thus  establishing an electrochemical gradient across the cell  membrane, with a net positive charge in the ECF. The  loss of K + from cells down the concentration gradient is  opposed by this electrochemical gradient. Potassium is  also exchanged for H + (Fig. 5.1).   A small shift of K + out of cells may cause a significant  rise in plasma concentrations, whether in vivo or in vitro.  In the latter situation, the artefactual hyperkalaemia due  to haemolysed or old specimens may be misinterpreted  and should be avoided. Usually the shift of K + across  cell membranes is accompanied by a shift of Na + in  the opposite direction, but the percentage change in  extracellular [Na + ] is much less than that of K + .   Increased uptake and net gain of K + by cells may  occur in alkalosis due to increased uptake by cells and  increased urinary loss. Insulin enhances the cellular  uptake of glucose and potassium. Hyperkalaemia  stimulates insulin secretion and hypokalaemia inhibits  it. This effect may be used to treat hyperkalaemia  by giving exogenous insulin/glucose infusion (see  Treatment of hyperkalaemia). It is the main cause of the  change from hyperkalaemia to hypokalaemia during  the treatment of diabetic ketoacidosis.   Catecholamines have a similar action, and it has been  suggested that this may contribute to the hypokalaemia  sometimes found after the stress of myocardial   infarction. (3-adrenergic stimulation increases cellular  potassium uptake by stimulating the Na + /K + -ATPase  ‘pump’. (3-blockade increases plasma potassium  concentration and (3-agonists decrease it, an effect that  is independent of body potassium stores.   Synthesis of Na + /K + -ATPase is stimulated by  thyroxine, which may contribute to the hypokalaemia  sometimes associated with hyperthyroidism.   The extracellular [H + ] affects the entry of potassium  into all cells. Changes in the relative proportions of K +  and H + in distal renal tubular cells affect the urinary  loss of potassium. There is a reciprocal relationship  between K + and H + :   • In acidosis, increased loss of potassium from cells  into the ECF coupled with reduced urinary secretion  of potassium causes hyperkalaemia.   • In alkalosis, net increased uptake of potassium into  cells and increased urinary potassium loss cause  hypokalaemia.   In the kidney, Na + derived from the luminal fluid  is pumped through the cell in exchange for either K +  or H + . If K + is lost from the ECF, it passes down the  increased concentration gradient, so reducing its    Figure 5.2 Exchange of Na + for either K + or H + in the  renal tubules. Eh, non-bicarbonate base; CD, carbonate  dehydratase.   intracellular content. Unless there is renal tubular cell  damage, Na + are reabsorbed in exchange for fewer K +  and more H + than usual. For each H + formed within the  tubular cell by the CD mechanism, one bicarbonate ion  (HC0 3 “) is produced:   As more H + is secreted into the urine, the reaction  is increased and more HC0 3 “ is generated, passing into  the ECF accompanied by the reabsorbed Na + . The result  is an extracellular alkalosis and acid urine. Therefore,  chronic potassium depletion is usually accompanied  by a high plasma bicarbonate concentration. The  combination of hypokalaemia and a high plasma  bicarbonate concentration is more likely to be due to  K + depletion than to metabolic alkalosis.   Potassium and diuretic therapy  Diuretics may be used to treat hypertension as well as  oedema, for example in cardiac failure. They can be  divided into two principal groups, based upon their site  of action.   • Potassium-losing diuretics increase the sodium load  on the distal tubules and collecting ducts with  enhanced Na + /K + exchange. This may result in  hypokalaemia.   - Loop diuretics, such as furosemide or  bumetanide, inhibit sodium reabsorption from  the ascending limb of the loops of Henle.   - Thiazides act at the junction of the loops and  the distal tubules, which are sometimes called  the ‘cortical diluting segments’.   - Carbonate dehydratase inhibitors, such as  acetazolamide, are rarely used as diuretics but  are still used to treat glaucoma. They may cause  a hypokalaemic hyperchloraemic acidosis.   - Diuretics that inhibit either aldosterone directly  or the exchange mechanisms in the distal  tubules and collecting ducts cause potassium  retention and may lead to hyperkalaemia,  especially if glomerular function is impaired.  Potassium-sparing/-retaining diuretics include  spironolactone, a competitive aldosterone  antagonist.   - Other potassium-sparing diuretics include  amiloride and triamterene, which are direct  inhibitors of the Na + /K + exchange mechanism  in the distal tubules.   Potassium-sparing diuretics can be used together  with those causing potassium loss when hypokalaemia  cannot be controlled by potassium supplementation.  Used alone, they have only a weak diuretic action. Beware  of giving potassium supplements to patients taking  potassium-sparing diuretics or angiotensin- converting  enzyme (ACE) inhibitors or angiotensin II receptor  blockers (ARBs) because of the risk of hyperkalaemia.   In rapidly changing clinical states frequent estimation  of plasma potassium is the best way of assessing therapy.  Plasma potassium is best measured in a freshly collected,  unhaemolysed venous sample as a corresponding  serum concentration is higher owing to the release of  intracellular potassium as part of the clotting process.  In chronic potassium depletion, measurement of the  plasma bicarbonate concentration ([HC0 3 “]) may help  to indicate the state of cellular repletion (intracellular  potassium depletion causes extracellular alkalosis).   Urinary potassium (kaluria) estimations to  determine the primary cause of potassium depletion  may be useful diagnostically to help find the cause of  hypokalaemia. Most extrarenal causes of potassium loss  are associated with volume depletion and therefore with  secondary hyperaldosteronism. Excretion of less than  about 20 mmol/day can be expected only in the well-  hydrated hypokalaemic patient with extrarenal losses,  in whom aldosterone secretion is inhibited. Therefore  a low potassium excretion confirms extrarenal loss, but  a high one does not prove that it is the primary cause.  High urinary potassium concentration (for example  more than 20mmol/L) in the face of hypokalaemia  suggests inappropriate K + renal loss as a cause of  the hypokalaemia, for example due to renal tubular  problems or mineralocorticoid excess syndromes.   The transtubular potassium gradient (TTKG) is an  estimate of the potassium concentration at the end  of the cortical collecting duct beyond the site where  aldosterone influences potassium secretion. This can  be calculated on a spot urine (which should have an  osmolality greater than that of the plasma) and plasma  samples from the following equation:   urinary [potassium] X plasma osmolality (5.2)  plasma [potassium] X urine osmolality   The TTKG may be useful diagnostically in the  investigation of hyperkalaemia, when a TTKG less than  3 implies that the kidneys are not excreting potassium  appropriately (i.e. a lack of aldosterone effect).   Hypokalaemia is often the result of potassium depletion,  but if the rate of loss of potassium from cells equals or  exceeds that from the ECF, potassium depletion may  exist without hypokalaemia. Hypokalaemia can also  occur without depletion if there is a shift of K + into cells.   The causes of hypokalaemia (summarized in Box 5.1)  may be classified according to the predominant cause as  either renal or non- renal. Pseudo hypokalaemia is defined  as the in vitro uptake of K + from the plasma into blood  cells, primarily erythrocytes. This sometimes occurs if  the blood sample is kept in a warm environment. This  contrasts with the more usual hyperkalaemia found in  specimens stored in the refrigerator.   • Enhanced renal secretion of potassium due to  increased activity of the pump in distal renal tubules  by mineralocorticoid activity. This is associated with  a hypokalaemic alkalosis.   - Primary hyperaldosteronism (Conn’s syndrome)  due to adrenal adenoma or hyperplasia (see  Chapter 8).   - Secondary hyperaldosteronism often aggravates  other causes of potassium depletion (see  Chapter 2).   - Cushing’s syndrome and steroid therapy.  Patients secreting excess of, or on prolonged  therapy with, glucocorticoids tend to become  hypokalaemic due to the mineralocorticoid  effect on the distal renal tubules (see Chapter 8).   - ‘Ectopic’ adrenocorticotrophic hormone  (ACTH) secretion, which stimulates cortisol  secretion (see Chapter 8).   - Bartter’s syndrome is a rare autosomal recessive  condition in which there is hyperplasia of the  renal juxtaglomerular apparatus with increased  renin and therefore aldosterone secretion.  Angiotensin II activity is impaired and therefore  there is no vasoconstriction; consequently the  patient is normotensive. This latter finding  helps to distinguish the syndrome from that of  primary hyperaldosteronism, in which there can  also be a hypokalaemic alkalosis. Individuals may  have polyuria, polydipsia, short stature, learning  difficulties and maternal polyhydramnios. The  defect is in the epithelial ion channels of the  ascending limb. The biochemical features mimic   Box 5.1 Some causes of hypokalaemia  classified into predominantly non-renal  and renal causes   Decreased Na + /H + exchange  Carbonate dehydratase inhibitors  Renal tubular acidosis (types i and il)   those of high-dose loop diuretic intake. Treatment  consists of potassium supplementation, often  with a potassium-sparing diuretic such as  spironolactone or amiloride. By suppressing  prostaglandin-stimulated renin release, non-  steroidal anti-inflammatory drugs may also  improve the condition.   - The very rare Gitelman’s syndrome, in contrast  to Bartter’s syndrome, is often asymptomatic  until adulthood. It is an autosomal recessive  condition due to a mutation in the thiazide-  sensitive Na + /Cf co-transporter in the  distal convoluted tubules. Unlike Bartter’s,  there is more severe hypomagnesaemia and  hypocalciuria. Once thiazide use has been  excluded, a hypokalaemic hypomagnesaemic  alkalosis in the presence of a urinary calcium to  creatinine molar ratio less than 0.2 is suggestive  of Gitelman’s syndrome.   - Liddle’s syndrome, also very rare, is an  autosomal dominant condition with  hypertension and hypokalaemic metabolic  alkalosisis, in which suppressed plasma  aldosterone and renin concentrations are  associated with cerebrovascular disease.  Mutations in the selective sodium channels  cause increased sodium reabsorption through  the collecting duct epithelial sodium channel,  causing hypertension. Treatment involves  sodium restriction and potassium-sparing  diuretics such as amiloride or triamterene;  spironolactone is ineffective.   - Carbenoxolone therapy is occasionally used  to accelerate the healing of peptic ulcers. It  potentiates the action of aldosterone and can  cause hypokalaemia by inhibiting the enzyme  ll-(3-hydroxysteroid dehydrogenase (see  Chapter 8).   - Liquorice and tobacco contain glycyrrhizinic acid,  which also inhibits the enzyme 1 1-(3 -hydroxysteroid  dehydrogenase. Overindulgence in liquorice-  containing sweets or habitual tobacco chewing  can rarely cause hypokalaemia (see Chapter 8).   • Increased renal potassium loss may also result from  impaired proximal tubular potassium reabsorption  as in:   - renal tubular dysfunction (for example the  recovery phase of acute oliguric kidney injury),   - hypercalcaemia, which may impair the  reabsorption of potassium even if there is no  renal damage,   - hypomagnesaemia can also reduce the  intracellular potassium concentration and  causes renal potassium wasting - it may be a  cause of a refractory hypokalaemia unless the  plasma magnesium is corrected,   - a number of drugs can cause increased renal  loss of potassium, usually from distal tubules,  including diuretics (non-potassium sparing),  cisplatin, aminoglycosides, amphotericin and  foscarnet, and also CD inhibitors, for example  acetazolamide.   • Decreased renal Na + /H + exchange because of  impaired generation of H + within the renal tubular  cells, which favours Na + /K + exchange associated  with a hypokalaemia. Causes include renal tubular  acidosis types I and II (see Chapter 4).   • Excess sodium available for exchange in the distal  renal tubules can also increase renal potassium loss.  These include:   - diuretics inhibiting the pump in the loops  of Henle - the activity of the distal pump is  enhanced by secondary hyperaldosteronism.   - Glucose and insulin therapy. This may be used  to treat severe hyperkalaemia.   - Increased secretion of catecholamines, such  as may occur from the stress of myocardial  infarction. The use of |3-agonists such as  salbutamol in inhalers may also stimulate this  secretion.   - Rapidly growing tumours, such as certain  leukaemias, can evoke hypokalaemia, probably  by potassium uptake into the proliferating  cells.   - Familial hypokalaemic periodic paralysis is  rare and is an autosomal dominant defect of  the dihydropyridine receptor, a voltage-gated  calcium channel. In this condition episodic  paralysis is associated with entry of potassium  into cells, which can be provoked by a high  carbohydrate intake. Thyrotoxic periodic  paralysis is a similar condition, but genetically  different, that is found in hyperthyroidism.   - Treatment of pernicious anaemia with vitamin  B p , by rapid cellular uptake of potassium into  cells (see Chapter 15).   - Chronic starvation. If water and salt intake are  also reduced, secondary hyperaldosteronism  may aggravate the hypokalaemia.   - Inadequate potassium replenishment  in patients receiving intravenous fluid  replacement or total parenteral nutrition.   - Diarrhoea, whether due to infections,  malabsorption states, radiation or  chemotherapy.   - Habitual purgative users may present with  hypokalaemia. It may be possible to detect  laxatives in the stools to confirm this.   - The concentration of potassium in fluid from  a recent ileostomy and in diarrhoea stools may  be 5-10 times that of plasma, but a prolonged  drain of any intestinal secretion, even if its  potassium concentration is not very high,  causes depletion, especially if urinary loss is  increased by secondary hyperaldosteronism.   - Mucus-secreting villous adenomas of  the intestine are very rare but may cause  considerable potassium loss. Zollinger-Ellison  syndrome and VIPomas [tumours that secrete  vasoactive intestinal polypeptide (VIP)] are  other rare causes.   - Rare chloride -losing diarrhoea (intestinal ion  transport defect) (see Chapter 16).   - The unusual habit of geophagia (earth eating) can  also cause hypokalaemia, possibly by chelating K + .   - Remember that hypokalaemia can be exacerbated  by magnesium loss and hypomagnesaemia.   Clinical features of hypokalaemia  The clinical features of disturbances of potassium  metabolism are due to changes in the extracellular  concentration of the ion. Hypokalaemia, by interfering  with neuromuscular transmission, causes muscular  weakness, hypotonia and cardiac arrhythmias,  and may precipitate digoxin toxicity. Patients with  mild hypokalaemia (3.0-3.5mmol/L) may have  no symptoms, although those in which it is more  severe may show generalized weakness, lassitude and  constipation. If the plasma potassium concentration is  below 2.5 mmol/L, muscle necrosis and rhabdomyolysis  may rarely occur. Hypokalaemia may also aggravate  paralytic ileus and hepatic encephalopathy.   Cardiac symptoms depend on the rate of change  of potassium loss and any other concomitant heart  disease. Hypokalaemia can increase the risk of digoxin  toxicity and causes prolongation of the P-R interval,   Figure 5.3 Typical appearance of hypokalaemia on an  electrocardiogram. Adapted with kind permission from  Houghton AR and Gray D. Making Sense of the ECG: A  Hands-on Guide, 3rd edition. London: Hodder Arnold,  2008.   flattening of the T waves and prominent U waves on  the electrocardiogram (ECG) (Fig. 5.3).   Intracellular potassium depletion causes extracellular  alkalosis, and severe hypokalaemia can be associated with  a metabolic alkalosis. This reduces the plasma free ionized  calcium concentration and, in long-standing depletion of  gradual onset, the presenting symptoms may be muscle  cramps and even tetany. This syndrome is accompanied  by high plasma [HC0 3 “] . Prolonged potassium depletion  impairs the renal concentrating mechanism and may  cause polyuria with further potassium depletion.   The following procedure should help elucidate the  cause of the hypokalaemia in conjunction with plasma  potassium and bicarbonate determinations (see Box  5.1). Raised plasma [HC0 3 “], in the absence of chronic  pulmonary disease, is indicative of a metabolic alkalosis,  and low plasma [HC0 3 “] is suggestive of a metabolic  acidosis. Therefore, hypokalaemia can be divided into  alkalosis and acidosis groups.   Diuretic therapy is the most common cause of  hypokalaemic alkalosis in hypertensive patients.   • Take a careful drug history, with special reference to  potassium-losing diuretics, purgatives and steroids.  Rare causes of a steroid-like effect are ingestion of  carbenoxolone or liquorice.   • Check the potassium concentration in a spot urine  sample. If less than or equal to 20 mmol/L, it is suggestive  of gastrointestinal loss or transcellular potassium shift  or poor potassium intake. If it is more than 20 mmol/L,  it is indicative of renal potassium loss such as occurs  in the very rare Bartter’s and Gitelman’s syndromes  and other mineralocorticoid excess disorders.   more than 20 mmol/L in Bartter’s or Gitelman’s  syndrome. This is useful diagnostically to distinguish  them from other causes of hypokalaemia such as  gastrointestinal loss of potassium, for example  vomiting or diarrhoea in which urinary chloride is  usually less than 20 mmol/L. If purgative abuse is  suspected, an estimation of the drug levels in stool  samples may be indicated.   • Exclude hypomagnesaemia, which may be associated  with hypokalaemia (see Chapter 6).   • Fortheinvestigationofprimaryhyperaldosteronism  (Conn’s syndrome), Cushing’s syndrome and  other mineralocorticoid excess syndromes, see  Chapter 8.   A 17-year-old student presented to her general  practitioner with generalized muscle weakness and  tiredness. Her blood pressure was 1 16/70 mmHg.  The following biochemical results were obtained:   The results suggest a hypokalaemic metabolic  alkalosis. There is also hypochloraemia, due to  chloride loss from chronic vomiting. It transpired that  the patient had anorexia nervosa with bulimia and  had been self-inducing vomiting for many months.  This had resulted in a metabolic alkalosis (note raised  plasma bicarbonate) and severe hypokalaemia. The  low urinary chloride concentration helps distinguish  this from the much rarer Bartter’s syndrome, in  which the urinary chloride concentration would be  expected to be more than 20 mmol/L.   Hypokalaemic acidosis, which may be associated  with hyperchloraemia (hyperchloraemic acidosis),  is relatively rare and the cause is usually obvious (see  Chapter 4). Having checked plasma potassium, chloride  and bicarbonate concentrations, consider the following:   • Loss of potassium through fistulae in the proximal  small intestine may be accompanied by significant  bicarbonate loss.   • Certain drugs should be considered. These include  azetazolamide, a carbonic dehydratase inhibitor  sometimes used in the treatment of glaucoma and  altitude sickness or as a diuretic.   • If neither of these causes is present, a diagnosis of  renal tubular acidosis should be considered (see  Chapter 4).   Mild hypokalaemia can be treated with oral potassium  supplements, which can be continued until plasma   potassium concentrations have returned to normal.  The normal subject loses about 60 mmol of potassium  daily in the urine, much larger amounts being excreted  during diuretic therapy. By the time hypokalaemic  alkalosis is present, the total deficit is probably 100-  200 mmol. A patient with hypokalaemia should usually  be given about 80 mmol/ day, but more may be needed  if plasma potassium concentrations fail to rise.   It may sometimes be dangerous to give potassium,  especially intravenously, if there is renal impairment.  One of the most common forms of hyperkalaemia in  hospital patients results from supplemental potassium  administration. Generally the plasma potassium  decreases by 0.3 mmol/L for each 100 mmol reduction  in total body stores. Sometimes, 40-100 mmol of  supplemental potassium is needed per day to maintain  potassium concentrations within the reference range in  patients receiving diuretics.   • potassium chloride effervescent tablets, containing  6.5 mmol K + per tablet,   • Sando-K: 12 mmol K + per tablet (as bicarbonate and  chloride).   Oral therapy may be safer, as potassium enters the  circulation more slowly. However, oral potassium salts  can cause gastrointestinal upset and are not adequate  if there is life-threatening hypokalaemia (for example  less than 2.0 mmol/L or clinical symptoms and signs).  The slow- release forms maybe better tolerated but may  cause gastrointestinal ulceration.   In severe hypokalaemia, particularly if the patient is  unable to take oral supplements, intravenous potassium  should be given cautiously. Diarrhoea not only reduces  the absorption of oral potassium supplements, but  may also be aggravated by them; this too may be an  indication for intravenous therapy.   Intravenous potassium should be given with care,  especially in the presence of poor glomerular function;  ECG monitoring is wise, along with regular monitoring  of plasma potassium and renal function. Potassium  chloride is the usual intravenous form. The following  rules should be observed:   • Unless the deficit is unequivocal and severe,  intravenous potassium should not be given if there  is oliguria.   • Potassium in intravenous fluid should not usually  exceed a concentration of 40 mmol/L.   • Intravenous potassium should not usually be given  at a rate of more than 20mmol/h and should be  given by infusion pump. In very severe depletion this  dose may have to be exceeded; in such cases frequent  plasma potassium estimations must be performed.   • Twenty millimoles of potassium chloride can be  added to a full bag (500 mL) of intravenous fluid  and mixed well, although pre-mixed bags are now  becoming standard. Potassium chloride solution  should never be given undiluted.   • Always check the labelling of vials carefully when  giving potassium, as fatalities have occurred due to  errors in administration.   If hypomagnesaemia is present, this should also be  treated, as it can be a cause of refractory hypokalaemia.   Plasma potassium results should not be reported  if the specimen is haemolysed or if plasma was not  separated from cells within a few hours after the  blood was taken. Pseudohyperkalaemia, due to in  vitro leakage of potassium from cells into plasma,  is often misinterpreted, sometimes with dangerous  consequences. This may occur if there has been a delay  in separating the plasma from the cells, particularly  if the blood sample has been refrigerated, when the  activity of the Na + /K + -ATPase pump is slowed. A rare  familial form of pseudohyperkalaemia is thought to  be due to defective red cell membranes in hereditary  spherocytosis. Thrombocytosis and leucocytosis can  also result in pseudohyperkalaemia. Also, beware of  blood tubes contaminated with potassium-EDTA  (ethylenediamine tetra-acetic acid): blood samples for  potassium assay should usually be collected in lithium  heparin tubes.   The causes of hyperkalaemia are summarized in  Box 5.2, and can be classified into renal and non-renal  causes.   - Angiotensin-converting enzyme inhibitors,  such as ramipril and enalapril inhibit the  conversion of angiotensin I to angiotensin II  and therefore aldosterone secretion. They may  cause hyperkalaemia, especially if glomerular  function is impaired or in presence of non-  steroidal anti-inflammatory drugs (NSAIDs).   Box 5.2 Some causes of hyperkalaemia  classified into predominantly renal and  non-renal causes   Renal glomerular dysfunction, e.g. acute kidney  injury or chronic kidney disease  Sodium depletion  Decreased Na + /K + exchange  Mineralocorticoid deficiency  Hypoaldosteronism (Addison's disease)   - Angiotensin II receptor blockers, for example  losartan and candesartan, and diuretics that  either act on the distal tubules and antagonize  aldosterone activity (spironolactone) or  inhibit the pump (amiloride). NSAIDs may  also cause hyperkalaemia, as can heparin, the  former by reducing renin and aldosterone  secretion.   • Acute kidney injury (AKI) or chronic kidney disease  (CKD ) involves impaired renal secretion of potassium  and decreases the activity of the Na + /K + -ATPase  pump in the distal renal tubules when fewer Na +   reach the distal convoluted tubules to be exchanged.  This is especially true when the glomerular filtration  rate (GFR) is less than 25 per cent of normal (see  Chapter 3).   • Another mechanism is gain of K + by ECF via more  than one route with reduced renal excretion.   • Acidosis. In diabetic ketoacidosis in poorly  controlled type 1 diabetes mellitus (Chapter 12),  potassium enters the ECF from cells because  normal action of the pump depends on the energy  supplied by glucose metabolism. If glomerular  function is relatively normal, urinary potassium  loss is increased, resulting in total body potassium  depletion. Loss from the plasma into the urine  rarely keeps pace with the gain from cells, and  mild hyperkalaemia is common. As the condition  progresses, two other factors contribute to  hyperkalaemia, namely volume depletion causing a  reduced GFR and ketoacidosis.   All these factors are reversed during insulin and  fluid therapy. As potassium re-enters cells, extracellular  potassium concentrations fall and the depletion is  revealed. Plasma potassium concentrations must be  monitored regularly during therapy and potassium  must be given as soon as the concentration begins  to fall.   • Hypoxia causes impairment of the pump in all cells,  with a net gain of potassium within the ECF. Such  impairment in the distal tubules causes potassium  retention. If hypoxia is severe, lactic acidosis  aggravates hyperkalaemia (see Chapter 4).   • Mineralocorticoid deficiency: mineralocorticoids  such as aldosterone have a sodium-retaining  and potassium-losing action (see Chapter 2).  Thus deficiency of these hormones can cause  hyperkalaemia. This is dealt with in more detail in  Chapter 8, but examples include:   - adrenal insufficiency (Addison’s disease) due to  absence of or damage to the adrenal glands,   - hyporeninaemic hypoaldosteronism, presenting  in the elderly, often with renal impairment and  glucose intolerance or type 2 diabetes mellitus;  it is associated with hyperkalaemia and type IV  renal tubular acidosis (see Chapter 4),   - rare causes include pseudohypoaldosteronism,  for example Gordon’s syndrome (discussed in  Chapter 8).   These are due to the gain of potassium by the body  from the intestine or by the intravenous route:   • Excess potassium therapy, especially in patients  with impaired glomerular function, either orally as  potassium supplementation or, more commonly,  intravenously, for example with potassium chloride-  containing fluids.   - Severe tissue damage, for example haemolysis,  burns, crush injury, tumour lysis syndrome,  rhabdomyolysis and severe exercise. In these  cases intracellular potassium moves out of cells.   - Familial hyperkalaemic periodic paralysis is  exceedingly rare and is associated with release  of potassium from cells.   - Various drugs, such as digoxin, (3-adrenergic  blockers and suxamethonium (succinylcholine),  can cause hyperkalaemia by reducing the entry  of potassium into cells (see Box 5.2).   Clinical features of hyperkalaemia  Hyperkalaemia, particularly if severe, carries the danger  of cardiac arrest. Changes are also seen on ECG, such as  widening of the QRS complex and tall, ‘tented’ T waves  (Fig. 5.5).    Figure 5.5 Typical appearance of hyperkalaemia on  electrocardiogram. Adapted with kind permission from  Houghton AR and Gray D. Making Sense of the ECG: A  Hands-on Guide, 3rd edition. London: Hodder Arnold,  2008.   • Exclude artefactual (pseudohyperkalaemia) causes of  hyperkalaemia such as haemolysis, thrombocytosis,  leucocytosis or delayed separation of plasma from  blood cells or potassium-EDTA-contaminated tubes.   • Is there a cause for cell damage or transcellular  potassium shifts, such as hypoxia, rhabdomyolysis  or severe trauma?   • What are the plasma urea and creatinine  concentrations? Severe renal glomerular dysfunction  is a common cause of hyperkalaemia.   • Measure the plasma bicarbonate concentration.  An acidosis, whether metabolic (with a low plasma  bicarbonate concentration) or respiratory (with a  normal or high plasma bicarbonate concentration),  may cause mild hyperkalaemia even if glomerular  function is normal. A cause of respiratory acidosis is  usually obvious clinically (see Chapter 4).   • Measurement of the TTKG (described above) may  help to determine whether there is a potassium renal  excretory effect.   • Is there evidence of mineralocorticoid deficiency?  This should not be forgotten, as adrenal insufficiency  can be life threatening. If there is any doubt, consider  performing a short Synacthen test (see Chapter 8 for  details of the investigation of adrenal insufficiency).   • Rare causes of hyperkalaemia, such hyporeninaemic  hypoaldosteronism, pseudohypoaldosteronism and  type IV renal tubular acidosis, are shown in Box  5.2. The investigation of renal tubular acidosis is  described in Chapter 4.   gluconate, 10 mL of a 10 per cent solution, is given   A 64-year-old patient was being treated for  hypertension. Her medications were enalapril and  amiloride. The results were:   The hyperkalaemia was found to be due to the  injudicious use of an angiotensin-converting enzyme  inhibitor and potassium-sparing diuretic. This drug  combination can lead to severe hyperkalaemia,  especially in someone with impaired renal function.   slowly intravenously with ECG monitoring. This  antagonizes the effect of hyperkalaemia on heart muscle  but does not alter potassium concentrations. However,  calcium should never be added to bicarbonate solutions  because calcium carbonate is poorly soluble in water. If  too much calcium is given, hypercalcaemia may cause  cardiac arrest.   Intravenous 50 mL glucose 50 per cent with 10  units of soluble insulin by intravenous injection  over 15-30 min starts to lower plasma potassium  concentrations for a couple of hours by increasing  potassium entry into cells. Insulin enhances the activity  of the pump and must be given with glucose to prevent  hypoglycaemia.   More rarely, 50-100 mL of 8.4 per cent sodium  bicarbonate, usually via a central line, may be infused  over 30 min if a severe acidosis (for example pH less  than 7.1) is present, or 1.26 per cent solution in isotonic  saline if the situation is less urgent. Remember that   8.4 per cent sodium bicarbonate is hyperosmolar.  Infusing bicarbonate increases the rate of potassium  entry into cells.   Sometimes, if the hyperkalaemia is not too severe, 1 0—  20 mg nebulized salbutamol can be used to lower plasma  potassium as (3,-catecholamines cause redistribution of  potassium into cells. Reductions in plasma potassium of  0.5-1.0mmol/L can occur and last for 2-3h. In severe  hyperkalaemia with renal dysfunction haemofiltration  or dialysis may be indicated.   Sodium or calcium polystyrene sulphonate  (Resonium-A or Calcium Resonium) 15 g orally three  or four times a day can remove potassium from the  body but may take at least 24 h to work. Type IV renal  tubular acidosis is a chronic cause of hyperkalaemia  and may be treated with fludrocortisone and a thiazide  diuretic or furosemide.   Disorders of potassium homeostasis are common  in clinical practice and can be life threatening.  Potassium is a predominantly intracellular cation,  and thus plasma levels do not adequately reflect  total body potassium stores.   Potassium ions are controlled by renal excretion,  which is increased by aldosterone and reduced by H + .   Hypokalaemia can cause weakness, paralytic ileus  and ECG changes; common causes are potassium  loss from the kidneys or gastrointestinal tract,  cellular redistribution and poor intake.  Hyperkalaemia can cause lethal cardiac arrhythmias  and is commonly due to renal retention, movement  out of cells or increased intake.   Disorders of calcium metabolism are common in  clinical practice and may result in hypocalcaemia or  hypercalcaemia as well as bone abnormalities. Intimately  associated with calcium disorders are disorders involving  phosphate and magnesium metabolism.   The total body calcium depends upon the calcium  absorbed from dietary intake and that lost from the  body (Fig. 6.1). Ninety-eight per cent of body calcium  is found in the skeleton. The extraosseous fraction,  although amounting to only 1 per cent of the total,  is essential because of its effect on neuromuscular  excitability and cardiac muscle. An important mediator  of intracellular calcium is calmodulin, a calcium-  binding regulatory protein.   Factors affecting calcium intake  About 25 mmol (lg) of calcium is ingested per day,  of which there is a net absorption of 6-12 mmol  (0.25-0.5 g). The active metabolite of vitamin D,   1.25- dihydroxycholecalciferol (l,25-(OH) 2 D 3 , also  called calcitriol), is needed for calcium absorption.   Factors affecting calcium loss  Calcium is lost in urine and faeces. Urinary calcium  excretion depends on the amount of calcium reaching  the glomeruli, the glomerular filtration rate (GFR)  and renal tubular function. Parathyroid hormone and   Faecal calcium is derived from the diet and that  portion of the large amount of intestinal secretions  that has not been reabsorbed. Calcium in the intestine   may form insoluble, poorly absorbed complexes with  oxalate, phosphate or fatty acids. An excess of fatty acids  in the intestinal lumen in steatorrhoea may contribute  to calcium malabsorption.   The mean plasma calcium concentration in healthy  subjects is tightly controlled, at around 2.15-  2.55 mmol/L, and is present in two main forms:   • Calcium bound to proteins, mainly albumin: this  accounts for a little less than half the total calcium  concentration as measured by routine analytical  methods and is the physiologically inactive form.   • Free ionized calcium (Ca 2+ ), which comprises most  of the rest. This is the physiologically active fraction.   Changes in plasma protein concentration,  particularly of albumin, the principal plasma protein,  alter the most commonly measured concentration,  that of plasma total calcium, but not that of the free  ionized fraction. The plasma total (but not free  ionized) calcium concentration is lower in the supine  than in the erect position because of the effect of  posture on fluid distribution and therefore on plasma  protein concentration. The direct measurement of the  physiologically active free calcium ionized fraction is,  for technical reasons, confined to special cases such as  acid-base disturbance.   Formulae incorporating the albumin concentration  have been devised in an attempt to calculate the active  fraction of the plasma total calcium concentration,  but, because binding is not simple, these are not always  reliable, particularly if extremes of plasma albumin  concentration occur. The following is a commonly used  formula:   plasma albumin-adjusted or ‘corrected’ calcium (mmol/L)  = plasma measured calcium  + (40 - plasma [albumin]) (g/L) X 0.02 (6.1)   Changes in plasma hydrogen ion concentration  ([H + ]) affect the binding of calcium to plasma  proteins because H + competes with Ca 2+ for binding  sites. The plasma total calcium concentration is  unaltered by changes in [H + ]. If [H + ] falls, as in an  alkalosis, tetany may occur, despite a normal plasma  total calcium concentration. Conversely, an acidosis  decreases binding and so increases the proportion  of plasma calcium in the free ionized form. Also, by  increasing calcium solubility, it increases the rate of  release of calcium from bones into the extracellular  fluid (ECF). The increased load reaching the kidneys  increases the renal calcium loss. Prolonged acidosis  may cause osteomalacia, partly due to the buffering  effect of bone.   A 45-year-old man was in the intensive care unit for  multiple trauma following a road traffic accident.  Some of his biochemistry results were as follows:   Note that the plasma calcium now adjusted falls  within the reference range and does not require  specific treatment. Remember this if the patient has  hypoalbuminaemia.   There are a number of mechanisms by which plasma  calcium concentrations are controlled. Calcium  homeostasis follows the general rule that extracellular  concentrations are controlled rather than the total  body content. The effectiveness of this control depends  upon:   If any one of these factors is impaired, calcium leaves  bone by passive physicochemical diffusion, and plasma  concentrations may be maintained at the expense of  bone calcification.   Parathyroid hormone (PTH) is a single-chain  polypeptide containing 84 residues, with its 34  N-terminal amino acids largely determining its  biological activity. It is metabolized by renal, hepatic  and bone cells. Renal clearance from plasma of the  physiologically inert C-terminal fragment is slower  than that of the N-terminal fragment, which may  accumulate in plasma in renal glomerular dysfunction.  The biological actions of PTH include:   • stimulation of osteoclastic bone resorption, so  releasing both free ionized calcium and phosphate  into the ECF; this action increases the plasma  concentrations of both calcium and phosphate,   • decreased renal tubular reabsorption of phosphate,  causing phosphaturia and increased reabsorption  of calcium; this action tends to increase the plasma  calcium concentration but to decrease the phosphate.   The control of PTH secretion depends on the  concentration of free ionized calcium in blood  circulating through the parathyroid glands. A fall  increases the rate of PTH secretion, which, under  physiological conditions, continues until the calcium  concentration returns to normal. The secretion of  PTH is also affected by the extracellular magnesium  concentration, being decreased by severe, chronic  hypomagnesaemia.   Detectable plasma PTH, even if the concentration  is within the reference range, is inappropriate in the   presence of hypercalcaemia and is consistent with  primary or, more rarely, tertiary hyperparathyroidism.   Parathyroid hormone-related protein (PTHRP) is a  peptide hormone that has a similar amino acid sequence  at the biologically active end of the peptide, therefore  activating the same receptors as PTH. The function of  PTHRP is uncertain, but it maybe important in calcium  metabolism in the fetus. The gene that codes for PTHRP  is widely distributed in body tissues but is normally  repressed. However, it may become derepressed in  certain tumours, causing humoral hypercalcaemia of  malignancy.   Calcitonin (produced in the C cells of the thyroid  gland) decreases osteoclastic activity, slows calcium  release from bone and has the opposite effect on  plasma concentrations of PTH. It is probably less  important than PTH in physiological homeostasis.  Plasma concentrations may be very high in patients  with medullary carcinoma of the thyroid, although  hypocalcaemia is not usually reported in this condition.  However, exogenous calcitonin has been used to treat  hypercalcaemia and Paget’s disease of bone.   Metabolism and action of vitamin D  Vitamin D is derived from:   • ergocalciferol (vitamin D 2 ), obtained from plants in  the diet,   • cholecalciferol (vitamin D 3 ) , formed in the skin by the  action of ultraviolet light on 7-dehydrocholesterol  (Fig. 6.2); this is the form found in animal tissues,  especially the liver.   In normal adults, much more cholecalciferol is  derived from the action of sunlight on skin (wavelength  270-3 10 nm) than from food. Dietary sources are  important when requirements are high, such as during  growth or pregnancy, or in those elderly or chronically  sick individuals who are confined indoors and not  exposed to the sun.   Vitamin D is transported in plasma bound to  specific carrier proteins. It is inactive until metabolized.  In the liver, cholecalciferol is hydroxylated to  25-hydroxycholecalciferol (25-OHD,) by the enzyme  25-hydroxylase. The rate of formation of 25-OHD 3  is affected by the supply of substrate in the form of  calciferol, whether derived from the skin or from the   Figure 6.2 Formation of the active vitamin D metabolite from 7-dehydrocholesterol. UV, ultraviolet.   diet. It is the main circulating form and store of the  vitamin. Other hydroxylated metabolites are found,  such as 24,25-(OH) 2 D 3 .   In the proximal renal tubular cells of the kidney,  25-OHD, undergoes a second hydroxylation, catalysed  by the enzyme 1-Ot-hydroxylase to form the active  metabolite l,25-(OH) 2 D 3 .   The activity of 1-OC-hydroxylase, and hence the  production of l,25-(OH) 2 D 3 , maybe stimulated by:   • an increase in plasma PTH concentration, possibly  because of its phosphate-lowering effect.   The kidney is an endocrine organ, synthesizing and  releasing the hormone l,25-(OH) D ; impairment of  the final hydroxylation helps explain the hypocalcaemia  of renal disease. This hormone increases calcium  absorption by intestinal mucosal cells. In conjunction  with PTH, it stimulates osteoclastic activity, releasing  calcium from bone.   The action of PTH on bone is impaired in the  absence of l,25-(OH) 2 D 3 . A fall in plasma free ionized  calcium concentration stimulates PTH secretion.  The PTH enhances 1 -a- hydroxylase activity and  therefore stimulates l,25-(OH),D 3 synthesis. The two  hormones act synergistically on the osteoclasts of bone,  releasing calcium into the circulation; l,25-(OH) 2 D 3  also increases calcium absorption from the intestinal  lumen. In the short term, the homeostatic mechanisms  involving the effects on bone are the more important; if   hypocalcaemia is prolonged, more efficient absorption  becomes important. Once the plasma free ionized  calcium concentration is adjusted, the secretion of both  PTH and l,25-(OH) 2 D 3 is suppressed.   Thus, 25-OHD 3 is the circulating, inactive form of  vitamin D and plasma concentrations fall in deficiency  states. The measurement of the biologically active  metabolite, l,25-(OH),D 3 , which circulates in plasma  bound to vitamin D-binding protein (VDBP) in very  low concentrations, is rarely indicated unless a defect in  the vitamin metabolic pathway is suspected, as it does  not reflect body stores.   The vitamin D receptor (VDR) is found in almost  all cell nuclei with various effector systems such as  endocrine, paracrine or autocrine. Calcitriol activates  the receptor, which forms a heterodimer with the  retinoid-X receptor and binds to hormone response  elements on deoxyribonucleic acid (DNA) and is  involved in the expression of various gene products.  These pathways not only involve bone metabolism  but also have implications for the immune system and  carcinogenesis.   The calcium-sensing receptor (CaSR) is a G protein-  coupled receptor. This allows the parathyroid cells and  the ascending loop of Henle epithelial cells to respond  to changes in extracellular calcium. The parathyroid  cell surface is rich in CaSR, which allows PTH secretion  to be adjusted rapidly depending on the calcium  concentration.   Defects in the CaSR gene are responsible for  various rare defects of calcium homeostasis.  Inactivating mutations include familial benign   hypocalciuric hypercalcaemia and neonatal  severe hyperparathyroidism; activating mutations  include autosomal dominant hypocalcaemia with  hypercalciuria. Calcimimetic agents have been  devised that bind and activate the CaSR, resulting in  decreased PTH release and reduced plasma calcium  concentrations.   Thyroid hormone excess may be associated with  the histological appearance of osteoporosis and  with increased faecal and urinary excretion of  calcium, probably following its release from bone.  Hypercalcaemia is a very rare complication of severe  hyperthyroidism. Unless there is gross excess of thyroid  hormone, the effects on plasma calcium are over-  ridden by homeostatic reduction of PTH secretion and  by urinary loss.   Other hormones influencing calcium metabolism  include oestrogens, prolactin and growth hormone.  These may increase l,25-(OH) 2 D 3 production and  increase calcium absorption during pregnancy,  lactation and growth.   The consequences of most disturbances of calcium  metabolism can be predicted from knowledge of the  actions of PTH on bone and on renal tubular cells, and  from plasma concentrations of calcium and phosphate.  A low plasma free ionized calcium concentration  normally stimulates PTH secretion, which results in  phosphaturia; the loss of urinary phosphate over-rides  the tendency to hyperphosphataemia due to the action  of PTH on bone.   Consequently, the plasma phosphate concentration  is usually low when the plasma PTH concentration  is increased. Conversely, a high plasma free ionized  calcium concentration, unless due to inappropriate  excess of PTH, inhibits PTH secretion and causes a high  plasma phosphate concentration. Therefore plasma  calcium and phosphate concentrations usually vary in  the same direction unless:   • renal glomerular dysfunction is severe enough  to impair the phosphaturic (and therefore  hypophosphataemic) effect of PTH or PTHRP,   • there is inappropriate excess or deficiency of PTH  due to a primary disorder of the parathyroid gland  or to secretion of PTHRP; in such cases calcium and  phosphate vary in opposite directions.   - Renal damage is one of the most serious clinical  consequences of prolonged hypercalcaemia.  Because of the high plasma free ionized calcium  concentration, the solubility of calcium  phosphate may be exceeded and precipitate  in extraosseous sites such as the kidneys (see  Chapter 3).   - Polyuria, characteristic of chronic  hypercalcaemia, may result from impairment  of renal concentrating ability owing to  calcification of the tubular cells; acute  hypercalcaemia may cause reversible inhibition  of the tubular response to anti diuretic hormone  rather than to cell damage. These effects can  lead to dehydration.   - Renal calculi, without significant parenchymal  damage, may be caused by precipitation of  calcium salts in the urine if the free ionized  calcium concentration is high in the glomerular  filtrate owing to hypercalcaemia (see Chapter   - Hypokalaemia, often with a metabolic alkalosis,  is associated with hypercalcaemia. Calcium  may directly inhibit potassium reabsorption  from the tubular lumen (see Chapter 5).   • High extracellular free ionized calcium concentrations  can depress neuromuscular excitability in both  voluntary and involuntary muscle. There may also  be muscular hypotonia.   • Depression, anorexia, nausea and vomiting,  associated with high plasma calcium concentrations,  are probably caused by an effect on the central  nervous system.   • Calcium stimulates gastrin (and therefore gastric  acid) secretion. There is an association between  chronic hypercalcaemia and peptic ulceration.  The patient may complain of constipation and  abdominal pain. Hypercalcaemia may also present  as an acute abdomen.   • Some patients with hypercalcaemia may be  hypertensive. If renal damage is not severe, the  hypertension may respond to reducing the plasma  calcium concentration.   • Severe hypercalcaemia causes characteristic changes  in the electrocardiogram (ECG), with shortening of   the Q-T interval and broadening of the T waves. If  plasma concentrations exceed about 3.5mmol/L,  there is a risk of sudden cardiac arrest or ventricular  arrhythmias. For this reason severe hypercalcaemia  should be treated as a matter of urgency.   ‘Bones, moans, groans and stones’ is a useful  mnemonic to remember some of these clinical  consequences of hypercalcaemia.   Overall, thiazides are one of the most common causes  of mild hypercalcaemia. However, most causes of  severe hypercalcaemia are related to either primary   Primary hyperparathyroidism (adenoma, hyperplasia,  carcinoma or associated with multiple endocrine  neoplasia)   hyperparathyroidism or malignancy. In the case of the  latter, 80 per cent are due to bony metastases, with the  remainder being mainly due to ectopic PTHRP. Some  causes of hypercalcaemia are depicted in Box 6.1.   True free ionized or albumin-adjusted hyper-  calcaemia with hypophosphataemia is usually caused  by inappropriate secretion of PTH or PTHRP. The term  ‘inappropriate secretion’ is used in this book to indicate  that the release of hormone into the circulation is not  adequately inhibited by negative feedback control.  Inappropriate PTH secretion occurs in the following  clinical situations:   If renal glomerular function is adequate, the high  circulating PTH or PTHRP concentrations cause  hypercalcaemia, which is associated with a low-normal  or low plasma phosphate concentration in relation  to GFR, and to phosphaturia. If glomerular damage  develops due to hypercalcaemia, the kidneys cannot  respond normally to the phosphaturic effect of PTH  and, because of impaired hydroxylation of 25-OHD 3 ,  plasma calcium concentrations may fall towards or  within the reference range as renal failure progresses.  Because plasma phosphate concentrations tend to rise,  diagnosis may be difficult at this stage.   A 53-year-old man saw his general practitioner  because of bone pain and constipation. A number  of laboratory tests were requested, the results for the  most relevant of which were as follows:   The patient has hypercalcaemia. Note also the  hypophosphataemia and inappropriately raised  PTH concentration. The diagnosis was subsequently  found to be primary hyperparathyroidism due to  a parathyroid adenoma associated with multiple  endocrine neoplasia (MEN) type I. His symptoms  are typical of chronic hypercalcaemia.   The clinical features of PTH- or PTHRP-induced  hypercalcaemia are due to:   • excess circulating concentration of free ionized  calcium that is the direct consequence of increased  osteoclastic activity and release of calcium from  bone, and enhanced absorption of calcium from the  intestinal lumen by vitamin D; PTH increases the  formation of l,25-(OH) 2 D 3 ,   • the effects of persistent PTH or PTHRP activity on  bone in the presence of a normal supply of vitamin  D and calcium (see Fig. 6.1).   The differences between the clinical presentations  associated with inappropriately high plasma PTH  concentrations depend on the duration of the disease.  The following effects on bone become evident only in  long-standing cases. Prolonged decalcification of bone  causes a secondary increase in osteoblastic activity.  Alkaline phosphatase-rich osteoblasts release the enzyme  into the circulation and, if the number of cells is greatly  increased, plasma alkaline phosphatase activity rises.   This is caused by inappropriate secretion of PTH by  the parathyroid glands, causing hypercalcaemia. It is  usually due to one or more parathyroid adenomas,  but occasionally to hyperplasia of all four parathyroid  glands or to carcinoma of one of the glands. Ectopic  parathyroid tumours do also occur. Primary  hyperparathyroidism may be associated with other  multiple endocrine neoplasias (MENs), such as  pituitary and pancreatic adenomas (MEN type I), or  with phaeochromocytomas and medullary carcinoma  of the thyroid (MEN type II). The incidence of primary  hyperparathyroidism increases with age, being most  common in elderly females.   The majority of cases of primary hyperthyroidism  are diagnosed after the chance finding of high  plasma calcium, usually with low plasma phosphate  concentrations.   Where there are clinical symptoms and signs at  presentation, these are due to hypercalcaemia and  include the following:   • Generalized ill health Depression, nausea, anorexia  and abdominal pain and polyuria.   • Renal calculi About 10 per cent of patients  who present with renal calculi have primary  hyperparathyroidism.   • Bone pain In most patients, subperiosteal bone  erosions or cysts may be seen on radiography of   the terminal phalanges. However, extensive, severe  bone disease, osteitis fibrosa cystica, is now a rare  presenting feature, as patients are usually diagnosed  before the disorder is extensive, and consequently  plasma alkaline phosphatase activity is usually  normal or only slightly increased. There are increased  numbers of osteoclasts and an increased risk of bone  fracture.   • Medical emergency Occasionally patients are  admitted as an emergency with abdominal pain,  vomiting and constipation. Severe hypercalcaemia is  a recognized cause of acute pancreatitis and should  be considered as one cause of an ‘acute abdomen’.   Recently, it has been reported that an incipient form  of primary hyperparathyroidism exists in which there  is initially normal plasma calcium but elevated PTH.   The treatment of primary hyperparathyroidism is  often surgical, with removal of the parathyroid gland(s).  However, this can render the patient hypocalcaemic,  and asymptomatic patients are sometimes treated  conservatively.   This may occur if the parathyroid glands have been  subjectedtolong-standingandsustainedpositivefeedback  by low plasma free ionized calcium concentrations  (hypocalcaemia) of secondary hyperparathyroidism  which have been subsequently corrected.   The parathyroid glands hypertrophy; PTH secretion  becomes partly autonomous and is not suppressed  by negative feedback by the hypercalcaemia. The  diagnosis is usually made when the cause of the original  hypocalcaemia is removed, for example by renal  transplantation or correction of long-standing calcium  or vitamin D deficiency as in malabsorption. A history  of previous hypocalcaemia and the finding of a very  high plasma alkaline phosphatase activity due to the  prolonged osteomalacia distinguish it from primary  hyperparathyroidism. In some cases, the glandular  hypertrophy gradually regresses and the plasma calcium  concentration returns to normal.   Unlike primary or tertiary hyperparathyroidism, in  which plasma PTH concentration is increased, there are  other causes of hypercalcaemia where plasma levels of  PTH are reduced or suppressed. These are now discussed.   Some patients with multiple bony metastases (from,  for example, breast, lung, prostate, kidney and    thyroid tumours) or with multiple myeloma show  hypercalcaemia. Here there is usually a parallel rise  of plasma phosphate. The hypercalcaemia is caused  by direct bone breakdown due to the local action of  malignant deposits and cytokine activation.   Malignant deposits in bone stimulate a local  osteoblastic reaction and hence a rise in plasma alkaline  phosphatase activity. This osteoblastic reaction does  not occur in bone eroded by the marrow expansion  of myelomatosis. Therefore, in the latter condition  the plasma concentration of alkaline phosphatase of  bony origin is relatively normal, despite extensive bone  involvement with osteolytic lesions.   Parathyroid hormone-related protein is synthesized  by some malignant tumours of non-endocrine tissues  and is not subject to normal feedback control by the  high plasma free ionized calcium concentration. Bony  lesions due to circulating PTHRP are not normally  present because the underlying disease is usually either  fatal or successfully treated in a relatively short time.  It is of note that PTHRP is not usually detected by the  PTH assay.   However, the plasma alkaline phosphatase activity may  be raised because of secondary deposits in bone or the  liver, or both. In humoral hypercalcaemia of malignancy,  the plasma calcium concentration may rise from normal  to dangerously high very rapidly, in contrast to primary  hyperparathyroidism. Ectopic hormone production is  discussed more fully in Chapter 24.   A 76-year-old woman with known breast carcinoma  was admitted to hospital drowsy, with weight loss  and backache. The following results were returned.   A bone scan subsequently showed the patient to  have widespread bone metastases. Note the severe  hypercalcaemia and the appropriately suppressed  plasma PTH, suggesting a non-parathyroid source of  the hypercalcaemia. Various tumours are associated  with bone metastases, including breast tumours.   Various medications can evoke hypercalcaemia, such as  thiazides (decreases calcium renal excretion), lithium,  and vitamin A excess.   This rare condition occurs with the excessive use of  calcium-containing antacids for dyspepsia. It is also  associated with a metabolic alkalosis. Milk-alkali  syndrome is now rarely seen, since the advent of proton  pump inhibitors and H, receptor antagonists for the  treatment of dyspepsia.   Vitamin D excess may be caused by overvigorous  treatmentofhypocalcaemia.Increasedintestinalcalcium  absorption may cause dangerous hypercalcaemia.  Vitamin D therapy, with either ergocalciferol or the  active metabolite 1,25-dihydroxyvitamin D, should  always be monitored by frequent estimation of plasma  calcium concentrations and, if there is osteomalacia, by  measuring plasma alkaline phosphatase activity. If the  cause of hypercalcaemia is obscure, a careful drug history  should be taken. Occasionally patients inadvertently  overdose themselves with vitamin D, which is available  in many countries without prescription.   Hypercalcaemia is a rare complication. 1,25-Dihy-  droxycholecalciferol is synthesized in the granuloma  tissue and increases calcium absorption from the  intestinal tract. Chronic beryllium poisoning produces a  granulomatous reaction very similar to that of sarcoidosis  and may also be associated with hypercalcaemia. The  same is possibly also true of tuberculosis, histoplasmosis  and leprosy.   Prolonged excess of thyroid hormone in severe  hyperthyroidism may be associated with the histological  appearance of osteoporosis and a consequent increase  in urinary calcium excretion. Hypercalcaemia is a very  rare complication.   These include acromegaly (see Chapter 7), Addison’s  disease (see Chapter 8) and phaeochromocytoma (see  Chapter 24).   Hypercalcaemia with an inappropriately high plasma  PTH concentration in the presence of hypocalciuria   has been reported in some families, in none of whom  was a parathyroid adenoma found at operation. The  condition is inherited as an autosomal dominant trait.  The aetiology of the condition is thought to be due a  defect on the CaSR (see above). Low urinary calcium  concentration in the face of hypercalcaemia points to  the diagnosis. A useful test is the calcium excreted per  litre of glomerular filtrate (CaE):   Hypocalciuric hypercalcaemia is likely if this  is less than 0.015 (in mmol/L). It is important to  exclude this condition, as it can mimic primary  hyperparathyroidism.   Idiopathic hypercalcaemia of infancy includes a number  of conditions that cause hypercalcaemia during the first  year of life. Excessive vitamin D supplementation of  cow’s milk is now a very uncommon cause. Williams’  syndrome is a rare familial disorder associated  with increased intestinal calcium absorption and  hypercalcaemia. Clinical features include growth  retardation, mental deficiency and characteristic ‘elfin’  facies. Congenital heart disease may also be present (see  Chapter 26). There is also a rare neonatal autosomal  dominant hyperparathyroidism   Establish whether the high plasma total calcium  concentration is due only to a high protein-bound  fraction. Two groups of causes should be differentiated  for raised plasma albumin-adjusted calcium  concentration:   1. raised albumin-adjusted calcium concentration  due to inappropriately high PTH and usually  hypophosphataemia,   2. raised albumin-adjusted calcium concentration  due to other causes and associated with low PTH  concentrations and often hyperphosphataemia.   The following procedure may be useful to find the  cause of hypercalcaemia, although the diagnosis may  be obvious before all the steps have been followed:   • Check the albumin-adj usted calcium. Take a specimen  without venous stasis (preferably without a tourniquet)  to eliminate artefactual haemoconcentration and   repeat the plasma calcium and albumin assays. If true  hypercalcaemia is confirmed, a cause must be sought.   • Take a careful history, with special reference to  the drug history, such as vitamin-D-containing  preparations and thiazide diuretics. Is there evidence  of milk-alkali syndrome, albeit rare now? If so, check  acid-base status.   • Is the plasma phosphate concentration low in relation  to the renal function? Hypophosphataemia suggests  the diagnosis of primary hyperparathyroidism.   Apart from thiazide usage, the most common  causes of hypercalcaemia are either primary  hyperparathyroidism or malignancy; the latter may be  obvious following clinical examination and radiological  and haematological tests, for example anaemia, and  raised erythrocyte sedimentation rate (ESR) and  biochemical investigations.   It is essential that primary hyperparathyroidism  and malignant hypercalcaemia are distinguished.  In the case of malignancy, pay special attention for  breast, kidney, lung or prostate carcinoma. A raised  plasma PTH concentration is usually seen in primary  hyperparathyroidism; conversely, suppressed levels are  found in malignant states and indeed in hypercalcaemia  of many other causes. Very rarely PTHRP should be  measured if ectopic secretion of this is suspected, for  example by a tumour.   If primary hyperparathyroidism due to an adenoma  is found, exclude MEN syndrome (see Chapter 24).  Imaging of the parathyroid glands is often needed  to distinguish adenoma from hyperplasia of the  parathyroid glands. Isotope subtraction scanning or  ultrasound of the neck may help to localize the adenoma,  as may venous sampling for PTH levels.   Very high plasma alkaline phosphatase activity  is unlikely to be due to uncomplicated primary  hyperparathyroidism; near-normal activity is usual,  although it maybe raised if there is radiological evidence  of bone involvement. If it is very high, it suggests either  malignancy or some concurrent disease such as Paget’s  disease.   Perform serum and urinary protein electrophoresis  if a multiple myeloma is suspected (see Chapter 19).   • Look for evidence of sarcoidosis; plasma angiotensin-  converting enzyme (ACE) concentration is often  raised (see Chapter 18) and a chest radiograph may  be useful.   • Is there acromegaly, Addison’s disease or  thyrotoxicosis (see Chapters 7, 8 and 11)?   • A urinary calcium determination (CaE) is useful to  help exclude hypocalciuric hypercalcaemia.   A steroid suppression test is rarely necessary to  identify the cause of hypercalcaemia because of the  development of robust PTH assays. Briefly, this test   relies on the fact that hypercalcaemia of primary  hyperparathyroidism is not usually suppressed by  steroids, unlike many of the other causes of a raised  calcium concentration such as malignant disease.   If the plasma albumin-adjusted calcium concentration  is below about 3.5mmol/L, and if there are no  significant clinical symptoms or signs such as changes  attributable to hypercalcaemia, there is no need for  urgent treatment. However, therapy should be started  as soon as the abnormality is found and preliminary  investigations have been performed because of the  danger of renal damage. If possible, the primary cause  should also be diagnosed and treated.   The patient should be fluid volume repleted, if  necessary by intravenous infusion of saline. The  plasma total calcium concentration will often fall as  the plasma albumin concentration is diluted, but the  plasma free ionized calcium concentration is probably  little affected. Correcting haemoconcentration enables  a more realistic assessment to be made of the degree of  true hypercalcaemia.   Bisphosphonates are first-line agents in the medical  management of hypercalcaemia. These are structurally  similar to pyrophosphate. They bind to hydroxyapatite  in bone, thus inhibiting bone turnover and the  mobilization of calcium. They are poorly absorbed  from the intestinal tract and may have to be given  intravenously to patients with severe hypercalcaemia.  Cyclical administration may prevent the long-term  complication of osteomalacia.   The management of apparently asymptomatic mild  hypercalcaemia due to primary hyperparathyroidism  is controversial. It has been suggested that prolonged  hypercalcaemia does not always cause obvious renal  dysfunction and that the risk of parathyroidectomy  (e.g. rendering the patient hypocalcaemic) may be  greater than that of mild hypercalcaemia. The decision  whether to operate must be made on clinical grounds;  of particular importance are the fitness of the patient  for operation, plasma calcium concentration greater  than 3.0mmol/L, deteriorating renal function, renal  calculi, poor bone mineral density or 24-h urinary  calcium concentration greater than lOmmol/L.   The plasma albumin-adjusted calcium concentration  at which urgent treatment is indicated because of the   danger of cardiac arrest is usually about 3.5mmol/L. If  in doubt, abnormalities associated with hypercalcaemia  should be sought on the ECG. Consider the following:   • Rehydration The patient should be volume repleted,  intravenously with saline if necessary. Furosemide  may also be given in an attempt to increase urinary  calcium clearance and avoid fluid overload. Check  electrolytes and renal function carefully.   • Bisphosphonates After rehydration and correction of  any electrolyte abnormalities, bisphosphonates such  as pamidronate are usually the treatment of choice.   • Steroids May sometimes lower the plasma calcium  concentration in malignancy and almost always in  cases of sarcoidosis and vitamin D intoxication.   • Calcitonin Sometimes used to treat severe  hypercalcaemia. The effect lasts about 3 days;  repeated doses are often less successful in maintaining  a ‘normal’ plasma calcium concentration.   Steroids and calcitonin usually have no significant  effect for about 24 h.   As with most other extracellular constituents, rapid  changes in plasma calcium concentration may be  dangerous because time is not allowed for equilibration  across cell membranes. The aim of emergency treatment  should be to lower the plasma concentration to one  that is not immediately dangerous, while initiating  treatment for mild hypercalcaemia as outlined above.  Too rapid a reduction, even to only normal or slightly  raised concentrations, may cause tetany.   Low plasma albumin-adjusted calcium concentrations,  including those associated with a normal total  calcium concentration of alkalosis, cause increased  neuromuscular activity eventually leading to tetany and  carpopedal spasm, generalized seizures, laryngospasm,  hyper-reflexia, paraesthesiae and hypotension.   Prolonged hypocalcaemia, even when mild,  interferes with the metabolism of the lens in the eye  and may cause cataracts. Because of this, asymptomatic  hypocalcaemia should be sought when there has been  a known risk of parathyroid damage, such as after  partial or total thyroidectomy, and, if found, treated.  Hypocalcaemia may also cause depression and other  psychiatric symptoms as well as cardiac arrhythmias,  including prolonged Q-T interval on ECG.   A 72-year-old woman presented to her general  practitioner with tiredness, muscle aches and  difficulty in standing up. The following test results  were found.   Alkaline phosphatase 763 U/L (<250)  25-hydroxyvitamin D 5 pg/L (> 75)   The patient has osteomalacia, as evidenced by the low  plasma 25-hydroxyvitamin D concentration. Note  also the hypocalcaemia with hypophosphataemia  and raised alkaline phosphatase and secondary  appropriate elevation of PTH. The symptoms are  typical of osteomalacia, which can lead to proximal  myopathy and bone pain. The elderly are particularly  prone to osteomalacia, in part related to poor dietary  vitamin D intake.   Latent neuromuscular hyperactivity, carpopedal  spasm and tetany (Trousseau’s sign) can be evoked by  inflating a blood pressure cuff to 10-20mmHg above  systolic blood pressure for 3-5 min. Chvostek’s sign  can be elicited by tapping the facial nerve anterior to  the ear, when ipsilateral facial muscle contraction may  occur, although this can also occur in about 10 per cent  of individuals without hypocalcaemia.   It is sometimes useful to divide hypocalcaemia into  those cases with a low plasma phosphate concentration  (hypophosphataemia) and those with high plasma  phosphate concentration (hyperphosphataemia),  although not all cases of hypocalcaemia fall neatly into  this classification. The causes of hypocalcaemia are  given in Box 6.2.   In steatorrhoea, fat (and therefore vitamin D)  absorption is impaired; this malabsorption may be  aggravated if calcium combines with unabsorbed fatty  acids to form insoluble soaps in the lumen. Deficiency  due to undernutrition is more commonly caused by  deficiency of vitamin D than of calcium.   In relatively affluent countries, malabsorption is  the most common cause of calcium and vitamin D   Hypoparathyrodism (low parathyroid hormone levels)  Idiopathic or autoimmune  Surgical removal of parathyroid glands  Congenital absence of parathyroid glands, e.g.  DiGeorge’s syndrome   deficiencies. Worldwide dietary deficiency is important.  The following groups are at risk of developing  osteomalacia or rickets:   • children and pregnant women, in whom increased  needs may not be met by the normal supply from  the skin,   • people such as the elderly and chronically sick who  are less exposed to sunlight because they are confined  indoors.   There is a relatively high incidence of osteomalacia  and rickets among the Asian community in some urban  Western societies. The causes are unclear but probably  include dietary habits, relative lack of sunlight and  possibly genetic factors.   Chronic liver disease may occasionally be associated  with mild osteomalacia, especially if there is cholestasis  causing malabsorption due, for example, to primary  biliary cirrhosis. However, it is unlikely that significant  impairment of vitamin D hydroxylation is the cause.   Prolonged anticonvulsant therapy, especially if  both barbiturates and phenytoin are taken, may be  associated with hypocalcaemia and even osteomalacia.  These drugs probably induce the synthesis of hepatic  enzymes which catalyse the conversion of vitamin D to  inactive metabolites.   In all these conditions, the low plasma free ionized  calcium concentration stimulates PTH secretion;  the plasma phosphate concentration tends to be low  relative to the GFR. However, if there is renal glomerular  dysfunction, the plasma phosphate concentration may  be increased but tends to be lower than in those patients  with the same concentration of plasma urea but normal  plasma PTH concentrations. In chronic cases a rising  plasma alkaline phosphatase activity indicates the onset  of the bone changes of osteomalacia or rickets.   Type 1 vitamin-D-dependent rickets is due to  1-ot-hydroxylase deficiency. It is an autosomal  recessive disorder and results in low l,25-(OH) 2 D 3  concentrations.   Type 2 vitamin-D-dependent rickets is also an  autosomal recessive disorder and causes a vitamin D  resistance and is a defect of the vitamin D or calcitriol  receptor. Thus plasma l,25-(OH) 2 D 3 concentrations  are elevated.   The most common causes of hyperphosphataemia  include acute kidney injury and chronic kidney disease  (dysfunction) (see Chapter 3).   Renal disease such as chronic kidney disease  causes relative resistance to vitamin D because of the  direct effect of the disease on the functioning renal  tubular cells and therefore on 1 -a.-hydroxylation of  25-OHD 3 and inhibition of 1 -a.-hydroxylation by  hyperphosphataemia associated with the low GFR of  renal glomerular dysfunction.   Hypocalcaemia may develop within a few days  of the onset of renal damage. Low plasma protein  concentrations often contribute to the reduction in the  total calcium concentration.   Tetany is rare in renal disease, probably because the  accompanying acidosis increases the plasma free ionized   calcium concentration above tetanic concentrations.  Treatment of the metabolic acidosis with bicarbonate  is usually contraindicated because a rise in blood pH  may cause precipitation of calcium phosphate in  extraosseous sites. In the kidney it may aggravate renal  dysfunction.   Hypoparathyroidism is usually caused by surgical  damage to the parathyroid glands, either directly  or indirectly by impairment of their blood supply  during partial thyroidectomy. Total thyroidectomy  or laryngectomy is often associated with removal or  damage to the parathyroid glands and it is important  to monitor plasma calcium concentrations. Post-  thyroidectomy hypocalcaemia is not always due to  damage to the glands and may be transient.   Parathyroidectomy, carried out to treat primary  or tertiary hyperparathyroidism, also carries a risk  of damage to the remaining parathyroid tissue.  However, there are several causes of temporary  hypocalcaemia.   If the hypercalcaemia has been severe and  prolonged, the remaining parathyroid tissue may have  been suppressed by negative feedback for so long that  there may be true hypoparathyroidism evidenced by a  rising plasma phosphate concentration, which usually  recovers within a few days. Unless transient it should be  treated if symptomatic.   In rare cases of primary hyperparathyroidism with  overt bone disease, rapid entry of calcium into bone,  when plasma PTH concentrations fall, may cause true,  but temporary, post-operative hypocalcaemia.   Although early post-operative parathyroid  insufficiency may recover, a low plasma calcium  concentration persisting for more than a few weeks  should usually be treated.   Autoimmune hypoparathyroidism is rare. It may  occur as a familial disorder, presenting either during  childhood or in adults or as part of the polyendocrine  syndrome, with antibodies against parathyroid  tissue and sometimes other tissues, for example the  adrenal glands and pancreatic islets. Autoimmune  hypoparathyrodism can also be associated with the  MEDAC syndrome (multiple endocrine deficiency,  Addison’s disease and candidiasis).   Congenital absence of the parathyroid glands is also  rare; it is associated with impaired cellular immunity,  a characteristic facial appearance and cardiac  abnormalities known as DiGeorge’s syndrome.   This is a very rare inborn error associated with an  impaired response of both kidneys and bone to PTH,  that is, end-organ resistance to circulating PTH. Thus  plasma PTH concentration is raised with hypocal-  caemia. Type 1 is a defect proximal to cyclic adenosine  monophosphate (cAMP) tissue generation, and thus  urinary cAMP is low; type 2 pseudohypoparathyroid-  ism is due to a defect distal to cAMP production,  so urinary cAMP levels are normal. The associated  phenotype may show short stature, obesity, mental  retardation and short third and fourth metacarpals.   Box 6.2 shows some miscellaneous rarer causes of  hypocalcaemia that do not necessarily present with  either hypophosphataemia or hyperphosphataemia.   High plasma PTH concentrations cause phosphaturia  with hypophosphataemia if glomerular function is  normal.   Secondary hyperparathyroidism (‘appropriate’  secretion of PTH) occurs in response to a low plasma  free ionized calcium concentration. The parathyroid  glands respond with appropriate secretion of PTH.  If the response is effective, the plasma calcium  concentration returns to normal, the stimulus to  secretion is removed and hormone production is  then inhibited by negative feedback. Preservation of  plasma calcium concentrations occurs at the expense  of bone mineralization and therefore decalcification  may result. Parathyroid hormone cannot act effectively  on bone in the absence of l,25-(OH) D . In cases of  vitamin D deficiency with hypocalcaemia, plasma PTH  concentrations may be very high.   Without an adequate supply of calcium and  phosphate, osteoid cannot be calcified despite marked  osteoblastic proliferation. The histological finding of  uncalcified osteoid is characteristic of osteomalacia in  adults or rickets in children. Osteomalacia, before fusion  of the epiphyses, may present with a slightly different  clinical, radiological and histological picture and is  called rickets. Plasma alkaline phosphatase activity is  increased because of osteoblastic proliferation.   to long-standing deficiency of calcium, phosphate  and vitamin D. Predisposing factors include:   - reduced dietary intake of vitamin D, calcium  and phosphate in undernutrition,   - impaired absorption of vitamin D, for example  in steatorrhoea or hepatobiliary disease,   - impaired metabolism of vitamin D to 1,25-  (OH), D 3 due to renal disease,   • Without osteomalacia or rickets: if PTH action is  inadequate to correct the abnormality, the plasma  calcium concentration remains low and bone disorders  are not present. Predisposing factors include:   In secondary hyperparathyroidism the plasma  calcium concentration is never high, and usually  the plasma calcium and phosphate concentrations  tend to be low. High-normal or high plasma calcium  concentrations in renal glomerular dysfunction suggest  either that primary hyperparathyroidism has caused the  renal disease or that prolonged calcium deficiency has  led to the development of tertiary hyperparathyroidism.   As in the case of hypercalcaemia, the causes of  hypocalcaemia fall into two main groups:   • reduced albumin-adjusted calcium concentration  due to primary PTH deficiency and associated with  hyperphosphataemia,   • reduced albumin-adjusted calcium concentration  due to other causes and associated with  appropriately high PTH concentrations and usually  hypophosphataemia.   Determine first if the fall in plasma total calcium  concentration (albumin-adjusted calcium) is due to a  low protein-bound fraction.   Patients with a low albumin concentration should  not be given calcium and/or vitamin D unless there is  clinical evidence of a low albumin-adjusted calcium  concentration.   • Is the patient on drugs or chemicals that may cause  hypocalcaemia (see Box 6.2)?   • Is the plasma phosphate concentration high? If  the plasma urea and/or creatinine concentration   is high, renal dysfunction is the likely cause (see  Chapter 3).   • Is the plasma phosphate concentration low? If so,  calcium deficiency with normal secretion of PTH in  response to feedback is likely. At this point a plasma  PTH assay may be useful. Is the plasma alkaline  phosphatase activity high? This finding may suggest  prolonged secondary hyperparathyroidism due to  calcium deficiency.   • If indicated, do relevant bone radiographs show  signs of rickets or osteomalacia? These may confirm  prolonged calcium deficiency. Check plasma  25-hydroxyvitamin D levels; if the plasma levels  are low, look for causes of undernutrition and  malabsorption states.   In the rare hypocalcaemia of type 1 vitamin-D-  dependent rickets there are low plasma l,25-(OH) 2 D 3  concentrations, whereas type 2 vitamin-D -dependent  rickets causes a vitamin D resistance and plasma  l,25-(OH) 2 D 3 concentrations are elevated.   Raised plasma phosphate in the face of  hypocalcaemia and low plasma PTH concentration  suggests hypoparathyroidism.   Is there a history of neck surgery which has led to  hypoparathyroidism? Hypoparathyroidism may also  be of autoimmune origin and associated with other  autoimmune disorders. It needs to be distinguished  from the even more rare ‘pseudohypoparathyroidism’  by measuring plasma PTH concentrations. Parathyroid  hormone concentrations will be low in true  hypoparathyroidism but high if there is the end-organ  unresponsiveness of pseudohypoparathyroidism.   Check plasma magnesium, as severe hypomagnesaemia  can cause hypocalcaemia by reducing the action of PTH.   A raised urinary calcium concentration may help  diagnose the rare autosomal dominant hypercalciuric  hypocalcaemia.   Apparent hypocalcaemia, due to low plasma albumin  concentrations, should not be treated. Always look at  the albumin-adjusted calcium value.   Asymptomatic true hypocalcaemia, or that causing  only mild clinical symptoms, can usually be treated  effectively with oral calcium supplements and vitamin  D supplements. It is difficult to give enough oral calcium  by itself to make a lasting and significant difference  to plasma calcium concentrations. If a normal diet is    Figure 6.4 Algorithm for the investigation of hypocalcaemia. EDTA, ethylenediamine tetra-acetic acid.   being taken, vitamin D, by increasing the absorption of  calcium from the intestine, is usually adequate without  calcium supplements. 1,25-Dihydroxycholecalciferol  and alfacalcidol (1-a-hydroxycholecalciferol) are most   commonly used as they have short half-lives, particularly  if there is hypoparathyroidism or a defect in vitamin  D metabolism. It is important to monitor the plasma  calcium closely to avoid inducing hypercalcaemia and   Because of the danger of ectopic calcification by  precipitation of calcium phosphate, hypocalcaemia  with hyperphosphataemia in renal disease should be  treated cautiously. The plasma phosphate concentration  should first be lowered by giving a phosphate-binding  agent that binds phosphate in the intestinal lumen.  1,25-Dihydroxycholecalciferol and alfacalcidol, the  active vitamin D metabolites, have been used to treat  hypocalcaemia in renal disease.   If there are cardiac arrhythmias, seizures or severe  tetany including laryngospasm shown to be due to  hypocalcaemia, intravenous calcium, usually as 10 mL  of 10 per cent calcium gluconate, should be given  over about 5 min. Treatment can then begin as above,  depending upon the aetiology of the hypocalcaemia.   Hypocalcaemia during the first week after a  thyroidectomy or parathyroidectomy should be  treated only if there is tetany, and usually with calcium  replacement, which, unlike vitamin D supplements,  has a rapid effect and a short half-life. Persistent  hypocalcaemia may indicate that the parathyroid glands  are permanently damaged and that long-standing, or  even life-long, vitamin D supplementation is necessary.  Parathyroid bone disease may result in ‘hungry bones’  and prolonged post-operative hypocalcaemia.   Hypercalciuria in the absence of hypercalcaemia  (hypercalciuria normocalcaemia) may predispose to  the formation of renal calculi (see Chapter 3) and may  occur in:   • some cases of osteoporosis in which calcium cannot  be deposited in normal amounts because the bone  matrix is reduced,   • acidosis, in which the release of free ionized calcium  from bone is increased.   Hypercalciuria can broadly be divided into absorptive  hypercalciuria, type I (hyperabsorption of calcium),  type II (diet-responsive hypercalciuria) and type III  (renal phosphate leak resulting in decreased calcium  resorption), and renal hypercalciuria (decreased renal  calcium resorption). These can be distinguished by  tests of oral calcium absorption.   Hypercalciuria can, of course, also occur in the face  of hypercalcaemia, such as resorptive hypercalciuria  associated with primary hyperparathyroidism.   Estimation of urinary CaE is rarely diagnostic in  the differential diagnosis of hypercalcaemia except  familial hypocalciuric hypercalcaemia. If glomerular  function is normal, all other causes of hypercalcaemia  usually increase the calcium load on the glomeruli  and evoke hypercalciuria. If renal glomerular function  is impaired, calcium excretion is low even if there is  hypercalcaemia.   Some disorders of bone rarely alter plasma calcium  concentrations but are important in the differential  diagnosis of changes in mineral metabolism.   Osteoporosis is not a primary disorder of calcium  metabolism. The reduction of bone mass is due to  thinning of the protein on which the calcium is usually  deposited. A slight increase in urinary calcium loss  is secondary to this. Disorders associated with an  increased incidence include the following:   • low plasma oestrogen concentrations, such as after  the female menopause (the most common cause)  and prolonged amenorrhoea; also low testosterone  concentrations in males,   • elderly subjects in whom there may also be mild  osteomalacia because of impaired renal production  of l,25-(OH) 2 D 3 ,   - drugs such as heparin, and anticonvulsants  such as phenytoin and carbamazepine,   In osteoporosis plasma calcium and phosphate  concentrations do not fall and, because there is no  increase in osteoblastic activity, the plasma total alkaline  phosphatase activity does not rise. These findings are  invaluable in distinguishing between osteomalacia and  osteoporosis.   Concentrations of new bone markers, such as bone-  specific alkaline phosphatase (bone formation), plasma  osteocalcin (bone formation), type 1 procollagen  peptides (bone formation), urinary deoxypyridinoline  and cross-linked N-terminal telopeptide and C-terminal  telopeptide of type 1 collagen (bone resorption),  urinary hydroxyproline (bone resorption) and bone-  resistant or tartrate-resistant acid phosphatase (bone  resorption) are raised in osteoporosis and may be  useful markers of the disease process. However, these  bone markers do not give information about exact  bone anatomy, for which imaging studies are necessary.   Sometimes radiographs are useful, especially if  fractures are suspected (Fig. 6.5). Bone mineral density  (BMD) is often used and reported as a T-score that  compares the patient’s BMD with that of a healthy  control. Normal BMD is within -1 standard deviation  (SD) of this, whereas osteopenia is defined as between  -1 and -2.5 SD and osteoporosis has a T-score of less  than -2.5 SD.   Treatment consists of adequate calcium and  vitamin D intake. The bisphosphonates increase BMD  and inhibit bone resorption, probably by inhibiting  osteoclast activity. Strontium ranelate is a duel action    Figure 6.5 Radiograph showing osteoporosis;  note compression of several vertebral bodies and  compression fractures of T12 and LI. Reproduced  with kind permission from Solomon L, Warwick D  and Nayagam S. Apley’s System of Orthopaedics and  Fractures, 9th edition. London: Hodder Arnold, 2010.   bone agent (DABA) that stimulates osteoblasts and  inhibits osteoclasts. Calcitonin inhibits osteoclast  reabsorption and recombinant PTH analogues such as  teriparatide stimulate osteoblasts. In women, hormone  replacement therapy is also used if indicated, but this  may have side effects. Raloxifene has been used and is a  selective estrogen receptor modulator (SERM).   Paget’s disease is more common in the elderly, possibly  being present in about 5 per cent of people over 60 years  old. There is increased bone turnover and remodelling  due to increased osteoclastic and osteoblastic function.  It may be asymptomatic or may present with bone  pain, pathological fractures, deafness (due to bone  overgrowth) and high-output cardiac failure due to  increased vascularity within the bone. Enlargement of  bones such as the skull (osteoporosis circumscripta),  femur and tibia (sabre tibia) can occur.   Diagnosis may necessitate radiographs and/or bone  scanning. Plasma calcium and phosphate concentrations  are rarely affected unless the patient is immobilized,  in which case the plasma calcium concentrations may  rise. Plasma alkaline phosphatase activity is typically  very high. Less than 1 per cent of patients may develop  osteosarcomas, and this complication maybe associated  with rapidly rising plasma alkaline phosphatase activity.  Bisphosphonates are often used to treat Paget’s disease;  although calcitonin has been tried, it is probably less  effective.   Rickets or osteomalacia caused by renal tubular  disorders of phosphate reabsorption   In a group of inborn errors of renal tubular function,  less phosphate than normal is reabsorbed from  the glomerular filtrate. The consequent rickets or  osteomalacia, unlike the usual form, responds poorly  to vitamin D therapy. The syndrome has therefore been  called ‘resistant rickets’. Familial hypophosphataemia  is an X-linked dominant trait; the syndrome may also  be part of a more generalized reabsorption defect in  Fanconi’s syndrome (see Chapter 3).   In these disorders, failure to calcify bone is probably  due to phosphate deficiency, although there may  also be impaired vitamin D metabolism. Plasma  phosphate concentrations are usually very low and fail  to rise when vitamin D is given; there is phosphaturia  inappropriate to the plasma concentration. The high  plasma alkaline phosphatase activity reflects increased  osteoblastic activity. The usually normal plasma calcium   concentrations differ from the findings in the ‘classic’  syndrome. As the free ionized calcium concentration is  normal, the parathyroid glands are not overstimulated  and therefore evidence in the bone of high PTH  concentrations is rare. The conditions respond to large  doses of oral phosphate and to a small dose of the active  metabolite of vitamin D.   Phosphate is a divalent anion, approximately  80 per cent of which is found in the bony skeleton  and 20 per cent is distributed in the soft tissues and  muscle. Phosphate is the major intracellular anion  and shifts between the intracellular and extracellular  compartments. Such transcellular movement can  result from the ingestion of carbohydrate or lipid,  as well as from acid-base alterations - for example,  acidosis can result in shifts of phosphate out of cells  into the plasma.   The daily phosphate intake is about 30 mmol,  with approximately 80 per cent being absorbed in  the jejunum. Protein-rich food is a major source of  phosphate intake, as are cereals and nuts. The output  is largely renal, with more than 90 per cent being  excreted by this route. Most of the phosphate filtered  at the glomeruli is reabsorbed by the proximal tubules.  Gastrointestinal loss of phosphate accounts for only  10 per cent of the body’s phosphate excretion.   Urinary phosphate excretion falls as the plasma  phosphate, and therefore glomerular filtrate,  concentrations decrease in response to reduced  dietary phosphate intake. The measurement of urinary  phosphate concentration may occasionally be useful to  distinguish hypophosphataemia due to true depletion  (low urinary phosphate) and the increased urinary  phosphate excretion found in renal tubular disorders,  such as X-linked hypophosphataemic rickets.   The urinary phosphate excretion threshold can be  derived from nomograms, and a low value implies renal  phosphate loss. Another way to assess renal phosphate  loss is to calculate the fractional phosphate excretion  (FEPi%):   An FEPi% of more than 10 per cent implies a renal  phosphate loss.   Phosphate is an important intracellular buffer as well  as being essential for buffering hydrogen ions in urine.  In addition, it has a structural role as a component of  phospholipids, nucleoproteins and nucleic acids.   Phosphate plays a central role in cellular metabolic  pathways, including glycolysis and oxidative  phosphorylation. A by-product of glycolysis is  2,3-diphosphoglycerate (2,3-DPG). This is a regulator  of haemoglobin oxygen dissociation. Nucleotides such  as adenosine triphosphate consist of phosphate. Other  actions include excitation-stimulus response coupling  and nervous system conduction. Phosphate also has a  role in the optimal function of leucocytes, for example  chemotaxis and phagocytosis, and for platelets in clot  retraction.   The causes of hyperphosphataemia are listed in Box  6.3. The majority of the clinical effects are the result of  hypocalcaemia, particularly if the plasma phosphate  concentration is more than 3.0mmol/L. The reason  for this is that calcium/phosphate precipitation into  the tissues can ensue when the phosphate and calcium  plasma concentrations exceed their solubility product.  Thus, metastatic calcification is a clinical consequence  of hyperphosphataemia.   The treatment for hyperphosphataemia is with oral  phosphate-binding agents, for example magnesium  hydroxide or calcium carbonate. These agents have  been used in the management of patients with chronic   Artefact due to in vitro haemolysis or old blood sample  Inappropriately high phosphate intake, usually  intravenously   kidney disease (see Chapter 3). There has been recent  controversy about the toxicity of aluminium-containing  phosphate-binding agents in view of possible side effects,  including nausea, vomiting, constipation and, more  seriously, microcytic anaemia and encephalopathy.  In the face of acute kidney injury or chronic kidney  disease and persistently severe hyperphosphataemia,  haemodialysis or peritoneal dialysis may be indicated.   Hypophosphataemia associated with disturbances of  calcium metabolism is usually due to high circulating  PTH concentrations. In such conditions, and in renal  tubular disorders of phosphate reabsorption, phosphate  is lost from the body in urine. Hypophosphataemia  may also be caused by severe and prolonged dietary  deficiency; urinary phosphate excretion is then usually  significantly reduced.   The redistribution of phosphate is a common cause  of hypophosphataemia in patients receiving parenteral  or enteral nutrition. Long-term parenteral feeding  without phosphate supplementation may cause true  phosphate depletion, as may feeding after prolonged  starvation (refeeding syndrome; see Box 6.4 and  Chapter 14).   Phosphate, like potassium, enters cells from the ECF  if the rate of glucose metabolism is increased. This may  be associated with glucose infusion during, for example,  the treatment of diabetic ketoacidosis with insulin.   Severe hypophosphataemia, often quoted as a  plasma inorganic phosphate concentration less than  0.30mmol/L, can result in a plethora of clinical  features. Rhabdomyolysis has been described, as has  impaired skeletal muscle function, including weakness  and myopathy. Hypophosphataemia is also reported  to impair diaphragmatic contractility, which may  help to explain the difficulty in weaning patients off  mechanical ventilators. Cardiomyopathy is another  possible complication of severe hypophosphataemia.   Hypophosphataemia can evoke seizures, perturbed  mental state and paraesthesiae, as well as renal tubular  impairment. If prolonged, it can lead to osteomalacia.  The haematological effects caused by severe  hypophosphataemia include thrombocytopenia,  impaired clotting processes and also reduced  leucocyte function. Haemolysis can also occur, as can  erythrocyte 2,3-DPG depletion, resulting in a shift  in the haemoglobin/oxygen dissociation curve to the  left, that is, haemoglobin has a greater affinity for  oxygen.   Isolated phosphate disorder  Hypophosphataemic osteomalacia  X-linked hypophosphataemia  Oncogenic hypophosphataemia  Paracetamol poisoning  As part of Fanconi’s syndrome  Miscellaneous  Liver disease  Septicaemia   Treatment of hypophosphataemia is usually not  necessary unless the plasma phosphate concentration  is less than 0.30mmol/L or the patient is symptomatic.  Sometimes oral phosphate salts have been used, although  diarrhoea can be a problem. If intravenous phosphate  replacement is indicated, the following regimens can  be used, namely 9 mmol of monobasic potassium  phosphate in half-normal saline by continuous  intravenous infusion over 12 h, or Polyfusor phosphate  50 mmol over 24 h. They should not be given to patients  with hypercalcaemia, because of the risk of metastatic  calcification, or to patients with hyperkalaemia.   Magnesium is predominately an intracellular divalent  cation and is important for optimal cell function. It is  an essential cofactor to many enzymes, as well as being  important for membrane function. Furthermore, it can  act as an antagonist to calcium in cellular responses and  has a structural role within the cell.   The body contains about 1 mol (approximately 25 g)  of magnesium, mostly in the bone and muscle. The   recommended daily allowance of magnesium for adults  is about 4.5 mg/kg; rich dietary sources include cereal,  nuts and vegetables.   Magnesium is largely absorbed in the upper small  intestine but the large intestine may also be important;  unlike calcium, its absorption is not vitamin D  dependent. As much as 70 per cent of magnesium  from dietary intake is not absorbed but eliminated  in the faeces. The major excretory route is via the  kidneys, and about 65 per cent of glomerular-filtered  magnesium is reabsorbed in the loop of Henle. The  exact mechanisms of magnesium homeostatic control  are unclear, although PTH, insulin and calcitonin  are important. Parathyroid hormone can increase  magnesium reabsorption, although hypercalcaemia  can increase the renal excretion of magnesium. About  35 per cent of plasma magnesium is protein bound, and  the plasma concentration is normally 0.7-1.2mmol/L.   Clinical consequences of hypermagnesaemia  Hypermagnesaemia can result in cardiac arrhythmias,  such as heart block and inhibition of atrioventricular  conduction leading to cardiac arrest, seizures, altered  nerve conduction, reduced tendon reflexes, paralytic  ileus, nausea, respiratory depression and hypotension.  Clinical features do not usually manifest until the  plasma magnesium concentration exceeds 2 mmol/L.   Acute kidney injury and chronic kidney disease  Familial hypocalciuric hypercalcaemia  Lithium treatment   If there is severe hypermagnesaemia, 10 mL of  10 per cent calcium gluconate given slowly intravenously  may relieve symptoms. Analogous to the treatment  of hyperkalaemia, insulin and glucose infusion can  be used in severe hypermagnesaemia (see Chapter 5).  Failing this, and if there is impaired renal function,  dialysis may be indicated (see Chapter 3).   Some causes of hypomagnesaemia are shown in  Box 6.6. The symptoms of hypomagnesaemia are  very similar to those of hypocalcaemia. If the plasma  calcium concentrations (allowing for that of albumin)  and blood pH are normal in a patient with tetany, the  plasma magnesium concentration should be assayed.   Hypomagnesaemia can result in cardiac arrhythmias,  including torsade de pointes, and digoxin sensitivity.   In addition, abdominal discomfort and anorexia have  been described, as well as neuromuscular sequelae  including tremor, paraesthesiae, vertigo, tetany,  seizures, irritability, confusion, weakness and ataxia.  Severe hypomagnesaemia can lead to hypocalcaemia  due to decreased PTH release and activity.   Long-term magnesium deficiency may be a risk  factor for coronary artery disease, perhaps increasing  atherosclerosis and platelet aggregation. There are  data suggesting that reduced magnesium intake is  associated with hypertension and insulin resistance.   Sometimes the symptoms and signs of magnesium  deficiency occur in the face of borderline magnesium  plasma concentrations, as plasma levels poorly reflect  intracellular magnesium stores. In such circumstances  the intravenous magnesium-loading test may be   useful: 30 mmol of magnesium (usually as sulphate)  is infused intravenously in 500 mL 5 per cent dextrose  over 4 h and urine is collected over 24 h for magnesium  determination. Magnesium depletion is unlikely if  more than 24 mmol of magnesium is excreted in 24 h.   Severe hypomagnesaemia, less than 0.5mmol/L  or if it is symptomatic, can be corrected by oral  magnesium salts, but these may be poorly absorbed  and lead to gastrointestinal upset. A possible regimen  would be oral magnesium gluconate 12mmol/day  to a maximum of 48 mmol/L, if required, in three or  four divided doses. Intravenous replacement is often  given as magnesium sulphate. Generally, 0.5 mmol/kg  per day can be given by intravenous infusion. Close  monitoring of plasma magnesium is necessary.  The treatment of hypomagnesaemia may facilitate  the treatment of refractory hypokalaemia and  hypocalcaemia.   Calcium, phosphate and magnesium metabolism  are closely related and abnormalities are clinically  relatively common.   Plasma calcium levels are controlled by PTH (raises  plasma calcium), vitamin D activity and optimal  renal and intestinal function.   Hypercalcaemia can result in various symptoms:  ‘bones, stones, moans and groans’. The causes  include malignant disease, primary or tertiary   hyperparathyroidism, certain drugs such as thiazides,  granulomatous disease such as sarcoidosis, milk-  alkali syndrome, thyrotoxicosis, Addison’s disease,  hypocalciuric hypercalcaemia and acromegaly.  Hypocalcaemia can present with paraesthesiae,  tetany, osteomalacia and seizures. The causes may  be due to poor diet, including vitamin D deficiency,  chronic kidney disease, malabsorption, certain drugs,  such as loop diuretics, and hypoparathyroidism.   A hormone can be defined as a substance secreted by an  endocrine gland that is transported in the blood, thereby  regulating the function of another tissue(s). Certain  hormones, such as growth hormone (GH, secreted  from the anterior pituitary gland), thyroxine (T , from  the thyroid gland) and insulin (from the pancreatic  islet cells), influence tissue metabolism directly.  Conversely, trophic hormones from the pituitary gland  stimulate target endocrine glands to synthesize and  secrete further hormones, which in turn partly control  trophic hormone release, usually by negative feedback  inhibition. For example, hypercalcaemia inhibits the  secretion of parathyroid hormone (PTH), and elevation  of plasma T 4 concentration inhibits the secretion of  thyroid- stimulating hormone (TSH).   Endocrine glands may secrete excessive or deficient  amounts of hormone. Abnormalities of target glands  may be primary or secondary to dysfunction of  the controlling mechanism, usually located in the  hypothalamus or anterior pituitary gland.   Hormone secretion may vary predictably over a 24-h  (circadian) or longer period. It may be episodic or may  respond predictably to physiological stimuli such as stress.  Simultaneous measurement of both the trophic hormones  and their controlling factors, whether hormones or  metabolic products, may be more informative than the  measurement of either alone. An important endocrine  principle is that an apparently ‘normal’ hormone result  should be interpreted in the context of the associated  hormone axis, for example a plasma PTH concentration  within the reference range may be abnormal if the plasma  calcium concentration is elevated.   It is also important to know about the assay’s  performance, as sometimes heterophilic interfering  antibodies may cross-react with various hormones, as can  certain immunoglobulins, for example macroprolactin  (see Chapter 9, Hyperprolactinaemia).   If the results of preliminary tests are definitely  abnormal, this maybe primary or secondary to a disorder  of one of the controlling mechanisms. Should the results  be equivocal when considered together with the clinical  findings, so-called ‘dynamic’ tests should be carried out.  In such tests the response of the gland or the feedback  mechanism is assessed after stimulation or suppression  by the administration of exogenous hormone.   Suppression tests are used mainly for the differential  diagnosis of excessive hormone secretion. The substance  (or an analogue) that normally suppresses secretion by  negative feedback is administered and the response is  measured. Failure to suppress implies that secretion is not  under normal feedback control (autonomous secretion).   Stimulation tests are used mainly for the differential  diagnosis of deficient hormone secretion. The trophic  hormone that normally stimulates secretion is  administered and the response is measured. A normal  response excludes an abnormality of the target gland,  whereas failure to respond confirms it.   Disorders of the pituitary gland and hypothalamus  are discussed in this chapter. Diseases of the target  endocrine organs, the adrenal cortex, gonads and  thyroid gland, are considered in Chapters 8, 9 and 1 1  respectively. The parathyroid glands and endocrine  pancreas are discussed in Chapters 6 and 12 respectively.   The anterior and posterior lobes of the pituitary gland  are developmentally and functionally distinct; both  depend on hormones synthesized in the hypothalamus  for normal function. The hypothalamus also has  extensive neural connections with the rest of the brain,  and stress and some psychological disorders affect the  secretion of pituitary hormones and of the hormones  from other endocrine glands; see also Chapter 9.   Control of posterior pituitary hormones  Two structurally similar peptide hormones, antidiuretic   hormone (ADH) - also called vasopressin or arginine  vasopressin (AVP) - and oxytocin, are synthesized in  the hypothalamus and transported down the nerve  fibres of the pituitary stalk attached to specific carrier  proteins - neurophysins. The hormones are stored in the  posterior pituitary gland and are released independently  of each other into the bloodstream under hypothalamic  control, together with neurophysin. Neurophysin has  no apparent biological function and is rapidly cleared  from plasma.   Antidiuretic hormone (arginine vasopressin) is mainly  synthesized in the supraoptic nuclei of the hypothalamus  and enhances water reabsorption from the collecting  ducts in the kidneys (see Chapters 2 and 3).   Oxytocin is synthesized in the paraventricular nuclei  of the hypothalamus. It controls the ejection of milk  from the lactating breast and may have a role in initiating  uterine contractions, although normal labour can  proceed in its absence. It may be used therapeutically  to induce labour.   Anterior pituitary hormones  There is no direct neural connection between the  hypothalamus and the anterior pituitary gland. The  hypothalamus synthesizes small molecules (regulating  hormones or factors) that are carried to the cells of  the anterior pituitary lobe by the hypothalamic portal  system. This network of capillary loops in the median  eminence forms veins, which, after passing down the  pituitary stalk, divide into a second capillary network in  the anterior pituitary gland, from where hypothalamic  hormones stimulate or inhibit pituitary hormone  secretion into the systemic circulation.   The cells of the anterior pituitary lobe can be  classified simply by their staining reactions as acidophils,  basophils or chromophobes. Immunohistochemistry  can identify specific hormone-secreting cells.   These hormones, which are simple polypeptides  with similar amino acid sequences, mainly affect  peripheral tissues directly. Stimulation and inhibition  of secretion via the hypothalamus is influenced by  neural stimuli.   Basophils secrete hormones that affect other  endocrine glands. The hypothalamic control is mainly  stimulatory. There are three cell types:   • Corticotrophs synthesize a large polypeptide  (pro-opiomelanocortin), which is a precursor  of both adrenocorticotrophic hormone (ACTH;  corticotrophin) and |3-lipotrophin (Fig. 7.1).  Secretion of these hormones occurs in parallel.   • Adrenocorticotrophic hormone stimulates the  synthesis and secretion of steroids, other than  aldosterone, from the adrenal cortex and maintains  adrenal cortical growth. Part of the molecule has  melanocyte-stimulating activity, and high circulating  concentrations of ACTH are often associated with  pigmentation.   • |3-Lipotrophin is inactive until rapidly converted  to endorphins. These are neurotransmitters which,  because they have opiate-like effects, help control  pain.   • Gonadotrophs secrete the gonadotrophins, follicle-  stimulating hormone (FSH) and luteinizing  hormone (LH), which act on the gonads.   • Thyrotrophs secrete TSH (thyrotrophin), which acts  on the thyroid gland.   • These hormones are structurally similar glycoproteins  consisting of two subunits, a and |3. The a -sub unit  is common to all three hormones; the |3-subunit is  important for receptor recognition and therefore in  specific biological activity.   Chromophobes, once thought to be inactive, do  contain secretory granules. Chromophobe adenomas  often secrete hormones, particularly prolactin.   Neural and feedback controls are the two most  important physiological factors influencing the  secretion of the anterior pituitary hormones (Fig. 7.2).    Figure 7.1 The products of pro-opiomelanocortin  (POMC): adrenocorticotrophic hormone (ACTH),  (3-lipotrophin (LPH), y-LPH, (3-melanocyte-stimulating  hormone (MSH) and [3- and y-endorphin. The numbers  indicate the amino acid sequence in POMC.   Extrahypothalamic neural stimuli modify, and at  times over-ride, other control mechanisms. Physical  or emotional stress and mental illness may give similar  findings to, and even precipitate, endocrine disease.  The stress caused by insulin-induced hypoglycaemia  is used to test anterior pituitary function. Stress may  also stimulate the secretion of ADH from the posterior  pituitary.   Feedback control is mediatedby the concentrations of  circulating target-cell hormones; a rising concentration  usually suppresses trophic hormone secretion. This  negative feedback may directly suppress hypothalamic  hormone secretion or may modify its effect on  pituitary cells (long feedback loop). The secretion of  hypothalamic hormones may also be suppressed by  rising concentrations of pituitary hormone in a short  feedback loop.   Inherent rhythms: hypothalamic, and consequently  pituitary, hormones are released intermittently, either  in pulses or in a regular circadian rhythm. Disturbances  of such rhythms maybe of diagnostic value. This subject  is considered further in the relevant sections.   Drugs may also stimulate or block the action  of neurotransmitters, such as catecholamines,  acetylcholine and serotonin, and influence the  secretion of hypothalamic, and consequently pituitary,  hormones. The following are some examples.   • Certain neuroleptic drugs, such as chlorpromazine  and haloperidol, interfere with the action of  dopamine. This results in reduced GH secretion  (reduced effect of releasing factor) and increased  prolactin secretion (reduced inhibition).   • Bromocriptine, which has a dopamine-like action,  and levodopa, which is converted to dopamine,  have the opposite effect in normal subjects.  Bromocriptine causes a paradoxical suppression of  excessive GH secretion in acromegalics; the reason  for this anomalous response is unknown.   All these effects have been used in both the diagnosis  and treatment of hypothalamic-pituitary disorders;  they are discussed in later sections.   Evaluation of anterior pituitary function  The interpretation oftheresultsofbasalpituitaryhormone  assays is often difficult. Low plasma concentrations are  not necessarily abnormal, and plasma concentrations  within the reference range do not exclude pituitary  disease. The diagnosis of suspected hypopituitarism is  best excluded by the direct measurement of pituitary  hormones after stimulation or by demonstrating  target gland hyposecretion after the administration  of the relevant trophic hormone. However, prolonged  hypopituitarism may result in secondary failure of the  target gland with diminished response to stimulation.   Laboratory tests establish only the presence or  absence of hypopituitarism, and the cause must be  sought by other clinical means such as radiological  imaging (see also Chapter 9).   It maybe difficult to distinguish between hypothalamic  and pituitary causes of pituitary hormone deficiency or,  more correctly, between deficient releasing factor and  a primary deficiency of pituitary hormone secretion.  Isolated hormone deficiencies are more likely to be of  hypothalamic than of pituitary origin. The coexistence  of diabetes insipidus suggests a hypothalamic disorder.   Some biochemical investigations evaluate both  hypothalamic and pituitary function and some only  the latter, although it may be possible to distinguish  the anatomical site of the lesion. For example, the TSH  response to thyrotrophin-releasing hormone (TRH)  may differ in hypothalamic and pituitary causes of  secondary hypothyroidism (see Chapter 11). In cases  of hypogonadism due to gonadotrophin deficiency,  differentiation on the basis of the response to   The main clinical syndromes associated with excessive  or deficient anterior pituitary hormone secretion are  shown in Table 7.1. Excessive secretion usually involves  a single hormone, but deficiencies are often multiple.  However, many pituitary tumours are non-secretory  and may present clinically with eye signs or headaches.   Growth hormone secretion from the anterior pituitary  gland is mainly controlled by hypothalamic GH-  releasing hormone (GHRH). After synthesis by the  hypothalamus, this is transported via the hypothalamic  portal system to the somatotrophs of the anterior  pituitary. Secretion of GHRH, and therefore of GH, is  pulsatile, occurring about seven or eight times a day,  usually associated with:   • in response to the falling plasma glucose  concentration about an hour after meals.   At other times, plasma concentrations are usually  very low or undetectable, especially in children.   Growth hormone release is inhibited in a negative  feedback pathway by another hypothalamic hormone,  somatostatin (GH-release inhibiting hormone).  Somatostatin is found not only in the hypothalamus and   Table 7.1 Disorders associated with primary  abnormalities of anterior pituitary hormone secretion   elsewhere in the brain, but also in the gastrointestinal  tract and pancreatic islet cells, where it inhibits the  secretion of many gastrointestinal hormones. Insulin-  like growth factor 1 (IGF-1) acts by feedback to inhibit  GHRH action.   All these stimuli have been used to assess GH  secretory capacity, which may also be impaired in  obese patients, in hypothyroidism and hypogonadism,  in some cases of Cushing’s syndrome and in patients  receiving large doses of steroids.   The main function of GH is to promote growth. Its  action is primarily mediated by IGFs, polypeptides  that are synthesized in many tissues, where they act  locally. Plasma concentrations of one of these, IGF-1  (also known as somatomedin C), correlate with GH  secretion.   Carbohydrate metabolism is affected by GH: GH  antagonizes the insulin-mediated cell uptake of glucose,  and excess secretion may produce glucose intolerance.   Fat metabolism is stimulated by GH: lipolysis  is stimulated, with a consequent increase in the  concentration of circulating free fatty acids. Free fatty  acid antagonizes insulin release and action. Growth  hormone enhances protein synthesis, in conjunction  with insulin, to stimulate amino acid uptake by cells.   The production of IGF-1 is also influenced by other  factors, the most important of which is nutritional  status. In undernutrition, plasma concentrations are  low, whereas GH concentrations are elevated, suggesting  that plasma IGF-1 may influence GH secretion by  negative feedback. Other factors, such as adequate  nutrition and T , are also needed for normal growth.  The growth spurt during puberty may be enhanced by  androgens.   Growth hormone excess causes gigantism during  childhood and acromegaly in adults.   Most patients with GH excess have acidophil  adenomas of the anterior pituitary gland, which may  be secondary to excessive hypothalamic stimulation.  Rarely, malignant tumours may release GH or GHRH.   Acromegaly is sometimes one of the manifestations of  multiple endocrine neoplasia (MEN).   The clinical manifestations of GH excess depend on  whether the condition develops before or after fusion of  the bony epiphyses. Gigantism is caused by excess GH  secretion in childhood before fusion of the epiphyseal  plates, which may be delayed by accompanying  hypogonadism. Heights of up to about 2 metres may be  reached. Acromegalic features may develop after bony  fusion, but these patients may die in early adult life  from infection or cardiac failure or as a consequence  of progressive pituitary tumour growth. The features of  acromegaly may include the following (Fig. 7.3):   • An increase in the bulk of bone and soft tissues with  enlargement of, for example, the hands, tongue,  jaw and heart. Changes in facial appearance are  often marked, due to the increasing size of the jaw  and sinuses; the gradual coarsening of the features  may pass unnoticed for many years. Thyroid gland  enlargement may be clinically detectable, but the  patient is usually euthyroid.   • Excessive hair growth, hyperhidrosis and sebaceous  gland secretion are common.   • Impaired glucose tolerance is present in about  25 per cent of patients, about half of whom develop  symptomatic diabetes mellitus. In most cases the  pancreas can secrete enough insulin to overcome the  antagonistic effect of GH.   • There is a predisposition to multiple pre-malignant  colon polyposis and hypertension.   Many of these features are due to the action of  IGF-1, which acts as a general growth factor.   A different group of symptoms may occur due to the  encroachment of a pituitary tumour on surrounding  structures:   • Compression of the optic chiasma may cause visual  field defects such as bitemporal hemianopsia.   • If destruction of the gland progresses, other anterior  pituitary hormones such as ACTH, LH, FSH and  TSH may become deficient (see above). Plasma  prolactin concentrations may, however, be raised as  prolactin differs from all other pituitary hormones  in its method of control. Secretion is inhibited, not  stimulated, by dopamine; therefore, impairment of  hypothalamic control causes hyperprolactinaemia.   The diagnosis of GH excess is suggested by the clinical  presentation, biochemical tests and radiological  findings of the pituitary. Magnetic resonance imaging  (MRI) is more sensitive than computerized tomography  (CT) scanning. Plasma GH concentrations are usually  higher than normal and may reach several hundred  milliunits per litre (mU/L), but, because of the wide  reference range, the results from ambulant patients may    Figure 7.3 Patient with acromegaly; note large hands and prominent mandible and supraorbital ridges. Reproduced  with kind permission from Rees PJ and Williams DG. Principles of Clinical Medicine. London: Hodder Arnold, 1995.   fail to distinguish those with only moderately raised  plasma concentrations from normal subjects. Random  GH measurements are often not diagnostic owing to  episodic secretion and a short half-life.   The diagnosis is confirmed by demonstrating a  raised plasma GH concentration that is not suppressed  by a rise in plasma glucose concentration. In normal  subjects, plasma GH concentrations fall to very low  levels - to below lpg/L after a 75 g oral glucose load.  In acromegaly, the secretion of GH is autonomous and  this fall may not occur or be only slight, or there may  even be a paradoxical rise. Growth hormone secretion  is inhibited by hyperglycaemia in the normal subject.   After an overnight fast, insert an indwelling intravenous  cannula. After at least 30 min, take basal samples for  plasma glucose and GH estimation. The patient should  drink 75 g of glucose dissolved in about 300 mL of  water, or an equivalent glucose load. Take samples for   A 48 -year- old man noticed that his hat size had  increased, and his wife thought that his appearance  had changed since their marriage, his features  becoming coarser and his hands larger. Plasma  insulin-like growth factor 1 (IGF-1) concentration  was raised and an oral glucose suppression test was  performed. The results were as follows:   0 minutes: GH 24.5 pg/L  30 minutes: GH 24.6 pg/L  60 minutes: GH 23.7 pg/T  90 minutes: GH 20.5 pg/T  120 minutes: GH 25.8 pg/L   The plasma growth hormone (GH) concentration  was not suppressed during the test in any of the  samples. These findings are indicative of acromegaly;  the clinical features are typical of acromegaly. This  case illustrates the principle of using a suppression  test when considering a condition involving a  hormone excess. In healthy individuals, plasma GH  concentration would be suppressed to less than   glucose and GH assays at 30, 60, 90 and 120 min after  the glucose load has been taken.   In normal subjects, plasma GH concentrations fall  to undetectable levels. Although failure to suppress  suggests acromegaly or gigantism, it may be found  in some patients with severe liver or renal disease, in  heroin addicts or in those taking levodopa. Fasting  plasma GH can be normal in 8 per cent of acromegalic  patients. The plasma glucose concentrations may  demonstrate impaired glucose tolerance or diabetes  mellitus in acromegaly. Note that the test is usually  unnecessary in patients who are diabetic, as GH should  already be suppressed.   If acromegaly is confirmed, it is wise to investigate  for other pituitary hormone defects, for example TSH,  LH, FSH and ACTH. Acromegaly can also be associated  with the MEN syndrome (see Chapter 24).   Plasma IGF-1 has a long half-life and may be used  in screening for acromegaly. Plasma concentrations  correlate with the activity of the disease. Measurement of  the plasma concentrations of GH, or of IGF-1, may be  used to monitor the efficacy of treatment. Remember that  pregnancy increases IGF-1 concentration, and starvation,  obesity and diabetes mellitus decrease it. Insulin-like  growth-factor-binding protein-3 is the main binding  protein for IGF-1 and its concentration is also increased  in acromegaly. Sometimes plasma GHRH concentrations  are useful and can be raised where there is an ectopic  source or may be suppressed in pituitary disease.  Computerized tomography or MRI body scanning may  help to find an ectopic source of GH or GHRH.   There are various approaches to treatment, often with  surgery to remove the adenoma, usually by trans-  sphenoidal hypophysectomy. Residual disease requires  medical therapy, usually with either bromocriptine  or cabergoline (dopamine receptor agonists) or  somatostatin analogues (somatostatin itself has  too short a half-life for effective therapeutic use).  Octreotide or lanreotide, which bind to somatostatin  receptors, can be used or pegvisomant (GH receptor  antagonist). Radiation therapy is sometimes used as an  adjuvant for large invasive tumours or when surgery is  contraindicated.   The aim of treatment is to ameliorate symptoms  and to obtain an oral glucose suppressed GH  concentration of less than lpg/L (this cut-off can be   GH assay dependent - conversion factor for units:  mU/L = 2.4 X |tg/L) and normalization of plasma IGF-  1 concentrations.   In adults, GH deficiency may cause clinical symptoms,  such as tiredness, dyslipidaemia and increased  cardiovascular disease.   Growth hormone deficiency can cause short stature  in children. It is present in a small percentage of  normally proportioned small children: the birthweight  may be normal but the rate of growth is subnormal.  Other causes of growth retardation and short stature  must be excluded before a diagnosis of GH deficiency  is made (Box 7.1).   Emotional deprivation may be associated with GH  deficiency that is indistinguishable by laboratory tests  from that due to organic causes. People with Laron-type  dwarfism have a GH receptor defect, and Pygmies have  a GH receptor defect and low IGF-1 concentrations.   It is important to investigate children with reduced  growth rate to identify those who may benefit from  recombinant human GH replacement treatment.   Isolated GH deficiency is most commonly secondary  to idiopathic deficiency of hypothalamic GHRH. In  some cases, the secretion of other hormones is also  impaired. Sometimes there may be an organic disorder  of the anterior pituitary gland or hypothalamus; rare  inherited forms have been described.   The clinical history should include information  about birthweight and whether intrauterine growth  retardation was an issue. The sex-adjusted mid-parental  height or target height is useful, and can be calculated  by adding 6.5 cm to the mean of the parents’ heights  for boys and subtracting 6.5 cm from the mean of the  parents’ heights for girls. Normal growth maybe defined  as more than 5 cm per year in mid-childhood. It is, of  course, important to exclude hypothyroidism, chronic  diseases and malabsorption states, poor nutritional  state and failure to thrive. Clinical examination should  assess for obvious syndromes, pubertal status, bone  age, growth or growth velocity (Fig 7.4), for example  Tanner-Whitehouse charts and proportionality of  limbs. Karyotyping may be indicated if a chromosomal  disorder such as Turner’s syndrome (45, XO) is suspected.   There is a physiological reduction in GH secretion  at the end of pre-puberty. Thus, in children with bone  age more than 10 years, priming with sex hormones  before investigation may be necessary. For example,  ethinyloestradiol may be given to girls and testosterone  to boys prior to testing.   There is no general agreement about the best way to  investigate such children biochemically. This is partly  because GH secretion is episodic, GH assays vary  between laboratories, and there is a variable response of  GH to provocative stimuli. Plasma GH concentrations  in normal children are often low and assays under basal   A 10-year-old boy was referred to the paediatric out-  patient clinic because of short stature. His height  was 1.08 m and he had normal body proportions.  Physical examination and preliminary biochemical  tests showed no obvious explanation for his small  stature. A random plasma GH was less than 2 ptg/L.  After a glucagon stimulation test, the plasma GH  concentration failed to increase above 2 ptg/L.  However, other pituitary hormone concentrations  were normal on biochemical testing.   A diagnosis of isolated GH deficiency was made.  Note the failure of GH concentration to increase  after stimulation by glucagon. This illustrates well  the concept of using stimulation dynamic tests when  considering a hormone deficiency state.   Familial short stature  Social/emotional deprivation  Undernutrition and chronic disease  Coeliac disease  Rickets   Growth hormone deficiency, congenital or acquired  Hypothyroidism  Cushing’s syndrome  Congenital adrenal hyperplasia  Chromosomal abnormalities  Turner’s syndrome (45, XO)    Figure 7.4 Graph showing extreme failure to thrive in a  young child; note growth retardation and flattening of  growth. Reproduced with kind permission from Nyhan  WL and Barshop BA. Atlas of Inherited Metabolic  Diseases, 3rd edition. London: Hodder Arnold, 2012.   conditions rarely exclude the diagnosis. A low plasma  IGF-1 concentration may be a useful screening test.  Urinary GH excretion, either in 24-h collections or timed  overnight, may offer a relatively safe screening test.   If blood is taken at a time when physiologically  high concentrations are expected, the need for the  more unpleasant stimulation tests may be avoided,  for example 60-90 min after the onset of sleep and  about 20 min after vigorous exercise. An adequate GH  response occurs with an absolute response of greater  than 20mU/L (7 pg/L), making GH deficiency unlikely.  It should be noted that these GH cut-offs may be age  and assay dependent.   If GH deficiency is not excluded by the above  measurements, it is necessary to perform one or more  stimulation tests. The response of GH to insulin may be  the most reliable to detect GH deficiency, but it is not   without the risk of fatal hypoglycaemia. Glucagon could  also be used as an alternative (see Glucagon stimulation  test of the hypothalamus-pituitary axis). A GH absolute  response of greater than 20mU/L (7 pg/L) makes GH  deficiency unlikely after the presentation of provocative  stimuli on two occasions. Other such stimuli include  arginine, clonidine or the GHRH test. (See below for a brief  summary of some of these tests. ) An unequivocally normal  response to a stimulation test excludes the diagnosis, and a  clearly impaired one confirms it. Once GH deficiency has  been established, a cause should be sought by appropriate  clinical and imaging means.   The following are second-line dynamic tests  sometimes used for suspected GH deficiency. Clonidine  at 0.15 mg/m 2 body surface area is given orally after  an overnight fast. Blood samples for plasma GH  are collected at 0, 30, 60, 90, 120 and 150 min. The  patient should be closely monitored for hypotension.  Arginine hydrochloride, like clonidine, is another agent  used in provocative dynamic tests for suspected GH  deficiency. Arginine should not be given to patients  with renal, hepatic or acid-base disorders or diabetes  mellitus. After an overnight fast, 0.5 g/kg body weight  to a maximum of 30 g is intravenously infused. Blood  samples for plasma GH are collected at 0, 30, 60, 90 and  120 min. Arginine may evoke allergic reactions and the  necessary precautions should be in place in case of this.   Figure 7.5 shows an algorithm for the investigation  of short stature.   Disorders of the posterior pituitary are rare compared  with those of the anterior pituitary. Deficiency of ADH in  diabetes insipidus may present as polyuria. In the syndrome  of inappropriate ADH, hyponatraemia due to water excess  occurs. (This is discussed in Chapters 2 and 3.)   Hypopituitarism is a syndrome of deficiency of pituitary  hormone production that may result from disorders of  the hypothalamus, pituitary or surrounding structures.  The anterior pituitary gland has considerable functional  reserve. Clinical features of deficiency are usually absent  until about 70 per cent of the gland has been destroyed,  unless there is associated hyperprolactinaemia, when  amenorrhoea and infertility may be early symptoms.  Panhypopituitarism alludes to the involvement of all  pituitary hormones; alternatively, only one or more  may be involved, as in partial hypopituitarism.   Some of the causes of hypopituitarism are shown in  Box 7.2.   Panhypopituitarism with the frill clinical picture  described below is uncommon. Suspicion of anterior  pituitary hypofunction usually arisesinpatients presenting  with various features such as clinical and radiological  evidence of a pituitary or localized brain tumour,  hypogonadism, adrenocortical insufficiency, short stature  caused by GH deficiency, and hypothyroidism.   Although isolated hormone deficiency, particularly of  GH, may occur, several hormones are usually involved. If a  deficiency of one hormone is demonstrated, it is important  to establish whether the secretion of others is also  abnormal. Gonadotrophins are often the first to decrease  in hypopituitarism and it is unusual for the post-pituitary  hormones such as ADH and oxytocin to be affected.   Progressive pituitary damage usually presents with  evidence of deficiencies of gonadotrophins and GH.  Plasma ACTH and/or TSH concentrations may remain  normal, or become deficient months or even years  later. The clinical and biochemical consequences of the  target-gland failure include the following:   • Growth retardation in children This may be due to  deficiency of GH; deficiency of TSH, and therefore  of thyroid hormone, may contribute.   Secondary tumour deposits  Infections  Tuberculosis  Meningitis  Syphilis  Infiltrations  Sarcoidosis  Haemochromatosis  Histiocytosis X  Vascular   • Secondary hypogonadism This is due to  gonadotrophin deficiency, presenting as  amenorrhoea, infertility and atrophy of secondary  sexual characteristics with loss of axillary and pubic  hair and impotence or loss of libido. Puberty is  delayed in children.   • Secondary adrenocortical hypofunction (ACTH  deficiency) In contrast to the primary form  (Addison’s disease), patients are not hyperpigmented  because ACTH secretion is not raised. The  sodium and water deficiency and hyperkalaemia  characteristic of Addison’s disease do not usually  occur because aldosterone secretion (which is  controlled by angiotensin and not by ACTH) is  normal. However, cortisol is needed for normal free  water excretion, and consequently there may be a  dilutional hyponatraemia due to cortisol deficiency.  Cortisol is also necessary for the maintenance  of normal blood pressure. Hypotension may be  associated with ACTH deficiency. Cortisol and/or  GH deficiency may cause increased insulin sensitivity  with fasting hypoglycaemia.   • Secondary hypothyroidism (TSH deficiency) This  may sometimes be clinically indistinguishable from  primary hypothyroidism.   • Prolactin deficiency Associated with failure to lactate,  this may occur after post-partum pituitary infarction  (Sheehan’s syndrome). However, in hypopituitarism  due to a tumour, plasma prolactin concentrations are  often raised and may cause galactorrhoea (secretion  of breast fluid).   Patients with hypopituitarism, like those with  Addison’s disease, may die because of an inability to  secrete an adequate amount of cortisol in response to  stress caused by, for example, infection or surgery. Other  life-threatening complications are hypoglycaemia and  hypothermia.   The clinical presentation of pituitary tumours  depends on the type of cells involved and on the size  of the tumour (microadenomas less than 10 mm and  macroadenomas more than 10 mm).   Tumours of secretory cells may produce the clinical  effects of excess hormone secretion:   • excess prolactin causes infertility, amenorrhoea and  varying degrees of galactorrhoea (see Chapter 9),   A 17-year-old woman presented to the endocrine  clinic because of headaches, weakness and  amenorrhoea. The following baseline biochemical  endocrine test results were obtained.   Luteinizing hormone 0.46 mU/L (1-25)  Follicle-stimulating hormone 0.87 mU/L (1-15)  09.00 h cortisol 56nmol/L (180-720)   The patient has panhypopituitarism. Note the  low concentrations of plasma gonadotrophins  and secondary hypogonadism. A low plasma  cortisol concentration at 09.00 h implies also  low adrenocorticotrophic hormone (ACTH)  concentration. The panhypopituitarism was  subsequentlyfoundtobeduetoacraniopharyngioma  that had infiltrated the pituitary gland. Note that  this case illustrates another important principle  of endocrine testing: that the plasma free T 4  concentration is low but the TSH concentration is  within the reference range, which is abnormal given  the hypothyroxinaemia.   • visual disturbances caused by pressure on the optic  chiasma or headache due to raised intracranial pressure,   • deficiency of some or all of the pituitary hormones  due to destruction of secretory cells in the gland.   Non-secreting tumours are difficult to diagnose  using biochemical tests, although the combined  pituitary stimulation test (see Investigation of  suspected hypopituitarism) may indicate subclinical  impairment of function. Hyperprolactinaemia, which  maybe asymptomatic, is a valuable biochemical marker  of the presence of a pituitary tumour. Prolactin may be  secreted by the tumour cells or by unaffected lactotrophs  if tumour growth interferes with the normal inhibition  of prolactin secretion (see Chapter 9).   Investigation of suspected hypopituitarism  The laboratory should always be consulted before any  complex investigation or uncommon test is performed,  in order to check the details of specimen collection and  handling.   Deficiency of pituitary hormones causes  hypofunction of the target endocrine glands.   Investigation aims to confirm such deficiency, to  exclude disease of the target gland and then to test  pituitary hormone secretion after maximal stimulation  of the gland.   • cortisol at 09.00 h, to assess the risk of adrenocortical   If the plasma concentration of the target gland  hormone is low and the concentration of trophic  hormone is raised, the affected target gland should be  investigated. Conversely, if the plasma concentrations  of both the target gland and trophic hormones are low  or low-normal, consider a pituitary stimulation test  (see below).   Investigation of the pituitary region using  radiological techniques such as CT or MRI scanning  may help elucidate a cause of the hypopituitarism.   Although some textbooks talk about the combined  pituitary stimulation test (insulin or glucagon plus TRH  and GnRH given as one test), this is rarely required, as  useful information can be obtained from the basal pituitary  hormones and, if indicated, an insulin stimulation/  hypoglycaemia test, although this is not without risk.   This test is potentially dangerous and must be done  under direct medical supervision. Fatalities have  been reported due to severe hypoglycaemia and the  test should be carried out only in specialist units by  experienced staff. It is contraindicated in the following  patient groups: the elderly, patients with ischaemic  heart disease, epilepsy or severe panhypopituitarism,  and patients in whom plasma cortisol at 09.00 h is less  than lOOnmol/L.   A resting electrocardiogram should be normal.  Hypothyroidism should be treated beforehand as this  can impair the cortisol and GH responses. However,   note that treatment with thyroxine can precipitate an  adrenal crisis in such patients and thus corticosteroid  replacement is also necessary.   • assessment of ACTH/cortisol reserve (although the  development of plasma ACTH assays has made such  testing less necessary),   • differentiation of Cushing’s syndrome from pseudo-  Cushing’s syndrome, for example depression or  alcohol excess.   Both ACTH and GH are released in response to the  stress of hypoglycaemia.   Fifty millilitres of 20 per cent glucose for intravenous  administration must be immediately available in case  severe symptomatic hypoglycaemia develops. Care  should be taken not to induce severe hyperglycaemia  during infusion, as it may cause hyperosmolality, which  can be dangerous. Plasma cortisol is usually measured  as an index of ACTH secretion. If glucose needs to be  given, continue with the sampling.   After an overnight fast, insert an indwelling intravenous  cannula, for example 19 gauge. After at least 30 min,  take basal samples at time Omin for cortisol, GH and  glucose.   Inject soluble insulin in a dose sufficient to  lower plasma glucose concentrations to less than  2.5mmol/L and evoke symptomatic hypoglycaemia.  The recommended dose of insulin must be adjusted  for the patient’s body weight and for the suspected  clinical condition under investigation. The usual  dose is 0.15 U/kg body weight. If either pituitary or  adrenocortical hypofunction is suspected, or if a low  fasting glucose concentration has been found, reduce  the dose to 0.1 or 0.05 U/kg. If there is likely to be  resistance to the action of insulin because of Cushing’s  syndrome, acromegaly or obesity, 0.2 or 0.3 U/kg may  be needed.   Take blood samples at 30, 45, 60, 90 and 120 min  after the injections for cortisol, GH and glucose assays.   Methods of hormone assay vary, and results should  not be compared with reference values issued by  other laboratories. Interpretation is not possible if  hypoglycaemia is not attained, and the dose of insulin  can cautiously be repeated if this is not attained in the  45-min blood sample.   If hypoglycaemia has been adequate, plasma  cortisol concentrations should rise by more than  200 nmol/L and exceed 580 nmol/L, and an adequate  GH response occurs with an absolute response of  greater than 20 mU/L ( 7 pig/L) . In Cushing’s syndrome,  neither plasma cortisol nor GH concentrations  rise significantly, although they usually do in cases  of pseudo-Cushing’s syndrome (Chapter 8). See  Chapters 9 and 1 1 for details of GnRH and TRH tests  if the combined pituitary test is used. After the test,  a supervised meal should be given and the patient  should not drive for at least 2 hours.   This test is useful if the insulin hypoglycaemic test is  contraindicated. However, it is essential that the test is  carried out in a specialist unit by experienced staff.   The basic principle is that glucagon stimulates GH  and ACTH release probably via a hypothalamic route.   The test is contraindicated if there is severe adrenal  failure, for example if cortisol at 09.00 h is less than  100 nmol/L or in hypothyroidism. It is also unreliable  in the presence of diabetes mellitus. Hypoglycaemia is  not normally provoked by the test.   Patients should fast overnight, although they can drink  water. An indwelling intravenous cannula, for example  gauge 19, is inserted. For adults, lmg of glucagon is  injected subcutaneously at 09.00 h.   Blood samples are taken at 0, 90, 120, 150, 180, 210  and 240 min for cortisol and GH.   Plasma cortisol should normally rise by at least  200 nmol/L to more than 580 nmol/L, and an adequate  GH response occurs with an absolute response of  greater than 20mU/L (7 pg/L).   Treatment of hypopituitarism  This consists of specific therapy depending on  its cause and may include surgical removal of a  large adenoma. If the ACTH axis is impaired, it is  essential to prescribe a glucocorticoid, for example  hydrocortisone in the acute situation or prednisolone  for maintenance. Secondary hypothyroidism will need  thyroid replacement.   Adrenal replacement should precede T 4 therapy  to avoid an Addisonian crisis (see Chapter 8).   Gonadotrophin deficiency may require testosterone in without progesterone as appropriate. In children and  males and oestrogen replacement in women, with or sometimes in adults, GH may be indicated.   The anterior pituitary gland releases a number of  peptide hormones, which are themselves regulated  by hypothalamus hormones that reach the pituitary  via the portal blood system. The anterior pituitary  hormones include ACTH, TSH, LH and FSH; their  respective target organs are the adrenal and thyroid  glands and the ovaries/ testes. Growth hormone  (GH) is also an anterior pituitary hormone but  does not have a specific target organ - instead it  influences most tissues.   Hypopituitarism can be due to many conditions,  such as pituitary infiltration or destruction, and   results in a deficiency of all (panhypopituitarism)  or some of the pituitary hormones.   Conversely, excess release of certain anterior  pituitary hormones can occur because of pituitary  tumours. For example, acromegaly is due to excess  GH, and Cushing’s disease to excess ACTH release.  The posterior pituitary releases oxytocin and ADH.  The former is involved in uterine contraction  during labour. Antidiuretic hormone controls  water elimination by changing the renal collecting  ducts’ permeability. Deficiency of ADH results in  diabetes insipidus (discussed in Chapter 2).   A number of endocrine abnormalities involve the  adrenal glands, and some of the more common  conditions are discussed in this chapter, which should  perhaps be read in conjunction with Chapter 7 (on  pituitary function) and Chapter 9 (which deals with  reproductive endocrinology).   The adrenal glands are divided into two  embryologically and functionally distinct parts. The  adrenal cortex is part of the hypothalamic-pituitary-  adrenal endocrine system. Morphologically, the adult  adrenal cortex consists of three layers. The outer thin  layer (zona glomerulosa) secretes only aldosterone.  The inner two layers (zona fasciculata and zona  reticularis) form a functional unit and secrete most  of the adrenocortical hormones. In the fetus there is a  wider fourth layer, which disappears soon after birth.  One of its most important functions during fetal life is,  together with the adrenal cortex, to synthesize oestriol,  in association with the placenta. The adrenal medulla is  part of the sympathetic nervous system.   Steroid hormones are derived from the lipid cholesterol.  Figure 8.1 shows the internationally agreed numbering  of the 27 carbon atoms of steroid molecules and the  lettering of the four rings. The products of cholesterol  are also indicated. If the molecule contains 21 carbon  atoms, it is referred to as a C21 steroid. The carbon  atom at position 21 of the molecule is written as C-21.  The side chain on C-17 is the main determinant of the  type of hormonal activity (Fig. 8.1), but substitutions  in other positions modify activity within a particular  group.   The first hormonal product of cholesterol is  pregnenolone. Several important synthetic pathways   diverge from it (Fig. 8.1). The final product is dependent  upon the tissue and its enzymes.   The zona glomerulosa secretes aldosterone,  produced by 18-hydroxylation. Synthesis of this steroid  is controlled by the renin-angiotensin system and not  normally by adrenocorticotrophic hormone (ACTH).  Although ACTH is important for maintaining growth of  the zona glomerulosa, deficiency does not significantly  reduce output.   The zonae fasciculata and reticularis synthesize and  secrete two groups of steroid:   • Cortisol, a glucocorticoid (the most important  C21 steroid), is formed by progressive addition of  hydroxyl groups at C-17, C-21 and C-ll.   • Androgens (for example androstenedione) are  formed after the removal of the side chain to produce  C19 steroids.   Adrenocorticotrophic hormone secreted by the  anterior pituitary gland stimulates synthesis of these two  steroid groups. Its secretion is influenced by negative  feedback from changes in plasma cortisol concentrations.  Impaired cortisol synthesis due, for example, to an  inherited 2 1 -a- hydroxylase or ll-|3-hydroxylase   deficiency (congenital adrenal hyperplasia) results in  increased ACTH stimulation with increased activity  of both pathways. The resultant excessive androgen  production may cause hirsutism or virilization.   The adrenocortical hormones can be classified into  groups depending on their predominant physiological  effects.   Cortisol and corticosterone are naturally occurring  glucocorticoids. They stimulate gluconeogenesis and   Figure 8.1 Numbering of the steroid carbon atoms of cholesterol and the synthetic pathway of steroid hormones;  the chemical groups highlighted determine the biological activity of the steroid.   the breakdown of protein and fat, that is, they antagonize  some of insulin’s action. Glucocorticoids in excess may  impair glucose tolerance and alter the distribution of  adipose tissue. Cortisol helps maintain the extracellular  fluid volume and normal blood pressure.   Circulating cortisol is bound to cortisol-binding  globulin (CBG; transcortin) and to albumin. At normal  concentrations, only about 5 per cent of the total is  unbound and physiologically active. Plasma CBG  is almost fully saturated, so that increased cortisol  secretion causes a disproportionate rise in the free  active fraction. Cortisone is not secreted in significant  amounts by the adrenal cortex. It is biologically   inactive until it has been converted in vivo to cortisol  (hydrocortisone) .   Glucocorticoids are conjugated with glucuronate  and sulphate in the liver to form inactive metabolites,  which, because they are more water soluble than  the mainly protein-bound parent hormones, can be  excreted in the urine.   In contrast to other steroids, aldosterone is not  transported in plasma bound to specific proteins. It  stimulates the exchange of sodium for potassium and  hydrogen ions across cell membranes and its renal   action is especially important for sodium and water  homeostasis. It is discussed more fully in Chapters 2,  3 and 4. Like the glucocorticoids, it is inactivated by  hepatic conjugation and is excreted in the urine.   There is overlap in the actions of C21 steroids.  Cortisol, in particular, may have a significant  mineralocorticoid effect at high plasma concentrations  when the free fraction is significantly increased.   The main adrenal androgens are dehydroepiandros-  terone (DHEA), its sulphate (DHEAS) and  androstenedione. They promote protein synthesis  and are only weakly androgenic at physiological  concentrations. Testosterone, the most powerful  androgen, is synthesized in the testes or ovaries but  not in the adrenal cortex. Most circulating androgens,  like cortisol, are protein bound, mainly to sex-  hormone-binding globulin and albumin.   There is extensive peripheral interconversion of  adrenal and gonadal androgens. The end products,  androsterone and aetiocholanolone, together with  DHEA, are conjugated in the liver and excreted as  glucuronides and sulphates in the urine.   The hypothalamus, anterior pituitary gland and adrenal  cortex form a functional unit - the hypothalamic-  pituitary-adrenal axis (see Chapter 7).   Cortisol is synthesized and secreted in response to  ACTH from the anterior pituitary gland.   The secretion of ACTH is dependent on corticotrophin-  releasing hormone (CRH), released from the  hypothalamus. High plasma free cortisol concentrations  suppress CRH secretion (negative feedback) and alter  the ACTH response to CRH, thus acting on both the  hypothalamus and the anterior pituitary gland (Fig. 8.2).   Figure 8.2 The factors controlling the secretion of cortisol from the adrenal gland, including the site of action  of dynamic function tests (shaded). +, stimulates; -, inhibits; ACTH, adrenocorticotrophic hormone; CRH,  corticotroph in-releasing hormone.   The melanocyte-stimulating effect of high plasma  concentrations of ACTH, or related peptides, causes  skin pigmentation in two conditions associated with  low plasma cortisol concentrations:   • Nelson’s syndrome: after bilateral adrenalectomy  for Cushing’s disease (see Causes of Cushing’s  syndrome), removal of the cortisol feedback causes  a further rise in plasma ACTH concentrations from  already high levels.   Inherent rhythms and stress  Adrenocorticotrophic hormone is secreted episodically,  each pulse being followed 5-10 min later by cortisol  secretion. These episodes are most frequent in the early  morning (between the fifth and eighth hour of sleep)  and least frequent in the few hours before sleep. Plasma  cortisol concentrations are usually highest between about  07.00 and 09.00 h and lowest between 23.00 and 04.00 h.   The secretion of ACTH and cortisol usually varies  inversely, and the almost parallel circadian rhythm of  the two hormones may be due to cyclical changes in  the sensitivity of the hypothalamic feedback centre to  cortisol levels. Inappropriately high plasma cortisol  concentrations at any time of day suppress ACTH  secretion. This effect can be tested by the dexamethasone  suppression test. Loss of circadian rhythm is one of  the earliest features of Cushing’s syndrome. Stress,  either physical or mental, may over-ride the first two  mechanisms and cause sustained ACTH secretion. An  inadequate stress response may cause acute adrenal  insufficiency. Stress caused by insulin-induced  hypoglycaemia can be used to test the axis.   Plasma cortisol is usually measured by immunoassay, but  the antibody may cross-react with other steroids or drugs.  Factors that may affect results include hydrocortisone  (cortisol), cortisone (converted to cortisolbymetabolism)  and prednisolone, which may contribute to the ‘cortisol’  concentration of some immunoassay methods. Thus,  it is recommended either that the patient is prescribed  dexamethasone (which is less likely to cross-react with  cortisol assays) or, if possible, that the prednisolone is  gradually reduced and then stopped for about 3 days  before sampling depending upon clinical context.  Oestrogens and some oral contraceptives increase the  plasma CBG concentration, and therefore the protein-  bound cortisol concentration.   Adrenocorticotrophic hormone is a single-chain  polypeptide made up of 39 amino acids with biological  activity at the N-terminal end of the peptide. A peptide  consisting of this sequence has been synthesized  (tetracosactide, Synacthen) and can be used for  diagnosis in place of ACTH. The ACTH stimulates  cortisol synthesis and secretion by the adrenal cortex.  It has much less effect on adrenal androgen production  and, at physiological concentrations, virtually no effect  on aldosterone production.   The main disorders of adrenocortical function are  shown in Table 8.1.   Cushing’s syndrome is mainly caused by an excess  of circulating cortisol but also other steroids such  as androgens. Many of the clinical and metabolic  disturbances can be explained by glucocorticoid excess.  The clinical and metabolic features may include the  following:   • Impaired glucose tolerance and hyperglycaemia.  Cortisol has the opposite action to that of insulin,  causing increased gluconeogenesis, and some  patients may have diabetes mellitus.    Figure 8.3 This depicts a patient before and after  corticosteroid therapy; note Cushingoid appearance.  Reproduced with kind permission from Kinirons M and  Ellis H. French’s Index of Differential Diagnosis, 15th  edition. London: Hodder Arnold, 2011.   • Increased protein catabolism, which also increases  urinary protein loss. Thus, there is a negative nitrogen  balance associated with proximal muscle wasting  with weakness, thinning of the skin and osteoporosis.  The tendency to bruising and the purple striae (most  obvious on the abdominal wall) are probably due to  this thinning.   • Hypertension, caused by urinary retention of  sodium and therefore of water, which are due to  the mineralocorticoid effect of cortisol. Increased  urinary potassium loss may cause hypokalaemia.   • Androgen excess, which may account for the common  findings of greasy skin with acne vulgaris and  hirsutism, and menstrual disturbances in women.   One of the most common causes of Cushing’s syndrome  is iatrogenic and related to excessive steroid treatment.  Increased endogenous cortisol production may be due to  hyperstimulation of the adrenal gland by ACTH, either  from the pituitary gland or from an ‘ectopic’ source,  or due to largely autonomous secretion by an adrenal  tumour such as an adenoma or carcinoma (Fig. 8.4).   • Cushing’s disease It is associated with bilateral  adrenal hyperplasia, often secondary to a basophil  adenoma of the anterior pituitary gland.   A 45-year-old woman was referred to the endocrine  clinic because of skin pigmentation, bruising, obesity,  hypertension and proximal muscle weakness. The  following biochemical results were returned:   The following were further results of an overnight  dexamethasone suppression test:   The raised 24-h urinary free cortisol and 09.00 h  cortisol concentrations are suggestive of Cushing’s  syndrome. This is supported by the fact that  there was a failure of cortisol suppression by low-  dose dexamethasone. The raised plasma ACTH  concentration could be indicative of either Cushing’s  disease or ectopic ACTH secretion. Further tests,  including computerized tomography scanning and  a corticotrophin-releasing hormone test, confirmed  ectopic ACTH secretion due to a lung tumour. Note  also the hypokalaemia and hyperglycaemia and the  other clinical features typical of Cushing’s syndrome.   • Ectopic ACTH secretion In this condition, usually  from a small-cell carcinoma of the bronchus, ACTH  concentrations may be high enough to cause skin  pigmentation. The patient may have weight loss  with cachexia. One metabolic complication is a  hypokalaemic alkalosis. The clinical features may be  indistinguishable from those of Cushing’s disease,  although sometimes patients do not have the  characteristic cushingoid features as the cortisol rises  so quickly.    Figure 8.4 Cushing’s disease, indicating excess cortisol production caused either by hyperstimulation of the  adrenal gland by adrenocorticotrophic hormone (ACTH), from either the pituitary or an ectopic source, or by  autonomous hormone secretion from an adrenal tumour.   The secretion of ACTH is appropriately suppressed  in primary cortisol-secreting tumours of the adrenal  cortex. The tumours may be benign or malignant and  are usually derived from the zona fasciculata/zona  reticularis of the adrenal cortex. These glucocorticoid-  secreting tumours do not normally secrete aldosterone,  which is produced in the zona glomerulosa layer of  the adrenal cortex. Benign adenomas occur and also  carcinomas. The latter secrete a variety of steroids,  including androgens, and thus may cause hirsutism or  virilization. In these cases plasma ACTH is suppressed  by the excess glucocorticoids.   Basis of investigation of suspected Cushing’s syndrome  The following questions should be asked:   • If so, does the patient have any other condition that  may cause it?   Plasma cortisol concentrations reflect ACTH activity  at that moment and, because of the episodic nature  of cortisol secretion, such isolated values may be   misleading. Indeed, cyclical Cushing’s syndrome may  require repeated investigation. The level of cortisol  measured in a 24-h urine sample reflects the overall  daily secretion.   One of the earliest features of Cushing’s syndrome  is the loss of the diurnal variation in cortisol secretion,  with high concentrations in the late evening, when  secretion is normally at a minimum. However, it is not  a diagnostic finding because it can also be caused by,  for example, stress and endogenous depression. The  assessment of diurnal rhythm is not a practical out-  patient procedure.   Only the unbound fraction of cortisol in plasma is  filtered at the glomeruli and excreted in the urine  (urinary ‘free’ cortisol). In Cushing’s syndrome, because  of the loss of circadian rhythm, raised plasma values  are present for longer than normal and daily urinary  cortisol excretion is further increased (i.e. there is a  disproportionately raised free fraction of cortisol).   Plasma and urinary cortisol concentrations are  usually much higher when Cushing’s syndrome is  due to adrenocortical carcinoma or overt ectopic   ACTH secretion. Determinations of 24-h urinary free  cortisol have about a 5 per cent false-negative rate, but  if three separate determinations are normal, Cushing’s  syndrome is most unlikely.   A small dose, for example 1 mg, of this synthetic steroid  inhibits ACTH, and thereby cortisol secretion by  negative feedback. This is usually given at midnight and  blood is taken for cortisol assay at 09.00 h the following  morning.   The overnight dexamethasone suppression test  is a sensitive, but not completely specific, test for  evaluating such patients. The false-positive rate is about  12 per cent and the false-negative rate about 2 per cent.  A normal fall in plasma cortisol concentrations makes  the diagnosis of Cushing’s syndrome very unlikely,  but failure to suppress plasma cortisol to less than  50 nmol/L does not confirm it with certainty.   There are some pitfalls, for example certain  anticonvulsant drugs, such as phenytoin, may interfere  with dexamethasone suppression tests, inducing liver  enzymes that increase the rate of metabolism of the  drug. Plasma concentrations may therefore be too low  to suppress the feedback centre.   In some cases, additional tests are needed to confirm the  diagnosis of excess cortisol production. The 48-h low-  dose dexamethasone suppression test may be useful as  it gives fewer false-positives than the overnight low-dose  dexamethasone test: 0.5 mg dexamethasone is given  orally at 6-h intervals from 09.00 h on day 1 for eight  doses, and then plasma cortisol is measured after 48 h  at 09.00 h. Plasma cortisol should normally suppress to  less than 50 nmol/L, but not in Cushing’s syndrome.   Various conditions can mimic Cushing’s syndrome  (pseudo-Cushing’s) and thus give false-positive results  for screening tests.   The following non-Cushing’s causes of abnormal  cortisol secretion are important to remember:   • Stress over-rides the other mechanisms controlling  ACTH secretion, with loss of the normal circadian  variation of plasma cortisol and a reduced feedback  response. Urinary free cortisol excretion may be  increased even in relatively minor physical illness or  mental stress.   • Endogenous depression may be associated with  sustained high plasma cortisol and ACTH  concentrations that may not be suppressed even  by a high dose of dexamethasone. However, these  patients often have a normal cortisol response to  insulin-induced hypoglycaemia, whereas those with  Cushing’s syndrome do not (see Chapter 7).   • Severe alcohol abuse can cause hypersecretion of  cortisol that mimics Cushing’s syndrome clinically  and biochemically. The abnormal findings revert to  normal when alcohol is stopped.   The following biochemical investigations may help to  elucidate the cause and ideally should be carried out on  a metabolic ward by experienced staff (Table 8.2):   • Plasma ACTH is detectable onlyin ACTH-dependent  Cushing’s syndrome, and plasma concentrations are  suppressed in patients with secreting adrenocortical  tumours. In patients with Cushing’s disease, plasma  ACTH concentration may be either high-normal or  moderately raised but inappropriate for the high   plasma cortisol concentration. Conversely, plasma  ACTH concentrations are markedly raised in  patients with ‘ectopic’ ACTH production.   • The high-dose dexamethasone suppression test  may be useful. The principle of this test is the same  as that of the low-dose test described above, but a  higher dose (2 mg) given over 48 h at 6-h intervals  may suppress the relatively insensitive feedback  centre of pituitary-dependent Cushing’s disease (this  occurs in about 90 per cent of cases). Plasma cortisol  concentration suppression by about 50 per cent is  usual. In the other two categories, ectopic ACTH  production or adrenal tumours, when pituitary  ACTH is already suppressed, even this high dose  will usually have no effect, although there may be  some suppression in some cases of ‘ectopic’ ACTH  secretion.   • If there is virilization, measure plasma androgens  such as testosterone, DHEA and DHEAS. High  concentrations suggest an adrenocortical carcinoma  (see Chapter 9).   • In cases of Cushing’s syndrome with raised plasma  ACTH concentration, the intravenous CRH  test (100 pg in adults or lpg/kg body weight in  children) has gained popularity. In Cushing’s disease  (pituitary disorder), an increase in baseline plasma  cortisol concentration of about 25 per cent occurs in  90 per cent of patients, whereas patients with ectopic  ACTH, for example with lung carcinoma, usually fail  to show this rise.   • The insulin tolerance test may be appropriate.  Elevated plasma cortisol concentrations suppress the  stress response to hypoglycaemia, and this test may  be of value in differentiating Cushing’s syndrome  from pseudo-Cushing’s syndrome due to depression  or obesity. This test is not without hazards due to  severe hypoglycaemia.   Confirmation of Cushing’s disease will require  pituitary imaging techniques, for example magnetic  resonance imaging (MRI), as well as pituitary hormone  assessment (see Chapter 7). Specialized units may  perform simultaneous petrosal sinus sampling in an  attempt to localize the pituitary tumour and confirm  differentiation from an ectopic ACTH source. A  peripheral blood sample to petrosal blood sample  ACTH ratio of more than 2 is indicative of Cushing’s  disease (ratio more than 3 if CRH stimulation used).   In cases of ectopic ACTH secretion, imaging  techniques are important to try to locate the tumour,   such as thin-slice MRI of the chest and abdomen.  Seventy per cent of these tumours co-secrete other  peptide hormones such as glucagon, somatostatin,  calcitonin and bombesin. Ectopic ACTH-induced  Cushing’s syndrome is more likely to present with  hypokalaemia, skin pigmentation, short clinical history  and severe myopathy. Plasma ACTH concentrations  are also often much higher than those found in  Cushing’s disease. Sometimes venous sampling of  ACTH is necessary to locate the tumour. Rarely, a  tumour may secrete CRH and thus mimic Cushing’s  disease. Abdominal imaging, for example CT scanning,  may locate an adrenal tumour. Here, plasma ACTH is  usually suppressed and the tumour may also secrete  androgens.   The treatment of Cushing’s syndrome depends  upon the cause. Surgery is usually the treatment of  choice. In Cushing’s disease, pituitary removal by  trans-sphenoidal surgery with pituitary radiation as  an adjunct may be necessary. Adrenal tumours also  usually require surgery, although the prognosis may  be poor with carcinoma. Bilateral adrenal removal  can lead to skin pigmentation due to pituitary ACTH  release - Nelson’s syndrome. Similarly, if the source  of ectopic ACTH is found, surgery may be indicated  to remove the tumour, for example lung carcinoma.  Rarely, cortisol-inhibiting drugs are used such as  ketoconazole, mitotane and metyrapone, which inhibit  steroid ll-|3-hydroxylase (Fig. 8.5).   Addison’s disease is caused by bilateral destruction of  all zones of the adrenal cortex, usually as the result of  an autoimmune process. The association of Addison’s  disease with hypoparathyroidism and mucocutaneous  candidiasis is described as polyglandular autoimmune  syndrome type 1 and has autosomal recessive  inheritance. Polyglandular autoimmune syndrome type  2 occurs when Addison’s disease is associated with type  1 diabetes mellitus and autoimmune thyroid disease,  either Hashimoto’s thyroiditis or Graves’ disease, and is  also related to human leucocyte antigen (HLA)-B8 and  DR-3 types.   Tuberculosis affecting the adrenal glands is an  important cause in countries where this disease is  common. Other causes of bilateral destruction of the  adrenal glands include amyloidosis, mycotic infections,  acquired immunodeficiency syndrome (AIDS) and   An important cause of acute adrenal crisis is bilateral  adrenal haemorrhage, which can occur on warfarin  therapy or in patients with meningococcus septicaemia,  as in Waterhouse-Friderichsen syndrome. Certain  drugs can inhibit glucocorticoid synthesis, including  ketoconazole, aminoglutethimide, methadone and  etomidate. Glucocorticoid deficiency contributes to the  hypotension and causes marked sensitivity to insulin;  hypoglycaemia may be a presenting feature.   The clinical presentation of Addison’s disease  depends on the degree of adrenal destruction. In cases  of massive haemorrhagic adrenal destruction, as in  Waterhouse-Friderichsen syndrome of meningo-  coccaemia, the patient may be shocked, with volume  depletion; this adrenal crisis should be treated as a matter  of urgency. Conversely, sometimes the presentation  can be surreptitious; a prolonged period of vague ill  health, tiredness, weight loss, mild hypotension and   pigmentation of the skin and buccal mucosa may occur.  The cause of these vague symptoms may only become  evident if an Addisonian crisis is precipitated by the  stress of some other, perhaps mild, illness or surgery.   Androgen deficiency is not clinically evident because  testosterone production by the testes is unimpaired  in males and because androgen deficiency does not  produce obvious effects in women. The pigmentation  that develops in some cases of Addison’s disease is  due to the high circulating levels of ACTH or related  peptides resulting from the lack of cortisol suppression  of the feedback mechanism. Patients with primary  adrenal insufficiency, as in Addison’s disease due to  autoimmune disease, may also show vitiligo.   Patients with acute cortisol deficiency may present  with nausea, vomiting and hypotension. Dilutional  hyponatraemia may be present because cortisol is  needed for sodium-free water excretion by the kidneys.  The biochemical changes therefore resemble those of  inappropriate ADH secretion.   A 28-year-old woman attended the endocrine out-  patient clinic because of weight loss, weakness,  amenorrhoea, buccal pigmentation and vitiligo.  Some of her biochemical tests were as follows:   Cortisol 30 min later after 250 pg intramuscular  Synacthen 80 nmol/L   These studies support the diagnosis of adrenal  insufficiency (Addison’s disease), with a failure of  plasma cortisol to rise after Synacthen stimulation.  The symptoms and signs are typical of the condition.  The patient was subsequently shown to have  polyendocrine syndrome with premature ovarian  failure, vitiligo and autoimmune Addison’s disease.  The buccal mucosa pigmentation was due to increased  adrenocorticotrophic hormone (ACTH) concen-  tration. Note the hyponatraemia and hyperkalaemia.   Hypoaldosteronism hyporeninism is discussed in  Chapters 4 and 5 and is associated with type 2 diabetes  mellitus and type IV renal tubular acidosis.   Abnormalities of |3-oxidation of very long-chain  fatty acids (VLCFAs) are X-linked recessive defects in  which VLCFAs accumulate in the tissues, including  the adrenal glands, for example adrenoleucodystrophy,  which should be considered in all boys with possible  adrenal failure. Another, rarer, cause of hypoadrenalism  is Allgrove’s syndrome due to congenital adrenocortical  unresponsiveness to ACTH.   Pseudohypoaldosteronism type 1 usually presents in  infancy with failure to thrive and salt wasting associated  with hypotension, hyperkalaemia and large elevations in  plasma renin and aldosterone. Defects are either in the  epithelial sodium channel or in the mineralocorticoid  receptor. Treatment is with a high sodium diet and  high-dose fludrocortisone or carbenoxolone.   Pseudohypoaldosteronism type 2, or Gordon’s  syndrome, is an autosomal dominant condition   typified by hyperkalaemia, hypertension and a mild  hyperchloraemic acidosis. There is a mutation in the  distal tubule chloride channel, resulting in enhanced  distal chloride reabsorption in preference to excretion  of potassium. Treatment is with a low-potassium diet  and a thiazide diuretic.   Adrenocorticotrophic hormone release may be  impaired by disorders of the hypothalamus or the  anterior pituitary gland, most commonly due to a  tumour or infarction (see Chapter 7). Corticosteroids  suppress ACTH release and, if such drugs have been  taken for a long time, the ACTH-releasing mechanism  maybe slow to recover after the steroid is stopped. There  may be temporary adrenal atrophy after prolonged lack  of stimulation. Extensive destruction of the anterior  pituitary gland may cause panhypopituitarism (see  Chapter 7), but, if it is only partial, ACTH secretion  may be adequate for basal requirements.   Adrenocortical deficiency may then only become  clinically evident under conditions of stress, which  may precipitate acute adrenal insufficiency. The most  common causes of stress are infection and surgery.   Patients may present with non-specific symptoms  such as weight loss and tiredness. Hypoglycaemia may  occur because of marked insulin sensitivity. Unlike  primary adrenal hypofunction, pigmentation is absent  because plasma ACTH concentrations are not raised.   If a diagnosis of acute adrenal insufficiency is suspected  clinically, blood shouldbe taken so that the plasma cortisol  concentration can be measured later; steroid treatment  must be started immediately, as the condition can be  life threatening. Adrenocorticol hypofunction may not  be excluded on the results of a random plasma cortisol  concentration, as this gives little idea as to adrenal stress  reserve. Nor will plasma cortisol estimation distinguish  between primary and secondary adrenal failure.   The tetracosactide (Synacthen) stimulation test should  be performed as soon as possible. Tetracosactide has the  same biological action as ACTH but, because it lacks the  antigenic part of the molecule, there is much less danger  of an allergic reaction. If the patient cannot stop using  steroids, a steroid such as dexamethasone, which does not  normally interfere with the assay of cortisol, should be  prescribed. (See below for details of the Synacthen test.)   A plasma autoantibody screen including adrenal  antibodies may point to a primary adrenal autoimmune  problem.   Plasma ACTH assay may be of value when  inappropriately low plasma cortisol concentrations  have been found; a raised plasma ACTH concentration  indicates primary insufficiency, whereas a low ACTH  concentration suggests secondary insufficiency.   The essential abnormality in adrenocortical  hypofunction is that the adrenal gland cannot adequately  increase cortisol secretion in response to stress. This  may be due to adrenal (primary) or hypothalamic-  pituitary (secondary) pathology. In the case of the latter,  it may be necessary to test the whole axis (see Chapter  7). Insulin-induced hypoglycaemia normally causes  ACTH secretion from the anterior pituitary gland.  This can be assessed by demonstrating a rise in plasma  cortisol concentrations. An impaired response indicates  pituitary dysfunction only if the adrenal cortices have  already been shown to be capable of responding to  exogenous ACTH.   Before starting treatment, take blood for immediate  plasma urea, creatinine and electrolyte estimations as  well as plasma glucose, calcium and cortisol.   Hyponatraemia, hyperkalaemia, hypoglycaemia  and uraemia are compatible with an Addisonian crisis.  Hypercalcaemia may also occur (see Chapter 6).   Start steroid treatment once blood has been taken, as  the condition is life threatening.   If the plasma cortisol is very high, an Addisonian  crisis is unlikely. Conversely, if the plasma cortisol  concentration is very low or undetectable, and if  there is no reason to suspect severe CBG deficiency,  for example due to the nephrotic syndrome,  an Addisonian crisis is likely. Plasma cortisol  concentrations, which would be normal under basal  conditions, may be inappropriately low for the degree  of stress. To confirm this, perform a short Synacthen  test (see below).   Measure the plasma cortisol concentration at 09.00 h.  If more than 580 nmol/L, Addison’s disease is unlikely,  but this does not provide information about the adrenal  glands’ reserve to respond to a stressful stimulus.   If the plasma cortisol concentrations are equivocal,  perform a short Synacthen test. A normal result makes  long-standing secondary adrenal insufficiency unlikely.   Send ablood specimen for plasma ACTH. If the plasma  ACTH concentration is high, this confirms primary  adrenal hypofunction. A low ACTH concentration  suggests secondary adrenal hypofunction, and pituitary  assessment is indicated (see Chapter 7).   If the diagnosis is still unclear, admit the patient to  a hospital metabolic ward and perform a prolonged  Synacthen test. A normal result excludes primary  adrenal hypofunction.   An increasing response over time to the short and  prolonged Synacthen tests indicates gradual recovery  of the adrenal cortex and suggests hypothalamic or  pituitary hypofunction (Fig. 8.6; see also Chapter 7).   The patient should be resting quietly but not fasting. It  is recommended that the test is done in the morning.  Resuscitation facilities should be available in case of an  allergic reaction.   Blood is taken for basal cortisol assay. Synacthen  250 pg is given by intramuscular or intravenous  injection. Blood is taken for cortisol assay at baseline,  30 and 60 min.   Normally the plasma cortisol concentration increases by at  least 200 nmol/L, to a concentration of at least 580 nmol/L.  The peak is usually at 30 min, although a steady increase  at 60 min may imply secondary hypoadrenalism due to  pituitary or hypothalamic disease.   Depot or prolonged Synacthen stimulation test  Repeated injections of depot Synacthen are painful and  may cause sodium and water retention. Therefore this  test is contraindicated in patients in whom sodium  retention may be dangerous, such as those with  congestive cardiac failure. The test is rarely indicated if  a basal plasma ACTH concentration is known. Its main  indication is to differentiate primary from secondary  adrenal insufficiency (due either to pituitary failure  or to prolonged corticosteroid therapy) when adrenal  failure has previously been confirmed by the short  Synacthen test, as above.   Depot Synacthen 1 mgis given at09.00hbyintramuscular  injection. Blood samples are then taken at baseline, 1, 2,  4, 8 and 24 h for plasma cortisol concentration.   Plasma cortisol values are usually between 600 nmol/L  and 1600 nmol/L.   The plasma cortisol concentrations should peak at  4-8 h. A delayed response peaking at 24 h or later is seen  in secondary adrenal insufficiency (ACTH deficiency or  corticosteroid treatment). Plasma cortisol <50 nmol/L  suggests primary adrenal failure.   There is a risk of adrenocortical hypofunction when  long-term corticosteroid treatment is stopped suddenly.  This may be due either to secondary adrenal atrophy  or to impaired ACTH release. Excess corticosteroid  therapy may cause side effects, including hypertension,  hyperglycaemia and osteoporosis.   It is probably best to give the replacement  hydrocortisone in three or more doses during the  day. Plasma cortisol day curves may also be useful  to help tailor the individual’s dose of replacement  glucocorticoid. Blood samples for cortisol can be  collected during the day, aiming for a 09.00 h cortisol  value of between 100 nmol/L and 700 nmol/L and in  the afternoon ideally more than 100 nmol/L.   Some patients require glucocorticoid therapy, for  example for acute episodes of asthma or inflammatory  conditions such as rheumatoid arthritis, but then need  their therapy withdrawn as their condition improves.  The main concern is that the hypothalamic-pituitary-  adrenal axis has been suppressed, thereby putting the  patient at risk of an adrenal crisis if treatment is stopped  too quickly. There are no universally agreed protocols  to investigate this, but the following may be useful.   If therapy is short term, that is, less than 3 weeks,  and the prednisolone dose equivalent is not higher  than 40 mg, the steroid can be stopped with little  likelihood of problems. In cases where a higher dose  of steroid equivalent has been used and for a longer  time period, particularly if the steroid has been given  in the evening or at night, gradual withdrawal is  important until 7.5mg/day (5-7. 5mg of prednisolone  is about an equivalent physiological glucocorticoid  dose) is reached. Afterwards, the steroid dose should  slowly be withdrawn at about 1 mg/ day per month.  Patients should be observed closely and made aware  that after withdrawal they are at a small risk of  adrenal insufficiency if they develop an infection  or undergo surgery, in which case steroid should be  resumed. If doubt persists once the steroids have been  totally withdrawn, a short Synacthen test may reveal  persistent adrenal insufficiency. Prednisolone lmg is  approximately equivalent to 4 mg hydrocortisone and  to 0.2 mg of dexamethasone.   The term congenital adrenal hyperplasia (CAH)  embraces various defects involving enzymes of cortisol  or aldosterone synthesis. Many of the enzymes involved  in cortisol and aldosterone pathways are cytochrome  p450 proteins designated CYP. CYP21 refers to  21-a-hydroxylase, CYP11B1 refers to ll-|3-hydroxylase  and CYP17 to 17-a-hydroxylase.   All forms of CAH are rare. An inherited deficiency  (usually autosomal recessive) of one of the enzymes  involved in the biosynthesis of cortisol, with a low plasma  concentration, causes a high rate of secretion of ACTH  from the anterior pituitary gland (Fig. 8.7). This results  in hyperplasia of the adrenal cortex, with increased  synthesis of cortisol precursors before the enzyme block.  The precursors may then be metabolized by alternative  pathways, such as those of androgen synthesis.   • female pseudohermaphroditism, by affecting the  development of the female genitalia in utero;  ambiguous genitalia may show phallic enlargement,  clitoromegaly and early pubic hair,   • virilization in childhood, with phallic enlargement  in either sex, development of pubic hair and a rapid  growth rate,   • milder virilization in females at or after puberty, with  amenorrhoea.   All female infants with ambiguous genitalia should  have plasma electrolytes estimated and evidence of  adrenocortical insufficiency looked for. In male infants  with no obvious physical abnormalities the diagnosis of  CAH may not be suspected.   The most common form of CAH is CYP2 1 deficiency,  which accounts for about 90 per cent of cases. Females  have ambiguous genitalia at birth (classic) or later  in adolescence (non-classic with milder enzyme  deficiency) and become virilized. Males with CYP21  deficiency are not usually diagnosed in the neonatal  period, as their genitalia are normal. However, if severe,  the infant may present with salt wasting with vomiting,  dehydration, failure to thrive and shock. Aldosterone  synthesis may be markedly reduced in more than half of  the infants with 21-a-hydroxylase deficiency and may  cause an Addisonian-like picture with marked renal   Figure 8.7 The abnormalities occurring in congenital adrenal hyperplasia. The substances highlighted are of diagnostic  importance; those shown in bold are increased in 21-hydoxylase deficiency. ACTH, adrenocorticotrophic hormone.   sodium loss during the first few weeks of life. Volume  depletion may be accompanied by hyponatraemia and  hyperkalaemia. Even if plasma sodium concentrations  are within the reference range, demonstrably increased  plasma renin activity may suggest lesser degrees of  sodium and water depletion.   CYP11B1 deficiency may also present with female  ambiguous genitalia and salt loss. The child may,  however, show hypertension and a hypokalaemic  alkalosis. The enzyme CYP 1 1 B 1 catalyses the conversion  of 11-deoxycortisol to cortisol in the glucocorticoid  pathway and the conversion of deoxycorticosterone  to corticosterone in the mineralocorticoid pathway.  CYP11B2 or aldosterone synthetase deficiency results  in hyponatraemia and hyperkalaemia, although normal  sexual differentiation occurs, as sex steroids are normal.   A 14-year-old woman with primary amenorrhoea  presented to the endocrine out-patient clinic. Physical  examination revealed hirsutism and clitoromegaly.  Some of her biochemistry tests were as follows:   Luteinizing hormone 5.2 U/L (1-25)  Follicle-stimulating hormone 4.3 U/L (1-15)  Testosterone 6.2nmol/L (1-3)   Sex-hormone-binding globulin 48nmol/L (20-90)  Oestradiol 464 pmol/L (70-880)  17-Hydroxyprogesterone 85nmol/L (<35)   The results are suggestive of congenital adrenal  hyperplasia. Note the raised plasma 17-hydroxypro-  gesterone and testosterone concentrations. The  patient was eventually found to have 2 1 -a-hydroxylase  deficiency, which was confirmed with a 24-h urinary  steroid profile and genetic tests. Raised adrenal  androgen concentrations help to explain her hirsutism  and clitoromegaly. Note also the hyponatraemia and  hyperkalaemia.   In CYP 17 deficiency, ambiguous genitalia or female  genitalia may be observed in male infants. A female  with CYP 17 deficiency appears phenotypically  female at birth but will fail to develop breasts or  menstruate due to inadequate oestradiol production.  Hypertension due to raised deoxycorticosterone  concentration may be present in CYP 17 deficiency  (Fig. 8.7).   Only the principles of the diagnosis of 2 1 -a-hydroxylase  deficiency are outlined here (see Fig. 8.7).   17-Hydroxyprogesterone can be metabolized  by the cortisol pathway only in the presence of  2 1 -a-hydroxylase, and plasma concentrations are raised  in patients with CAH. In some cases a short Synacthen  test may reveal elevated plasma 1 7 -hydroxyprogesterone  concentrations after stimulation. This test may be  useful, as sometimes cortisol precursors may be  within the normal range. Those with abnormalities of  21 -a-hydroxylase deficiency often showpost-Synacthen  17-hydroxyprogesterone values more than 35nmol/L.  However, those with either ll-|3-hydroxylase or other  enzyme deficiencies may show a normal response.   Plasma androstenedione concentrations may be  raised in those patients with excessive androgen  synthesis. Some indication of which enzyme is deficient  may be suggested by evaluating the pattern of steroid  excretion in a random or 24-h urine sample.   In ll-|3-hydroxylase deficiency there is a raised  concentration of 24-h urinary tetrahydrocortisol,  a metabolite of 11-deoxycortisol, and raised  deoxycorticosterone concentration. In the salt-losing  forms, plasma renin and aldosterone concentrations  may assist diagnosis.   If a diagnosis of CAH is confirmed, genetic  counselling may be necessary, with deoxyribonucleic  acid (DNA) and family studies.   Congenital adrenal hyperplasia is treated by giving  a glucocorticoid, for example hydrocortisone,  and, if necessary, a mineralocorticoid, for example  fludrocortisone. This treatment not only replaces the  deficient hormones but, by negative feedback, also  suppresses ACTH secretion and therefore androgen  production.   Measuring 17-hydroxyprogesterone and  androstenedione concentrations in plasma or saliva  enables treatment efficacy to be monitored. Salivary   steroid concentrations correlate well with those in  plasma, and saliva collection may be more acceptable  to some patients than repeated venepuncture.  Measuring the plasma renin activity may help assess  mineralocorticoid replacement.   Primary hyperaldosteronism (PH) is considered an  important cause of secondary hypertension in perhaps  as many as 5-15 per cent of cases, particularly in the face  of hypokalaemia and kaliuria (e.g. urinary potassium  more than 20mmol/L). (See Chapter 5 for a discussion  of hypokalaemia. )   The majority of cases of PH are due to adrenal  aldosterone-producing adenomas (APAs), which  produce 18-oxocortisol and 18-hydroxycortisol steroids  in excess, although 45 per cent of cases may be due to  bilateral idiopathic adrenal hyperplasia (IAH). There  is also a genetic-familial variety of PH. Type 1 familial  PH is glucocorticoid remediable aldosteronism (GRA),  in which the associated hypertension responds to small   A35-year-oldman attended the hospital hypertension  clinic (presenting blood pressure 184/100 mmHg)  with the following results (off antihypertensive  medication):   A random plasma aldosterone to renin activity ratio  was 984 (more than 750 is suggestive of Conn’s  syndrome).   The results suggest Conn’s syndrome (primary  hyperaldosteronism). Note the hypertension,  hypokalaemia and urinary potassium greater than  20 mmol/T. The patient also had a metabolic alkalosis.  Further investigations, including adrenal imaging,  showed an adrenal adenoma.   doses of glucocorticoids in addition to antihypertensives.  This condition is due to a chimeric gene product that  combines the promoter of the ll-|3-hydroxylase gene  with the coding region of the aldosterone synthetase  gene and is associated with haemorrhagic stroke.   The treatment of IAH is usually medical, and the  mineralocorticoid antagonist spironolactone has  proved useful, sometimes in conjunction with a thiazide  diuretic. The treatment of choice for unilateral variants  of PH such as APA is usually surgical by adrenalectomy.   Hypertension and a hypokalaemic metabolic alkalosis  in the face of kaliuria (urinary potassium more than  20 mmol/L) are suggestive of mineralocorticoid excess  such as Conn’s syndrome. A urinary potassium is thus a  useful screening test.   In PH, plasma aldosterone concentration will be  raised, with suppressed renin levels. Remember that, in  renal artery stenosis, Bartter’s or Gitelman’s syndromes,  renin-secreting tumours and pseudohypoaldosteron-  ism, both plasma aldosterone and renin concentrations  may be raised. Antihypertensive drugs may alter  renin and aldosterone levels but in some cases it may  not be safe to stop them, although, if there is severe  hypertension, a-adrenergic blockers may interfere less  than other antihypertensives.   A random plasma aldosterone to renin ratio of more  than 750, where aldosterone is expressed in pmol/L and  renin activity in pg/L per hour, is suggestive of PH and  may be used as a screening test. This ratio is increased  by |3-blockers and decreased by angiotensin-converting  enzyme (ACE) inhibitors, diuretics, calcium channel  blockers and angiotensin II receptor blockers. A raised  24-h urinary aldosterone excretion may also be a useful  screening tool.   In summary ACE inhibitors increase renin and reduce  aldosterone, (3-blockers reduce renin and aldosterone,  calcium channel blockers increase renin and reduce  aldosterone, and diuretics increase renin and aldosterone,  although a-blocking drugs may have minimal effect  upon renin and aldosterone concentrations.   Sometimes dynamic tests are needed to confirm the  diagnosis of PH, although in many cases the diagnosis  may be obvious. The basis of these additional tests is   that, in PH, aldosterone secretion is autonomous and  thus not suppressed by physiological or pharmaceutical  stimuli.   One such test is the captopril test (a single oral  dose of 25-50 mg), which normally suppresses plasma  aldosterone levels in healthy individuals after about 2 h,  but which does not do so in PH. Captopril is an ACE  inhibitor.   Alternatively, other dynamic tests that have been used  are saline infusion, or salt-loading, tests, including the  fludrocortisone suppression test. However, these tests  are not without risk, as blood pressure can rise during  them. Patients may need to be hospitalized during these  tests as they may evoke severe hypertension.   Patients are given intravenous 0.9 per cent isotonic  saline, infused over a 4-h period. They are usually in  the supine position. Plasma aldosterone is measured at  baseline and 4 h afterwards. In PH there is a failure to  suppress the plasma aldosterone.   Fludrocortisone suppression test  On day 1 the plasma aldosterone is measured. Then  fludrocortisone 0.1 mg is given every 6h for 4 days,  with three tablets of lOmmol/L Slow Sodium every  8h also for 4 days. Slow-K tablets are also given, to  maintain a normal potassium concentration during the  test period.   On day 4, plasma aldosterone is measured again.  Primary hyperaldosteronism is excluded if suppression  of plasma aldosterone occurs.   An investigation that is sometimes useful is the  postural test, which aims to distinguish between APAs  and IAH. With the former there is usually a fall in  plasma aldosterone on standing. Patients should be  normovolaemic and have a daily sodium intake of about  100 mmol. After they have been kept recumbent for at  least 8 h, blood is taken for plasma electrolyte, renin and  aldosterone assays. They then walk around for 30 min  and a further blood sample is taken for repeat plasma  renin activity and aldosterone assays.   The diagnosis can be made only if the plasma  aldosterone concentration is high and the renin activity  inappropriately low for the aldosterone concentration;  the latter does not increase significantly after 30 min  of walking around. Plasma aldosterone concentration  increases after ambulation in adrenal hyperplasia (as  angiotensin II responsive) and falls when there is an  adrenal adenoma.   Another biochemical assay that may help distinguish  adenomas from hyperplasia is based on the fact that  concentrations of plasma 18-hydroxy-corticosterone  are raised in adrenal adenomas compared with adrenal  hyperplasia. The rare renin-responsive aldostesterone-  producing adenoma, unlike APA, shows an increased  plasma aldosterone concentration on ambulation.   If familial type 1 PH (GRA) is suspected from  the family history, a dexamethasone suppression  test (1-2 mg/ day) may show improvement in blood  pressure as well as suppression of plasma aldosterone  concentration.   Diagnostic imaging of the adrenal glands by  MRI or CT scanning may demonstrate adrenal  adenoma or bilateral hyperplasia but gives no  functional information. However, remember that  about 10 per cent of normal individuals may have  false-positive results due to non-functioning adrenal  lesions. Adrenal scintigraphy using radiolabelled  iodomethyl-19 norcholesterol may also be useful  to locate the adrenal glands. Sometimes, measuring  aldosterone and renin concentrations in adrenal vein  sampling may help localize the adrenal pathology,  although this carries a risk of adrenal infarction.  Lateralized aldosterone overproduction is confirmed  if the aldosterone to cortisol ratio in one adrenal vein  when divided by that in the contralateral vein is more  than about 4.   Adrenal adenomas producing aldosterone are usually  surgically removed, sometimes by laparoscopic  adrenalectomy. Hyperplasia of the adrenal glands can  be treated with amiloride or spironolactone. Eplerenone  is a selective aldosterone receptor antagonist, which  may prove useful in preference to spironolactone. The  glucocorticoid-remediable form of aldosteronism may  respond to dexamethasone (Fig. 8.8).   This chapter discusses adrenal cortex steroid  biochemistry and abnormalities of adrenal cortical  function and how clinical biochemistry tests can be  used in their diagnosis and management. Disorders  of the adrenal medulla are discussed in Chapter 24.  The algorithms in Figures 8.5, 8.6 and 8.8 summarize  the investigation of Cushing’s syndrome, adrenal  insufficiency and hyperaldosteronism, respectively.   • The adrenal cortex produces three major groups of  hormones: glucocorticoids, mineralocorticoids and  androgens.   • Cortisol, a glucocorticoid, is a stress hormone  involved in metabolism. It is controlled by pituitary  adrenocorticotrophic hormone (ACTH).   • Aldosterone is a mineralocorticoid involved in  sodium retention and potassium excretion via the  kidneys. Aldosterone is controlled by the renin-  angiotensin axis.   adrenal cortex hormones and can be due to excess  ACTH from the pituitary (Cushing’s disease) or  ectopic ACTH release from certain tumours. Other  causes include adrenal tumours, hyperplasia and  excess glucocorticoid intake. Cushing’s syndrome  may result in hypertension, glucose intolerance,  hypokalaemia, weight increase and mood changes.   • Adrenal insufficiency is due to reduced adrenal  cortex hormone production; this can be due to  autoimmune disease or to adrenal gland infiltration.  Adrenal failure may be associated with weakness,  hypotension, hyponatraemia and hyperkalaemia  and is life threatening.   • Congenital adrenal hyperplasia, most commonly due to  2 1 -a.-hydroxylase deficiency, results in glucocorticoid  and mineralocorticoid underproduction but an  excess of adrenal androgens.   • Excess adrenal mineralocorticoid production can  result in hypertension, metabolic alkalosis and  hypokalaemia such as Conn’s syndrome.   The study of the endocrinology of the male and female  reproductive systems is a specialized field and overlaps  with urology, obstetrics and gynaecology. This chapter  looks at some of the more common conditions that  you are likely to encounter clinically and that have  particular relevance to clinical biochemistry. Pregnancy  and infertility are discussed in Chapter 10.   The reproductive system is responsible not only for the  production of hormones, but also for maturation of the  germ cells in the gonads. It is important to understand the  relationship between hormones with respect to these two  functions if the results of reproductive endocrinology  tests are to be interpreted correctly; see also Chapter 7.   The hypothalamus secretes: gonadotrophin-releasing  hormone (GnRH), which regulates the secretion of  the pituitary gonadotrophins, luteinizing hormone  (LH) and follicle-stimulating hormone (FSH); and  dopamine, a neurotransmitter, which also controls  prolactin secretion (see Chapter 7).   The gonadotrophins (LH and FSH), secreted by  pituitary basophil cells, control the function and  secretion of hormones by the testes and ovaries. The  secretion of GnRH is pulsatile and thus so, in turn, is  that of LH and FSH. Although there is only one releasing  hormone, secretion of LH and FSH does not always  occur in parallel and may be modified by feedback from  the circulating concentrations of gonadal androgens or  oestrogens. The actions of the gonadotrophins are:   • LH primarily stimulates the production of hormones  by the gonads,   Gonadotrophin-releasing hormone analogues, for  example goserelin, after an initial stimulation phase,  down-regulate gonadotrophin secretion and have  been used therapeutically for prostate carcinoma and  endometriosis.   Prolactin, secreted by acidophil cells, is important  during pregnancy and the post-partum period. It  stimulates breast epithelial cell proliferation and  induces milk production. Prolactin differs from all  other pituitary hormones in its method of control.  Secretion is inhibited, not stimulated, by dopamine  (prolactin inhibitory factor); therefore, impairment of  hypothalamic control causes hyperprolactinaemia. Its  secretion is regulated by a short feedback loop between  pituitary prolactin and hypothalamic dopamine via  dopamine-2-type receptors.   Oestrogens stimulate the proliferation of pituitary  lactotroph cells, although high oestrogen and  progesterone concentrations inhibit secretion, as in  pregnancy. Circulating prolactin concentrations are  normally higher in pregnancy and increase during  suckling as a result of the action of vasoactive intestinal  peptide and also high oestrogen concentrations in  pregnancy. Although thyrotrophin-releasing hormone  (TRH) stimulates the secretion of prolactin, as well  as of thyroid- stimulating hormone (TSH), this action  does not seem to be of physiological importance;  it may, however, be important in pathological  conditions. Similar factors affect prolactin and growth  hormone secretion. Secretion of both is pulsatile and  increases during sleep and in response to physical and  psychological stress.   Oestrogens, progesterone and androgens are secreted  by the ovarian follicles of the ovaries, which consist of  germ cells (ova) surrounded by granulosa and theca  cells. Androgens (C19 steroids), synthesized by theca   cells, are converted into oestrogens (Cl 8 steroids) in the  granulosa cells, a process that involves aromatization  of the A ring and the loss of the C-19 methyl group  (Fig. 9.1). Oestradiol is the most important ovarian  oestrogen. The liver and subcutaneous fat convert  ovarian and adrenal androgens to oestrone. Both  oestradiol and oestrone are metabolized to the relatively  inactive oestriol. Oestrogens are essential for the  development of female secondary sex characteristics  and for normal menstruation, and their concentration  in plasma in children is very low. Androstenedione is the  main androgen secreted by the ovaries. It is converted  to oestrone and to the more active testosterone in  extraovarian tissue. A small amount of testosterone is  secreted directly by the ovaries. Plasma concentrations  in women are about a tenth of those in men.   Progesterone is secreted by the corpus luteum  during the luteal phase of the menstrual cycle and by  the placenta. It prepares the endometrium of the uterus  to receive a fertilized ovum and is necessary for the  maintenance of early pregnancy. It also is pyrogenic and  increases the basal body temperature. In plasma, only  about 2 per cent of progesterone is unbound or free,  the majority being bound to albumin and transcortin.  Inhibin from granulosa cells inhibits FSH secretion  while activin enhances the action of FSH and LH.   Figure 9.1 Pathways of sex hormone synthesis. (In this  summary an arrow does not necessarily represent a  single reaction.) Reproduced with kind permission  from Candlish JK and Crook M. Notes on Clinical  Biochemistry. Singapore: World Scientific Publishing,  1993.   Testosterone is secreted by the Leydig cells, which  lie in the interstitial tissue of the testes between the  seminiferous tubules. The production of testosterone is  stimulated by LH and it, in turn, inhibits LH secretion  by negative feedback. Inhibin is a hormone produced  by the Sertoli cells, part of the basement membrane  of the seminiferous tubules, during germ cell  differentiation and spermatogenesis. These processes  require testosterone and are stimulated by FSH. Inhibin  controls FSH secretion by negative feedback (Fig. 9.2)  and activin enhances spermatogenesis. Testosterone  is involved in sexual differentiation, the development  of secondary sexual characteristics, spermatogenesis  and anabolism. In the male, the effects of testosterone  depend on intracellular conversion to the even more  potent androgen dihydrotestosterone by the enzyme  5-a-reductase in target cells (Fig. 9.3).   Luteinizing hormone stimulates testosterone  production from the Leydig cells. The Sertoli cells are  involvedin germ cell differentiation and spermatogenesis.  These functions depend on testosterone and are  stimulated by FSH.    Figure 9.2 The effect of the gonadotrophins luteinizing  hormone (LH) and follicle-stimulating hormone (FSH)  on testicular function. GnRH, gonadotrophin-releasing  hormone.   Figure 9.3 The relationship between the biological  actions of testosterone and di hydrotestosterone.   Testosterone and, to a lesser extent, oestradiol circulate  bound to a carrier protein, sex-hormone-binding  globulin (SHBG), as well as to albumin. As with other  hormones, only the free or unbound fraction (about  3 per cent of the total hormone concentration) is  metabolically active. Plasma SHBG levels in females  are about twice those in males. Changes in SHBG  concentrations change the ratio of free testosterone to  free oestrogen (see Table 9.1).   This is an important cause of amenorrhoea, sexual  dysfunction, osteoporosis, infertility and possibly  breast cancer. High plasma prolactin concentrations  inhibit the normal pulsatile release of GnRH and  inhibit gonadal steroid hormone synthesis directly.  Plasma gonadotrophin and oestrogen concentrations  are therefore low, and the symptoms of oestrogen  deficiency may occur. About a third of patients with  hyperprolactinaemia have galactorrhoea.   The finding of hyperprolactinaemia should be  interpreted with caution. As mentioned above, plasma  prolactin concentration is raised during pregnancy and  lactation. Plasma prolactin concentrations are higher  in females than in males and decrease post menopause.  Samples for prolactin estimations should be taken  at least 2-3 h after waking in order to eliminate the  misleading elevated plasma concentrations found  during sleep; the stress of venepuncture may also  cause prolactin secretion. A sustained increase of  more than about 700mU/L should be investigated.  Macroprolactinaemia, in which the raised plasma  prolactin concentration is due to a complex with  immunoglobulins, should be excluded, usually by being  precipitated in the plasma sample by polyethylene   A 34-year-old woman was seen in the endocrine clinic  because of galactorrhoea and oligomenorrhoea. She  was not taking any medication, and her renal function,  liver function and blood glucose were normal. Her  plasma biochemical results were as follows:   Luteinizing hormone 7.2 U/L (1-25)  Follicle-stimulating hormone 4.4 U/L (1-15)  Testosterone 2.2nmol/L (1-3)   The patient has hyperprolactinaemia, with a common  presentation. A pituitary magnetic resonance  imaging scan was suggestive of a microprolactinoma  (microadenoma). The other pituitary hormones are  normal, presumably because the microprolactinoma  had not encroached on the pituitary gland.  Dopamine receptor agonists such as bromocriptine  or cabergoline have been used to lower prolactin  concentration under these circumstances.   Hypothyroidism, polycystic ovary syndrome, chronic  kidney disease and certain drugs, e.g. antipsychotics,  opioids, estrogens, H 2 receptor antagonists and  antidepressants, can also evoke hyperprolactinaemia.  Antipsychotic drugs, such as chlorpromazine,  haloperidol, clozapine, olanzapine and aripiprazole  generally block dopamine receptors (D 2 ), although  the last may be associated with a low rate of  hyperprolactinaemia. Prolactin plasma concentrations  persistently greater than 1000 mU/L may need a change  in antipsychotic medication or a reduction in the dose;  concentrations over 2000 mU/L probably merit an  endocrinologist’s opinion to exclude a prolactinoma.   The pathological causes of hyperprolactinaemia  include a prolactin-secreting tumour of the pituitary  gland. If a pituitary tumour is found, it is usually  either a microadenoma (< 10 mm) or a macroadenoma  (>10mm). The higher the plasma prolactin  concentration, the greater the likelihood that a tumour  is present, with concentrations more than 2000 mU/L   suggestive of hypothalamic or microadenoma, whereas  concentrations more than 6000 mU/L are more likely  to indicate a macroadenoma. The latter are sometimes  associated with multiple endocrine neoplasia (MEN 1)  syndrome (see Chapter 24).   If hyperprolactinaemia is confirmed, pregnancy  must be excluded, and also renal impairment, relevant  drugs and hypothyroidism. Pituitary imaging with  computerized tomography or magnetic resonance  imaging may show a tumour. Dopamine receptor  agonists such as bromocriptine or cabergoline are used  to lower prolactin concentrations. Sometimes pituitary  surgery is needed to remove a pituitary tumour.   The causes of hyperprolactinaemia are shown in  Box 9.1 and a diagnostic algorithm in Figure 9.4.   The complex series of events leading to the development  of sexual competence depends on many steps occurring  at the correct time. The following simplified account  aims to provide a background for the discussion of  abnormalities of the system.   Damage to the pituitary stalk by non-prolactin-  secreting tumours of the pituitary gland or  hypothalamus   Surgical section of the pituitary stalk  Other pituitary tumours  Microadenomas  Macroadenomas  Drugs  Estrogens   Severe primary hypothyroidism (due to anterior  pituitary stimulation by high thyrotrophin-releasing  hormone concentrations).   Chromosomal sex is determined at fertilization by  the chromosomes present in the ovum and sperm,  each of which contributes 22 autosomes and one sex  chromosome, X or Y. Normal males have a 46, XY  karyotype, and normal females 46, XX. Abnormalities    Figure 9.4 An algorithm for the investigation of  hyperprolactinaemia. MRI, magnetic resonance  imaging.   occurring at this stage may result in defective gonadal  development, as occurs in Klinefelter’s syndrome  in males (47,XXY) or Turner’s syndrome in females  (45, XO).   The sex chromosomes determine whether the  primitive gonads become testes or ovaries. The  development and disorders of gonadal function in the  male and female are considered separately below.   Development of female characteristics  In the absence of a Y chromosome, the fetus starts to  develop female characteristics at about 12 weeks of  gestation. If androgens are produced at this stage, as,  for example, in congenital adrenal hyperplasia (CAH),  masculinization of the external genitalia may occur,  causing female pseudohermaphroditism (see Chapter  8). Proliferation of fetal germ cells produces several  million oocytes. By late fetal life, all the germ cells have  degenerated and no more oocytes can be produced.  Those oocytes that are present enter the first stage of  meiosis and their numbers decline throughout the  rest of the intrauterine period and childhood; the  inability to replenish them explains the limit to the  span of reproductive life in women, in contrast to  the continuous ability of men to produce sperm. An  abnormally high rate of decline leads to premature  menopause.   At the onset of puberty, gonadotrophin secretion  increases, as it does in the male. Ovarian oestrogen  secretion rises and stimulates the development of  female secondary sex characteristics and the onset of  menstruation (menarche).   At puberty the ovaries contain between 100000 and  200000 primordial follicles. During each menstrual  cycle a small number develop, but usually only one  reaches maturation, which is extruded from the ovary  as an ovum (ovulation), and is ready for fertilization.  The menstrual cycle is regulated by changing hormone  concentrations (Fig. 9.5) and by changing sensitivity of  ovarian tissue.   At the beginning of the menstrual cycle, ovarian follicles  are undeveloped and plasma oestradiol concentrations  are low. The secretion of LH and FSH increases    Figure 9.5 An example of plasma hormone  concentrations during the menstrual cycle. FSH,  follicle-stimulating hormone; LH, luteinizing hormone.   because of diminished negative feedback by oestrogens.  Together, LH and FSH cause the growth of a group of  follicles. By about the seventh day of the cycle, one follicle  becomes especially sensitive to FSH and matures, while  the rest atrophy. Luteinizing hormone also stimulates  oestradiol secretion, the plasma concentrations of  which rise steadily. This stimulates the regeneration  of the endometrium.   The rapid development of the dominant follicle and  the rise in plasma oestradiol concentration trigger a  surge of LH release from the anterior pituitary gland  by positive feedback. Ovulation occurs about 16 h later.  Combined oral contraceptives suppress gonadotrophin  plasma concentrations.   Luteal (post-ovulatory or secretory) phase  After ovulation, the high LH concentration stimulates  the granulosa cells of the ruptured follicle to luteinize  and form the corpus luteum, which synthesizes and   secretes progesterone and oestradiol. Progesterone  is the principal hormone of the luteal phase and  prepares the endometrium for the implantation of  the fertilized ovum. If the ovum is not fertilized, the  corpus luteum regresses and plasma ovarian hormone  concentrations fall; the menstrual cycle takes its  course, with sloughing of the endometrium and  menstrual bleeding.   As the plasma ovarian hormone concentrations fall,  the concentrations of LH and FSH in plasma begin to  rise and the cycle recommences. If fertilization does  occur, pregnancy may supervene (see Chapter 10).   Interpretation ofplasmasexhormoneconcentrations  must be made in relation to the stage of the cycle.  It may be important to establish whether a patient  who is complaining of infertility has ovulated, either  spontaneously or as a result of treatment to induce  ovulation. The plasma progesterone concentration  should be measured in a blood sample taken during  the second half of the menstrual cycle; usually day 21.  A value within the reference range for the time of the  cycle is good presumptive evidence of ovulation (see  Fig. 9.5), whereas a value in the range expected in  the follicular phase indicates the absence of a corpus  luteum, and therefore of ovulation. Ovulation may  also be detected by ultrasound examination of the  ovaries. Progesterone secretion is associated with a rise  in body temperature, which may be monitored serially  to determine the time of ovulation. Plasma prolactin  concentrations do not significantly change cyclically  during the menstrual cycle.   The menopause is defined as the time of permanent  cessation of menstruation, the average age being  about 50 years. The menopause occurs when all the  follicles have atrophied. Plasma concentrations of  oestrogens fall and those of FSH and, to a lesser extent,  LH increase after removal of the negative feedback  to the pituitary. These findings are therefore similar  to those of primary gonadal failure. Diagnosis of the  menopause is a clinical one, although a plasma FSH  consistently greater than 20-40 IU/L is suggestive of  ovarian failure but not a guarantee of sterility, and  thus suitable contraception is necessary for 1 year of  amenorrhoea in patients over 50 years and for 2 years  in those under 50 years.   It may sometimes be useful to measure plasma  oestradiol concentrations in women with estradiol  hormone replacement therapy (HRT) implants, as very   high levels may be associated with tachyphylaxis, that  is, tolerance to dose. There may also be a place for its  measurement in women on transdermal estradiol HRT  to ensure adequate absorption. Otherwise, measuring  plasma oestradiol is not generally useful, as the results  depend upon the type of estrogen used in the HRT  preparation and how this may cause assay cross-  reaction.   Gonadal dysfunction in women usually presents with  any or all of the following:   Amenorrhoea is defined as the absence of menstruation;  it may be due to hormonal abnormalities. If  there is ovarian failure, pituitary gonadotrophin  concentrations in plasma are high (hypergonadotrophic  hypogonadism); if the cause is in the hypothalamus  or anterior pituitary gland, gonadotrophin secretion  is reduced (hypogonadotrophic hypogonadism).  Amenorrhoea may be classified as either primary or  secondary (Box 9.2).   Primary amenorrhoea occurs when the patient has  never menstruated and is most commonly associated  with delayed puberty. The age of the menarche is very  variable. Extensive investigation should probably be  postponed until around the age of 16, unless there are  other clinical features of either endocrine disturbances,  such as hirsutism and virilism, or chromosomal  abnormalities. Turner’s syndrome (45, XO) and  testicular feminization syndrome may present with  primary amenorrhoea.   Secondary amenorrhoea occurs when previously  established menstrual cycles have stopped and is  most commonly due to physiological factors such as  pregnancy or the menopause. Other causes include  severe illness, excess or rapid weight loss for any  reason, including anorexia nervosa, or stopping oral  contraceptives. These should be considered before  extensive and potentially dangerous investigations  are started. A number of endocrine disorders, such  as hyperprolactinaemia, hyperthyroidism, Cushing’s  syndrome and acromegaly, may present with  amenorrhoea.   Hypogonadotrophic hypogonadism [low FSH/LH and  low testosterone (males) or oestrogen (females)]   GnRH receptor mutations  Isolated LH or FSH deficiency  PROP1 gene mutations that lead to absence of  some pituitary hormones  Secondary causes   Chronic systemic disease and undernutrition  Exercise-induced amenorrhoea (females)  Hyperprolactinaemia  Miscellaneous causes  Morbid obesity  Marijuana or opiate use  Prader-Willi syndrome  Laurence-Moon (Bardet— Biedl) syndrome   LH or FSH receptor mutations  Primary testicular failure, e.g. chemotherapy,  mumps orchitis, trauma, radiation, autoimmune,  LH resistance, anorchism/cryptorchidism,  testicular biosynthetic defects  FSH, follicle-stimulatinghormone; GnRH, gonadotrophin-  releasing hormone; LH, luteinizing hormone   Increased plasma free androgen concentrations, or  increased tissue sensitivity to androgens, produce effects  ranging from increased hair growth (hirsutism) to  marked masculinization, with virilism. Testosterone is  the most important androgen. In typical women about  half the plasma testosterone comes from the ovaries,  both by direct secretion and by peripheral conversion   Table 9.1 Conditions associated with altered sex-  hormone-binding globulin (SHBG) concentration   of androstenedione. The rest is derived from peripheral  conversion of adrenal androgens, androstenedione  and dehydroepiandrosterone (DHEA). Because of the  extensive interconversion of androgens, the source of  a slightly raised plasma testosterone concentration  may be difficult to establish. In general, markedly  raised plasma concentrations of DHEA or its sulphate  (DHEAS) indicate an adrenocortical origin.   The biological activity of testosterone depends on the  plasma free hormone concentration. The plasma total  concentration is also influenced by the concentration  of the binding protein SHBG. Some laboratories report  a free testosterone concentration or free androgen  index. Conditions associated with altered plasma  concentrations of SHBG are shown in Table 9.1.   Hirsutism is defined as an excessive growth of hair in  a male distribution and is common, possibly occurring  in 10 per cent of women. The Ferriman and Gallway  score can assess its severity. A common cause is familial  or racial hirsutism. For example, some Mediterranean  women have more terminal hair and fair- skinned  Europeans have the least. This difference may be due  to racial differences in 5-a-reductase activity in skin.  Plasma testosterone concentrations may be slightly  raised, but are often within the female reference range.  A raised testosterone concentration, particularly above  5nmol/L, may indicate a tumour of the adrenal or  ovary, which must be excluded. However, the plasma  concentration of free hormone may be significantly  increased if that of SHBG is low. A raised plasma  17-hydroxyprogesterone concentration may indicate  CAH (see Chapter 8). Some causes of hirsutism are  shown in Box 9.3.   Virilism is characterized by additional evidence  of excessive androgen secretion such as an enlarged  clitoris (clitoromegaly), increased hair growth of male   Congenital adrenal hyperplasia, e.g. 21-a-hydroxylase,  ll-(3-hydroxylase  Ovarian tumours  Arrhenoblastomas  Gonadoblastomas  Adrenal tumours  Adenomas  Carcinomas   distribution, receding temporal hair, deepening of  the voice and breast atrophy. It is always associated  with increased plasma androgen concentrations.  Plasma DHEA and DHEAS concentrations may also  be increased. Virilism usually implies an adrenal or  ovarian androgen source and can be a cause of female  pseudohermaphroditism (Box 9.4).   This is a condition showing features of hyperandro-  genism with anovulation and abnormal ovarian  morphology and is the most common cause of  anovulatory infertility. Presenting clinical symptoms  may also include hirsutism, menstrual disturbances,  enlarged polycystic ovaries and infertility. Plasma  testosterone and androstenedione concentrations are  often increased. The plasma LH may be elevated with  normal FSH. Because plasma SEIBG concentrations are  reduced in obese individuals, the plasma concentration  of free testosterone is often increased. The plasma  prolactin concentrations may also be high. Multiple   Ovarian tumours (such as arrhenoblastomas and  hilus cell tumours) that secrete androgens, mainly  testosterone   A 28-year-old woman attended the endocrine clinic  because of excessive facial hair. Her periods were  irregular and she was obese, with a body mass index  of 32.8 kg/m 2 . She was not taking any medication,  and her renal function, liver function and blood  glucose were all normal. Her plasma biochemical  endocrine results were as follows:   Luteinizing hormone (LH) 27.2 U/L (1-25)  Follicle-stimulating hormone 6.4 U/L (1-15)  Testosterone 2.7nmol/L (1-3)   The clinical features and results are suggestive of  polycystic ovary syndrome with raised plasma LH  and low plasma SHBG concentrations. Obesity is  associated with low SHBG concentrations. Polycystic  ovary syndrome was confirmedby vaginal ultrasound  scan of the ovaries, which showed large cysts.  The SHBG concentrations are reduced in obesity,  allowing increased free testosterone concentrations.   small subcapsular ovarian cysts may be demonstrated  on ultrasound scanning of the ovaries.   Polycystic ovary syndrome is also associated with  insulin resistance, obesity and elevated plasma insulin  concentrations, which may stimulate androgen  production from the ovarian theca interna cells.  Individuals may also have hyperlipidaemia, glucose  intolerance and hypertension.   Development of male characteristics  In the presence of the Y chromosome the fetal gonads  develop into testes at about 7 weeks’ gestation. The  Sertoli cells secrete anti-Miillerian hormone that  inhibits the Mullerian ducts in the male embryo.  The intracellular conversion of testosterone to  dihydrotestosterone by the enzyme 5-a-reductase  is essential for the development of male external  genitalia. If this enzyme is deficient, varying   degrees of feminization may occur, causing male  pseudohermaphroditism (see later). Male penis  formation is dependent upon testosterone.   During childhood, the rate of secretion of  gonadotrophins from the anterior pituitary gland  is low. As puberty approaches, the pulse amplitude  and frequency of LH secretion increase. Initially, this  occurs during sleep, but later continues throughout  the day. Leydig cell function and testosterone secretion  increase and stimulate the development of secondary  male characteristics. Gonadotrophin secretion also  stimulates meiosis of previously dormant germ cells in  the seminiferous tubules, and thus the production of  sperm. Deficient secretion of either pituitary or gonadal  hormones may cause delayed puberty.   The male germ cells produce spermatozoa  continuously after puberty. Spermatogenesis is  dependent on both normal Sertoli cell function and  testosterone secretion by Leydig cells. Therefore both  LH and FSH are needed for normal spermatogenesis,  but testosterone secretion can occur in the absence of  normal seminiferous tubules.   Gonadal dysfunction in men may present with the  symptoms of androgen deficiency or of infertility, or  both. Androgen deficiency is the result of impaired  testosterone secretion by Leydig cells. The patient  may present with delayed puberty or with regression  of previously established male characteristics that  are dependent on testosterone (hair distribution,  potency and libido). There may be primary testicular  dysfunction, in which case the low plasma testosterone  concentration is accompanied by raised plasma LH  concentrations (hypergonado trophic hypogonadism).  Dysfunction secondary to pituitary or hypothalamic  disease conversely results in low plasma LH  concentrations (hypogonadotrophic hypogonadism)  (see Box 9.2).   Infertility may be caused by androgen deficiency,  but most infertile men have normal plasma androgen  concentrations. Sertoli cell function is dependent  on both FSH and testosterone produced locally.  Semen analysis is an important investigation for male  infertility. However, biochemical tests may sometimes  help: if the cause is primary testicular failure, the  plasma testosterone concentration is low, and reduced  inhibin production by Sertoli cells causes a rise in   A 23-year-old man presented to his general  practitioner with erectile dysfunction. He was not  taking any medications, and his renal function, liver  function and blood glucose were normal. Some of  his plasma biochemical results were as follows:   Luteinizing hormone (LH) 27.2U/L (1-7)  Follicle-stimulating hormone (FSH) 41.4 U/L (1-8)  Testosterone 2.7nmol/L (10-30)   The diagnosis is hypergonadotrophic hypogonadism  - note the high plasma gonadotrophin (LH and  FSH) and low testosterone concentrations. Further  investigations, including karyotyping, showed  XXY chromosome complement (as opposed to the  normal XY male pattern), confirming Klinefelter’s  syndrome.   FSH concentrations. If there is evidence of a failure of  spermatogenesis, the plasma FSH concentration should  be measured. If the cause is secondary to anterior  pituitary failure, both plasma testosterone and FSH  concentrations are low.   Hyperprolactinaemia is much less common in  males than in females, but its presence may indicate a  pituitary tumour. Chromosomal abnormalities such  as Klinefelter’s syndrome (47, XXY) may also cause  abnormal male gonadal function.   This is the enlargement of male breast tissue (glandular  and not adipose tissue), which can be unilateral or  bilateral. Important in the aetiology is an increase in  the oestrogen to androgen ratio, which can be seen  physiologically at puberty or in the elderly. Certain  drugs, such as digoxin, spironolactone, phenothiazines  and cimetidine, may be implicated. However, other  causes include hyperthyroidism, human chorionic  gonadotrophin (hCG) -secreting tumours and  hyperprolactinaemia, as well as increased oestrogen  concentration (such as in liver disease and testicular  or adrenal tumours), low testosterone concentration  (in pituitary or gonadal failure) and Kallmann’s or  Klinefelter’s syndrome.   This can be defined as the appearance of secondary  sexual characteristics before the age of 8 years in either  females or males. It can be:   • true (central) precocious puberty caused by cerebral  tumours, infection or trauma, McCune-Albright  syndrome or hypothyroidism,   Ambiguous genitalia and intersexuality  During the fetus’s second month, its indifferent  gonad is stimulated to develop into a testis, initiated  by testis-determining factor derived from the  sex-determining region of the Y chromosome. If  this region is absent or abnormal, the indifferent  gonad develops into an ovary. In the gonadal  male, differentiation to male phenotype is  mediated by testosterone, which is converted to  dihydrotestosterone by 5-a-reductase.   Individuals with this condition usually show XX  chromosomes but male genitalia characteristics  and virilism. More commonly, it can be caused by  high concentrations of maternal androgens during  pregnancy or increased concentrations of fetal  androgens, such as in CAH, including deficiency of  21 -hydroxylase or 11 -hydroxylase, which result in  increased endogenous testosterone production (see  Chapter 8).   Individuals with this condition usually have  chromosomes XY and possess two testes but female  external genitalia. Causes include CAH such as  17-oc-hydroxylase deficiency or 17-|3-hydroxysteroid  dehydrogenase deficiency. Other causes are deficient  testosterone biosynthesis and androgen receptor  defects such as Reifenstein’s syndrome, testicular  feminization or 5-oc-reductase deficiency. The last  condition shows a high plasma testosterone to  dihydrotestosterone ratio.   This is very rare, with individuals having both testicular  and ovarian tissue.   In suspected cases, a careful clinical history, including  medications, and physical examination can be  useful. Measurement of plasma LH, FSH, TSH,  prolactin, oestradiol, testosterone and SHBG may  reveal a diagnosis. If intersex conditions are sought,  karyotyping maybe useful. (Chapters 7 and 8 should be  consulted if a pituitary or adrenal disorder, respectively,  is suspected.) Sometimes the more specialized tests  described below are necessary.   Gonadotrophin-releasing hormone (a decapeptide)  released by the hypothalamus stimulates the release  of the gonadotrophins LH and FSH from the normal  anterior pituitary gland. The test is used to diagnose  hypothalamic-pituitary disease in precocious and  delayed puberty with low basal gonadotrophin  concentrations.   Plasma LH and FSH concentrations are measured  in blood drawn before and at 20 and 60 min after the  injection.   Some patients show an allergic reaction, and therefore  resuscitation facilities should be at hand and the test  should be performed by experienced staff. Sometimes  nausea and abdominal pain are experienced.   The test can sometimes be combined with the TRH  and insulin or glucagon stimulation tests as part of  the triple pituitary test, although this is now rarely  performed (see Chapter 7).   In prepubertal children, plasma LH and FSH are  usually both less than 2.0 U/L. In normal subjects,  the levels of plasma LH and FSH at least double from  their basal levels at 20 min, but this rise fails to occur  in patients with pituitary hypofunction. Conversely,  an exaggerated response may be seen in patients with  hypothalamic disease. A normal response does not  exclude hypothalamic or pituitary disease.   Human chorionic gonadotrophin shares a common  subunit with LH and stimulates testicular Leydig cells  to release androgens. It has a long half-life and elicits a  rise in plasma testosterone after 72-120 h.   Some patients show an allergic reaction, and  therefore resuscitation facilities should be at hand and  experienced staff should perform the test.   On day 0, blood is taken for testosterone. Then  2000 hCG units are given on days 0 and 2 by  intramuscular injection.   On day 4, blood is again taken for testosterone,  androstenedione and dihydrotestosterone.   Normally there is at least a two-fold increase in  testosterone concentration, but in the absence of testes  there is no testosterone response.   • The gonads secrete sex hormones, although there is  also some adrenal gland production. In females, the  main sex hormone is the oestrogen 17-(3-oestradiol,  and in males testosterone.   • The anterior pituitary gonadotrophins FSH and  LH stimulate the gonads.   • Prolactin is released from the anterior pituitary  gland. Hyperprolactinaemia has many causes and  can result in infertility and galactorrhoea.   Plasma SHBG carries testosterone and oestrogen,  the free forms of which are biologically active.  Gonadal dysfunction in females usually presents with  any or all of the following: amenorrhoea, hirsutism,  virilism, infertility and ambiguous genitalia.   Gonadal dysfunction in males may present with  delayed puberty, decreased libido, gynaecomastia,  infertility or ambiguous genitalia.   This chapter discusses the biochemical changes that  occur in pregnancy and how clinical biochemistry tests  can be used in the investigation of infertility.   If the ovum is fertilized, it may implant in the  endometrium, which has been prepared by progesterone  during the luteal phase. The function of luteinizing  hormone (LH) is taken over by human chorionic  gonadotrophin (hCG), produced by the chorion and  developing placenta. Human chorionic gonadotrophin  is similar in structure and action to TH and prevents the  involution of the corpus luteum as circulating pituitary  gonadotrophin concentrations fall. Consequently, plasma  oestrogen and progesterone concentrations continue to  rise and endometrial sloughing is prevented. Progesterone  is initially produced by the corpus luteum of the ovary for  the first 8 weeks of pregnancy, then from implantation of  the embryo the placenta takes over progesterone synthesis.  During pregnancy the predominant oestrogen is oestriol  produced by the placenta. Prolactin concentration  gradually increases during the first two trimesters and then  rises steeply, to about 8000 mU/L, in the third trimester.   Prolactin, oestrogens, progesterone and (human)  placental lactogen stimulate breast development in  preparation for lactation. High plasma oestrogen  concentrations inhibit milk secretion; lactation can  start only when plasma concentrations fall after delivery  of the placenta. Initially lactation depends on prolactin.  Suckling stimulates secretion of the hormone, but, even  during lactation, plasma prolactin concentrations fall  progressively post partum and reach non-pregnant  levels after 2 or 3 months. Apart from the effects on  the breast, the high plasma concentration of prolactin  interferes with gonadotrophin and ovarian function  and produces a period of relative infertility.   Monitoring pregnancy (placental function)  Substances produced by the fetus or placenta  (fetoplacental unit) may be measured in maternal   plasma or urine to detect fetal abnormalities or to  monitor the progress of the pregnancy, for example  low urine unconjugated oestriol (E3) is associated  with poor pregnancy outcome. Such sampling is  relatively safe and simple, but occasionally more  invasive testing, such as of amniotic fluid obtained by  amniocentesis, may be needed. However, the ability to  visualize the fetus using ultrasound (Fig. 10.1) and the  use of cardiotocography for detecting fetal heart rate  have reduced the need for such tests.   The secretion of hCG by the placenta reaches a peak  (rising to about 500000U/L) at about 13 weeks of  pregnancy and then falls. The fetoplacental unit then  takes over hormone production, and the secretion of  both oestrogen and progesterone rises rapidly. Plasma  or urinary hCG concentrations, which give positive  results at 1 or 2 weeks after the first missed menstrual  period, are most commonly used to confirm pregnancy.   Figure 10.1 Ultrasound of 12 week pregnancy. Crown-  rump length measurement can be taken and also  nuchal translucency. Reproduced with kind permission  from Baker P. Obstetrics by Ten Teachers, 18th  edition. London: Hodder Arnold, 2006.   However, by using more sensitive immunoassay  techniques, plasma hCG may be detected soon after  implantation of the ovum and before the first missed  period.   Measurement of plasma hCG is also useful if an  ectopic pregnancy is suspected, in conjunction with  ultrasonography, or if the patient is being treated for  infertility. Serial hCG measurements may be used to  assess the progress of early pregnancy; single values are  difficult to interpret because of the wide reference range.  As a rough guide, plasma concentrations should double  every 2 days in a normal pregnancy. Raised plasma hCG  not due to pregnancy can be due to gestational or non-  gestational trophoblastic neoplasia or the menopause.   This is a peptide hormone synthesized by the placenta.  It is detectable in maternal plasma after about the  eighth week of gestation and has been used to assess the  likelihood of threatened miscarriage and to monitor  late pregnancy, but now is rarely used.   Some fetal abnormalities may be diagnosed by tests  carried out on maternal plasma or amniotic fluid.  Amniocentesis is a procedure by which amniotic  fluid is obtained through a needle inserted through  the maternal abdominal wall into the uterus and is  usually carried out after about 14 weeks’ gestation.  The procedure carries a small risk to the fetus. Both  the safety and the reliability of the procedure can be  improved if combined with ultrasound examination in   A 20-year-old woman attended the antenatal  clinic in the 17th week of pregnancy for a Down’s  syndrome screen test. A plasma a-fetoprotein (AFP)  was 67 kU/L (reference median being 38kU/L for 17  weeks). Her other blood tests were normal.   It was feared that the fetus had a neural tube defect  because of the raised plasma AFP concentration.  However, an ultrasound scan showed a twin  pregnancy and no evidence of a neural tube defect.  Other causes of a raised plasma AFP concentration  include incorrect gestational dating, fetal renal  disease, duodenal/oesophageal atresia and ventral  wall defects or exomphalos.   order to locate the position of the fetus, placenta and  maternal bladder.   Analytical results may be misleading if, for example,  the specimen is contaminated with maternal or fetal  blood or maternal urine, is not fresh or is not properly  preserved. Close liaison between the clinician and the  laboratory staff helps to ensure the suitability of the  specimen and the speed of the assay. Amniotic fluid is  probably derived from both maternal and fetal sources,  but its value in reflecting abnormalities arises from its  intimate contact with the fetus and from the increasing  contribution of fetal urine in later pregnancy.   a- Fetoprotein (AFP) is a low-molecular-weight  glycoprotein synthesized mainly in the fetal yolk sac  and liver. Its production is almost completely repressed  in the normal adult. It can diffuse slowly through  capillary membranes and appears in the fetal urine, and  hence in the amniotic fluid, and in maternal plasma.  Severe fetal neural tube defects, such as open spina  bifida and anencephaly, are associated with abnormally  high concentrations in these fluids. The reason for this  is not clear, but the protein may leak from the exposed  neural tube vessels.   As well as neural tube defects, the causes of raised  AFP concentration in amniotic fluid and maternal  plasma include:   In some countries, pregnant women attending for  antenatal care are offered plasma AFP assay at between  16 and 18 weeks’ gestation to screen for the presence  of a fetus with a neural tube defect (although high-  resolution ultrasound is beginning to replace this test).  The gestational age should be confirmed by ultrasound,  which should also exclude a multiple pregnancy as a  cause of high concentrations.   Positive results should be confirmed on a fresh  sample, which, if still high and if the diagnosis has not  been confirmed by ultrasound, may be followed by AFP  estimation on amniotic fluid. This is a more precise  diagnostic test and yields fewer false-positive results than  plasma assays if sampling is properly performed. It should  be reserved for subjects known to be at risk, either because  of a family history of neural tube defects or because of the  finding of a high concentration in maternal plasma with a  normal or equivocal ultrasound scan.   Amniotic fluid acetylcholinesterase assay is now  rarely used to detect certain fetal malformations,  including neural tube defects and exomphalos. It gives  reliable results up to about 23 weeks’ gestation. The  interpretation of the result is less dependent on fetal age  than AFP, but is equally invalidated by contamination  with fetal or maternal blood. The assay is less widely  available than that for AFP.   Low maternal plasma AFP and unconjugated oestriol,  and raised hCG and inhibin A concentrations, measured  between 15 and 20 weeks’ gestation, are associated with  an increased risk of the fetus having Down’s syndrome  (trisomy 21) and used as a second trimester screening  test. This combination of tests (quadruple or Quad  test) to screen for the congenital disorder is available  in specialized laboratories. Fetal nuchal translucency  determined by high-resolution ultrasound, along with  raised pre-|3-hCG and reduced pregnancy-associated  plasma protein A (PAPP-A), are used as a first trimester  ( 1 0-14 weeks’ gestation) screening test; increased nuchal  thickness (Fig. 10.1) is associated with chromosomal  abnormalities. A definitive test is amniocentesis, which  allows the collection of fetal cells for karyotyping. The  risk of Down’s syndrome increases with maternal age.   Chromosomal abnormalities and some inborn  errors of metabolism may be detected by cytogenetic,  biochemical or enzymatic assays on cells cultured  from amniotic fluid or after biopsy of chorionic villi.  These tests are performed only in special centres and  usually on individuals with a family history of a genetic  condition. Circulating fetal deoxyribonucleic acid  (DNA) in maternal blood may also prove to be useful.   Rhesus or other blood group incompatibility has  effects on the fetus, which may be assessed by  measuring amniotic fluid concentrations of bilirubin  in conjunction with maternal antibody titres. Normally  bilirubin concentrations in amniotic fluid decrease  during the last half of pregnancy. The concentrations at  any stage correlate with the severity of haemolysis. The  result is read off a Liley chart relating optical density  of amniotic fluid at 450 nm (an indirect measure of  bilirubin) against gestational time. Such tests may allow  the optimum time for induction of labour or the need  for intrauterine transfusion to be assessed.   The examination of amniotic fluid has also been used to  assess pulmonarymaturity. Immature lungs do not expand  normally at birth and may cause neonatal respiratory  distress syndrome (hyaline membrane disease), with the  need for respiratory support. It is therefore important to  have evidence of pulmonary maturity before labour is  induced. At about 32 weeks’ gestation, the cells lining the  fetal alveolar walls start to synthesize a surface-tension-  lowering complex (surfactant), 90 per cent of which is  the phospholipid lecithin, which contains palmitic acid.  Surfactant is probably washed from, or secreted by, the  alveolar walls into the surrounding amniotic fluid, in  which both lecithin and palmitic acid concentrations  steadily increase. The concentration of lecithin, relative  to another lipid, sphingomyelin, which remains constant  in amniotic fluid, can be measured. A rise in the lecithin  to sphingomyelin ratio may help determine pulmonary  maturity, and a ratio less than 2 implies immaturity. This  invasive test is rarely indicated now as steroids, which  induce surfactant synthesis, are sometimes given to  patients who have premature rupture of the membranes.   Weight gain of about 12 kg occurs due to increased  maternal fluid retention, increased maternal fat stores  and also the products of conception, including the  fetus, placenta and amniotic fluid.   Many plasma constituents are influenced by sex  hormones. For example, the reference ranges of plasma  urate and iron differ in males and females after puberty.  Therefore it is not surprising to find that during  pregnancy the very high circulating concentrations of  oestrogens and progesterone alter the concentrations of  many substances in plasma (Table 10.1).   The plasma concentrations of many specific carrier  proteins increase during pregnancy, accompanied by a  proportional increase of the substance bound to them,  without any change in the unbound free fraction.  Because the protein-bound fraction is a transport form  and because, in all cases, it is the free substance that is  physiologically active, this rise in concentration is of  importance in the interpretation of the results of such  assays as those of plasma thyroxine.   Other changes in maternal plasma are due to  progressive haemodilution by fluid retained during  pregnancy. This is maximal at about the thirtieth  week and the effects are most evident in reduced  concentrations of albumin, and of calcium, which is  bound to albumin. These changes are more marked in    A 35-year-old woman was 23 weeks pregnant and  was noted to have glycosuria. She had the following  biochemical results:   The results are typical of pregnancy; note the  low plasma urea and creatinine and albumin  concentrations, in part due to increased glomerular  filtration rate and plasma volume. Plasma alkaline  phosphatase concentration is elevated as a result of  placental isoenzyme release. Glycosuria was present  owing to a lowered renal threshold for glucose that  occurs during pregnancy and not necessarily as a  result of diabetes mellitus.   pre-eclamptic toxaemia, in which fluid retention may  be greater than normal. The glomerular filtration rate  (GFR) increases and creatinine clearance can be over  140mL/min by about 28 weeks. There is a reduced  renal threshold for glucose and increased excretion of  urate and some amino acids.   There is a mild increase in ventilation rate. Oxygen  consumption is increased, but Po 2 remains fairly  constant despite a small decrease in Pco 2 . There is  increased fasting glucose utilization, and therefore  fasting glucose concentration is lower.   Renal glycosuria is common in pregnancy and  sometimes in individuals taking oral contraceptives.  The GFR increases by about 50 per cent during  pregnancy, resulting in a reduced plasma creatinine.  Glycosuria may partly be due to an increased glucose  load in normal tubules. For gestational diabetes, see  Chapter 12. The positive protein and purine balance  during the growth of the fetus and the increase in GFR  that occurs during pregnancy result in lowered maternal  plasma urea and urate concentrations. Plasma alkaline  phosphatase activity rises in some women during the  last 3 months of pregnancy due to the presence of the  placental isoenzyme and should not be misinterpreted   “These changes may also occur in women taking oestrogen-  containing oral contraceptives.   FSH, follicle-stimulating hormone; GFR, glomerular filtration rate;  LH, luteinizing hormone; T 4 , thyroxine; TBG, thyroxine-binding  globulin; TIBC, total iron-binding capacity.   (see Chapter 18). Placental alkaline phosphatase does  not cross the placenta and therefore it is not present in  the plasma of the newborn infant.   During delivery, blood gases and lactate can be measured  in fetal blood to monitor for hypoxia. Capillary blood  samples may be collected from the fetal scalp during  delivery. Transcutaneous oxygen electrodes can  determine fetal Po r   Pregnancy-induced hypertension or pre-eclampsia is  associated with convulsions (as occurs in eclampsia),  oedema, impaired renal function, proteinuria and  maternal death as well as with placental insufficiency  and intrauterine fetal growth retardation.   Pre-eclampsia is defined as a blood pressure taken  on two occasions, at least 6h apart, of 140/90 mmHg  or greater (after 20 weeks’ gestation) in a woman  with previously normal blood pressure and who has  proteinuria (defined as >300 mg protein in 24-h urine  collection).   Plasma urate may rise in pre-eclampsia and is  predictive of maternal complications.   Trophoblastic tumours (hydatidiform mole,  choriocarcinoma), which may follow abnormal  pregnancy or a miscarriage, and some teratomas  secrete hCG, which can be estimated in plasma or  urine by sensitive tests allowing early detection and  treatment of recurrence. However, these tests will not  necessarily differentiate pregnancy or retained products  of conception from recurrence of a tumour, because  plasma hCG concentrations rise in both situations (see  Chapter 24). In monitoring trophoblastic tumours  it is important to monitor plasma hCG down to  undetectable levels.   Infertility can be defined as primary when conception  has never occurred despite at least 1 year of unprotected  coitus, and secondary when there has been a previous  pregnancy, either successful or not. Investigation earlier  than at 1 year maybe appropriate if the woman is more  than 35 years old or where pregnancy is associated with  other risks.   In cases of infertility, both partners should be  investigated. The history should include coital  frequency and success, serious illnesses, use of alcohol  and drugs, and sexually transmitted diseases.   Examination should include looking for anorexia  nervosa, hirsutism, virilism, galactorrhoea and  ambiguous genitalia. A history should also be taken for  medications and drugs (see Chapter 9).   A woman may be infertile despite having a clinically  normal menstrual cycle (about 95 per cent of such  cycles are ovulatory). Thus, even if the cycle seems to be  regular, it is important to determine whether ovulation  is occurring and if luteal development is normal.  Anovulatory infertility is probably the most common  form of female infertility and is associated with  oligomenorrhoea or amenorrhoea. (Discussion of the  complete investigation of female infertility, including  tests such as that for tubal patency, is beyond the scope  of this book.)   • If the patient is menstruating regularly, measure  plasma progesterone concentration during the  luteal phase on day 21 of the cycle. A normal  plasma concentration is strong evidence that the  patient has ovulated. A low plasma concentration  of <30nmol/L suggests either ovulatory failure or   A 28-year-old woman attended the gynaecology out-  patient clinic because of infertility. Her periods were  noted to be irregular. She was on no medication, and  her renal function, liver function and blood glucose  concentration were normal. Her plasma results were  as follows:   Luteinizing hormone 7.2 U/L (1-25)  Follicle-stimulating hormone 5.4 U/L (1-15)  Testosterone 1.7nmol/L (1-3)   The low 21 -day plasma progesterone concentration  suggests a poor luteal phase and possible anovulation.  The patient was having anovulatory menstrual cycles,  which explained her infertility.   impaired luteal function. This investigation should  be repeated on more than one occasion. The most  common cause of a low progesterone concentration  (< 30nmol/L) is inaccurate sample timing, although,  if authentic, it suggests lack of ovulation. However,  a plasma progesterone concentration of more than  lOOnmol/T suggests pregnancy.   • Follicular development and ovulation may be  monitored by ovarian ultrasound examination.  Polycystic ovary syndrome should be excluded  (see Chapter 9). Plasma follicle-stimulating  hormone (FSH), LH, oestrogen and testosterone  concentrations are useful, as is the exclusion of  thyroid disease.   • If there is primary amenorrhoea, consider  karyotyping the patient, for example Turner’s  syndrome (45, XO).   • Sometimes histological examination of an  endometrial biopsy specimen or the appearance of  cervical mucus can indicate whether luteal function  is normal. An oral progestogen challenge can be  used: a withdrawal bleed 5-7 days later implies  adequate endometrial oestrogen, whereas failure to  bleed despite oestrogen treatment implies uterine  disease.   • Hyperprolactinaemia should be excluded by  checking the plasma prolactin concentration (see  Chapter 9).   • Do the plasma FSH, TH and oestrogen results  suggest hypergonadotrophic hypogonadism or  hypogonadotrophic hypogonadism? In the presence  of amenorrhoea, a plasma FSH of more than 40U/L  is suggestive of ovarian failure. (See Chapter 9 for the  causes of hypergonadotrophic hypogonadism and  hypogonadotrophic hypogonadism.)   • Low concentrations of plasma gonadotrophins may  necessitate a gonadotrophin-releasing hormone  (GnRH) test to look for pituitary or hypothalamic  disease (see Chapter 9).   • Anti-Miillerian hormone (AMH) is released by  granulosa cells of the ovarian follicle and low serum  concentrations suggest poor ovarian ‘reserve’ (the  size of the ovarian ovum supply). Serum AMH  may, thus, have a place in the investigation of  infertility.   • Semen analysis: the volume should be at least 2mL.  There should be more than 20 x 10 9 /L spermatozoa,  more than 50 per cent being motile at 4h post  ejaculation and more than 30 per cent normal  morphology. A post-coital test is useful so that  cervical mucus can be examined for the presence  of spermatozoa and their activity. Sperm antibodies  may exist.   - Raised plasma FSH and LH concentrations  with a low testosterone concentration  (hypergonadotrophic hypogonadism) indicate  a testicular problem such as Leydig cell failure.  Low plasma FSH and LH and testosterone  concentrations suggest pituitary or hypothalamic  disease (hypogonadotrophic hypogonadism).  In the case of the latter, a GnRH test may be  required (see Chapter 9).   - A raised plasma FSH concentration in  comparison with LH may indicate seminiferous  tubular failure, irrespective of the plasma  testosterone concentration. There is usually  azoospermia or oligospermia. Oligospermia  with a low plasma FSH concentration suggests  pituitary or hypothalamic disease.   • If there is evidence of feminization, karyotype  should be considered. This should also be considered  if azoospermia is present, for example Klinefelter’s  syndrome (47,XXY).   • Plasma prolactin should be measured and  hyperprolactinaemia and thyroid disease excluded  (see Chapter 9).   • Defects of the male reproductive tract may also be  found and necessitate a urology opinion, but are  beyond the scope of this book.   • An hCG stimulation test may be indicated if absence  of testes is suspected or to assess Leydig cell reserve  (see Chapter 9).   • Some tumours can release |3-hCG or oestrogens and  may induce features of feminization. These can be  assayed in plasma if the cause of infertility is unclear.   Sometimes, despite both partners being investigated,  no cause for the infertility can be found. Some couples  decide to try in vitro fertilization (Fig. 10.2).   The combined oral contraceptive pills contain synthetic  estrogens and progestogens and suppress pituitary  gonadotrophin secretion and thus inhibit ovulation.  Withdrawal mimics involution of the corpus luteum and  results in menstrual bleeding. Progesterone or progestin  only (mini) pills may inhibit ovulation and also thicken  cervical mucus, thereby inhibiting sperm penetration.   Clomiphene blocks oestrogen receptors in the  hypothalamus and so prevents negative feedback. It may  stimulate gonadotrophin release, even when circulating  oestrogen concentrations are high. Clomiphene may be  used to induce ovulation in patients with amenorrhoea  or infertility. Gonadotrophin treatment may be used  if clomiphene fails to induce ovulation. This therapy  may cause dangerous follicular enlargement due to  hyperstimulation, or may stimulate many follicles  and so cause multiple pregnancy. Treatment must  therefore be monitored either by frequent plasma  oestradiol estimations (which would be expected to  be very high) or by ovarian ultrasound examination.  Ovulation may be assessed by demonstrating rising  plasma progesterone concentrations or by ultrasound.  Gonadotrophins may also be used to stimulate the  production of enough oocytes to enable them to be  ‘harvested’ for in vitro fertilization (Fig 10.2) or gamete  intrafallopian transfer.   Gonadotrophin-releasing hormone treatment  can be used for patients with infertility secondary  to hypogonadotrophic hypogonadism. It is given  subcutaneously in pulses, such as every 90 min, using  a portable syringe pump. Bromocriptine may reduce  high plasma prolactin concentrations, after which  menstruation may resume and fertility may be restored.   • A number of physiological changes may take  place during pregnancy that may affect laboratory  tests. In some cases this is due to increased  concentrations of hormone or transport proteins,  although the free hormone concentrations are  normal.   • Increased urinary (or plasma) hCG concentration  can be used to confirm pregnancy.   Biochemical tests can be used to monitor the  progression of pregnancy as well as to detect fetal  abnormality.   Infertility may be primary or secondary and can  be the result of problems with the man or woman,  or both. Biochemical tests can also be useful in the  diagnosis of infertility in conjunction with other  investigations.   It is important to understand thyroid biochemistry,  as it helps to interpret thyroid function tests, which  are among the most common endocrine requests in  clinical practice. Thyroid disease is relatively common,  and therefore is likely to be encountered clinically.   Briefly, thyroxine (T ), tri-iodo thyronine (T ) and  calcitonin are secreted by the thyroid gland. Both T 4 and  T 3 are products of the follicular cells and influence the  rate of all metabolic processes. Calcitonin is produced  by the specialized C cells and influences calcium  metabolism (see Chapter 6).   Thyroid hormones are synthesized in the thyroid gland  by the iodination and coupling of two molecules of the  amino acid tyrosine, a process that is dependent on an  adequate supply of iodide. Iodide in the diet is absorbed  rapidly from the small intestine. In areas where the  iodide content of the soil is very low, there used to be  a high incidence of enlargement of the thyroid gland  (goitre), but the general use of artificially iodized salt  has made this a less common occurrence. Seafoods  generally have a high iodide content. Therefore fish  and iodized salt are the main dietary sources of the  element. Normally about a third of dietary iodide is  taken up by the thyroid gland and the rest is renally  excreted.   Synthesis of thyroid hormones  Iodide is actively taken up by the thyroid gland under  the control of thyroid- stimulating hormone (TSH)  via a sodium/iodide symporter. Uptake is blocked by  thiocyanate and perchlorate. The concentration of  iodide in the gland is at least 20 times that in plasma  and may exceed it by 100 times or more.   Iodide is rapidly converted to iodine within the  thyroid gland, catalysed by thyroid peroxidase (TPO).  Iodination of tyrosine residues in a large 660-kDa  glycoprotein, thyroglobulin, takes place to form  mono-iodotyrosine (MIT) and di-iodotyrosine (DIT)   mediated by the enzyme TPO. This step is inhibited by  carbimazole and propylthiouracil.   Iodotyrosines are coupled to form T 4 (DIT and DIT)  and T ? (DIT and MIT) (Fig. 11.1), which are stored  in the lumen of the thyroid follicular cells. Normally  much more T 4 than T is synthesized, but, if there is  an inadequate supply of iodide, the ratio of T 3 to T 4  in the gland increases. The thyroid hormones, still  incorporated in thyroglobulin, are stored in the colloid  of the thyroid follicle.   Prior to the secretion of thyroid hormones,  thyroglobulin is taken up by the follicular cells, by a  process involving endocytosis and then phagocytosis,  and T 4 and T 3 are released by proteolytic enzymes  into the bloodstream. This process is stimulated by  TSH and inhibited by iodide. The thyroid hormones  are immediately bound to plasma proteins. Mono-  iodotyrosine and DIT, released at the same time, are  de-iodinated and the iodine is reused.   Each step is controlled by specific enzymes, and  congenital deficiency of any of these enzymes can lead  to goitre and, if severe, hypothyroidism. The uptake of  iodide, as well as the synthesis and secretion of thyroid  hormones, is regulated by TSH, secreted from the  anterior pituitary gland. About 10 times more T 4 than  T is formed, with most of the latter being formed by  de-iodination in the liver, kidneys and muscle.   Most of the plasma T 4 and T is protein bound, mainly  (70 per cent) to an a-globulin, thyroxine-binding  globulin (TBG), and, to a lesser extent (15 per cent),  transthyretin (previously called pre-albumin), with  about 10-15 per cent bound to albumin. In keeping with  many other hormones, the free unbound fraction is the  physiologically active form, which also regulates TSH  secretion from the anterior pituitary. Modern laboratory  assays tend to measure the free hormones. Changes in the  plasma concentrations of the binding proteins, particularly  TBG, alter plasma total T 4 and T 3 concentrations, but not  the concentrations of free hormones.   Some of the circulating T 4 is de-iodinated by enzymes  in peripheral tissues, especially in the liver and kidneys.  About 80 per cent of the plasma T 3 is produced by the  removal of an iodine atom from the outer ((3) ring; the  remaining 20 per cent is secreted by the thyroid gland.  De-iodination of the inner (a) ring produces reverse  T , which is probably inactive. The T binds more avidly  to thyroid receptors than T 4 and is the main active form.  The conversion of T 4 to T 3 may be:   • reduced by many factors, of which the most  important are:   - drugs such as (3-blockers, for example propranolol  or amiodarone (200 mg of this anti- arrhythmic  drug contains about 75 mg of iodine);   • increased by drugs that induce hepatic enzyme  activity, such as phenytoin.   The plasma T concentration is therefore a poor  indicator of thyroid hormone secretion because it  is influenced by many non-thyroidal factors and its  measurement is rarely indicated, except if thyrotoxicosis  is suspected.   Action of thyroid hormones  Thyroid hormones affect many metabolic processes,  increasing oxygen consumption. They bind to specific  receptors in cell nuclei and change the expression of  certain genes. Thyroid hormones are essential for normal  growth, mental development and sexual maturation  and also increase the sensitivity of the cardiovascular  and central nervous systems to catecholamines, thereby  influencing cardiac output and heart rate.   Thyroid-stimulating hormone stimulates the synthesis  and release of thyroid hormones from the thyroid  gland. Its secretion from the anterior pituitary gland is  controlled by thyrotrophin-releasing hormone (TRH)  and circulating concentrations of thyroid hormones.   Effect of thyrotrophin-releasing hormone  Pituitary TSH synthesis and release are stimulated by  TRH, a tripeptide produced in the hypothalamus and  released into the portal capillary plexus. The action  of TRH can be over-ridden by high circulating free T 4  (fT 4 ) concentrations, and therefore exogenous TRH has  little effect on TSH secretion in hyperthyroidism (see  later for TRH test). Once TRH reaches the pituitary,  it binds to TRH receptors, members of the seven-  transmembrane-spanning receptor family, which are  coupled to G proteins.   Effects of thyroid hormones in the control of thyroid-  stimulating hormone secretion   Thyroid hormones reduce TSH secretion by negative  feedback. Tri-iodothyronine binds to anterior pituitary  nuclear receptors. In the anterior pituitary gland, most  of the intracellular T 3 is derived from circulating fT 4 .  Therefore this gland is more sensitive to changes in  plasma T 4 than to T concentrations.   The secretion and control of thyroid hormones is  summarized in Figure 11.2.   Assessment of thyroid hormone secretion can be made  by measuring plasma TSH as well as either fT 4 or total T 4  [sometimes also free T 3 (fT 3 ) or total TJ. Each test has  its advantages and disadvantages, although probably  most laboratories now offer fT and fT assays rather  than total hormone concentrations. Thus, regarding  assays this chapter will mainly discuss free hormones.   Plasma thyroid-stimulating hormone  Concentrations of TSH are high in primary  hypothyroidism and low in secondary or pituitary  hypothyroidism. In hyperthyroidism, high plasma T 4  and T concentrations suppress TSH release from the  pituitary, resulting in very low or undetectable plasma  TSH concentrations. Plasma TSH assays are used as  first-line assays for thyroid function assessment. New-  generation assays have high sensitivity and have a  detection limit for plasma TSH of less than 0.1 mU/L.    Figure 11.2 Secretion and control of thyroid hormones.  Solid lines indicate secretion and interconversion of  hormones; dotted lines indicate negative feedback.   T 4 , thyroxine; T 3 , tri-iodothyronine; TRH, thyrotrophin-  releasing hormone; TSH, thyroid-stimulating hormone.   In normal individuals there is a log-linear relationship  between plasma fT and TSH concentrations; that is to  say, exponential increases in TSH concentration occur  with small incremental changes in fT concentration.   Plasma T 4 is more than 99 per cent protein bound;  therefore, plasma total T 4 assays reflect the protein-  bound rather than the free hormone fraction. Total  T, reflects fT, concentrations, unless there are  abnormalities of binding proteins.   • In the euthyroid state, about a third of the binding  sites on TBG are occupied by T 4 and the remainder  are unoccupied, irrespective of the concentration of  the binding protein.   • In hyperthyroidism, both plasma total and fT  concentrations are increased and the number of  unoccupied binding sites on TBG is decreased.   An increase in plasma TBG concentration causes  an increase in both bound T 4 and unoccupied binding  sites but no change in plasma fT concentrations. Such  an increase may occur because of:   • a high oestrogen concentration during pregnancy  or in the newborn infant,   • oestrogen therapy, for example certain oral  contraceptives or hormone replacement therapy,   A decrease in plasma TBG concentration decreases  both bound T 4 concentrations and unoccupied binding  sites, but does not alter the plasma fT concentration.  Such changes may occur because of:   • loss of low-molecular-weight proteins, usually in  the urine, for example nephrotic syndrome,   These changes might be misinterpreted as being  diagnostic of hyperthyroidism or hypothyroidism  respectively if only plasma total T 4 was assayed and it is  for this reason that fT concentrations are now generally  preferred.   Some drugs, such as salicylates and danazol, bind to  TBG and displace T . The change in unoccupied binding  sites is variable and TBG concentrations are unaffected.  Measurement of plasma TBG concentrations may  occasionally be indicated to confirm either congenital  TBG excess or deficiency.   Plasma total or free tri-iodothyronine  Total T 3 or fT, concentrations may help in the  diagnosis of hyperthyroidism but are not usually  used routinely to diagnose hypothyroidism because  normal plasma concentrations are very low. In  hyperthyroidism, the increase in plasma T, or fT  concentrations is greater, and usually occurs earlier  than that of T, or fT,.   Occasionally in hyperthyroidism the plasma T or  fT, concentrations are elevated but not those of T , or  fT (T toxicosis). Like T , T, is bound to protein. It is  usually preferable to measure the plasma concentration  of fT rather than total T , as the latter may be altered by  changes in the plasma concentrations of TBG.   Thyrotrophin-releasing hormone test  The TRH test is used to confirm the diagnosis of  secondary hypothyroidism, or occasionally to diagnose  early primary hypothyroidism. Since the development  of sensitive TSH assays, it is rarely used to diagnose  hyperthyroidism, although it may have a place in the  differential diagnosis of thyroid resistance syndrome  or TSH-secreting pituitary tumours (TSHomas).   It is sometimes used as part of the combined pituitary  stimulation test (see Chapter 7).   Allergic reactions may occur, and therefore  resuscitation facilities should be available and the test  should be carried out by experienced staff.   • 200 pg of TRH is injected intravenously over about  a minute.   • Further blood samples are taken 20 and 60 min after  the TRH injection, and TSH is measured in all samples.   Note that certain drugs, such as dopamine agonists  and glucocorticoids, reduce the response, and oestrogens,  metoclopramide and theophylline enhance it.   In normal subjects, plasma TSH concentration increases  at 20 min by at least 2 mU/L and exceeds the upper limit  of the reference range, with a small decline at 6 min.   • An exaggerated response at 20 min and a slight fall  at 60 min are suggestive of primary hypothyroidism.   • A normal or exaggerated increment but delayed  response, with plasma TSH concentrations higher  at 60 min than at 2 min, suggests secondary  hypothyroidism. If clinically indicated, pituitary and  hypothalamic function should be investigated.   • A flat response of TSH of less than 5mU/L is  compatible with primary hyperthyroidism, although  this may also occur in some euthyroid patients with  multinodular goitre.   Drugs may alter plasma T 4 and T. concentrations.  The more common effects are summarized in Table  11.1. If the primary change is in binding protein  concentrations, plasma free hormone concentrations  are usually normal.   Interference of assays by immunoglobulins  Anti-T 4 or anti-T, immunoglobulins or heterophilic  antibodies (induced by external antigens, e.g. derived  from other species that cross-react with self-antigens)  can cause a spurious elevation of T 4 or T 3 (or free  hormones) , respectively, when assayed by immunoassay.  This needs to be remembered when interpreting thyroid  function test results.   The most common presenting clinical features of  thyroid disease are the result of:   • goitre, either diffuse or due to one or more nodules  within the gland - there may or may not be abnormal  thyroid hormone secretion and thus the patient may  be euthyroid.   Hypothyroidism is caused by suboptimal circulating  concentrations of thyroid hormones. It becomes more   T 4 , thyroxine; T 3 , tri-iodothyronine; fT 4 , free thyroxine; fT 3 , free tri-iodothyronine; TBG, thyroxine-binding globulin.   A 57-year-old woman consulted her general  practitioner because of weight gain, constipation  and weakness. The following thyroid function test  results were returned:   The results show primary hypothyroidism with  high plasma TSH and low fT concentrations. The  symptoms are typical of hypothyroidism. The  patient was also shown to have positive thyroid  antibodies (anti-thyroid peroxidase). The thyroid  function tests normalized on treatment with 100 pg  a day of thyroxine.   prevalent with age, affecting about 6 per cent of people  over 60 years, and is more common in women.   The condition may develop insidiously and in its  early stages may cause only vague symptoms. There is a  generalized slowing down of metabolism, with lethargy,  bradycardia, depression and weakness.   If the hormone deficiency is caused by a primary  disorder of the thyroid gland, the patient may present  with weight gain, myopathy, menstrual disturbances,  such as menorrhagia, and constipation. The skin  may be dry, the hair may fall out and the voice may  be hoarse. Subcutaneous tissues are thickened; this  pseudo-oedema, with a histological myxoid appearance,  accounts for the term myxoedema, which is sometimes  used to describe advanced hypothyroidism. In severe  cases, coma with profound hypothermia may develop.   The following laboratory changes may be associated  with hypothyroidism, particularly if severe:   • Plasma cholesterol concentration. In hypothyroidism  the clearance of plasma low-density lipoprotein  (LDL) cholesterol is impaired and plasma cholesterol  concentrations may be moderately high.   • Plasma creatine kinase activity is often raised in  hypothyroidism, due to possible myopathy.   • Hyponatraemia may very rarely present in patients  with profound hypothyroidism or myxoedema  coma. It is caused by increased antidiuretic hormone  release with excessive water retention, occasionally   • A macrocytic anaemia may be observed, with raised  mean corpuscular volume (MCV).   • In severe hypothyroidism a reduced estimated  glomerular filtration rate may occur probably due to  impaired renal perfusion.   The most common cause of hypothyroidism  worldwide is iodine deficiency. In areas of adequate  iodine intake, acquired hypothyroidism is mainly due  to autoimmune thyroiditis or Hashimoto’s thyroiditis,  which is more frequently seen in women and the elderly.  About 90 per cent of patients have positive thyroid  antibodies, for example anti-thyroid peroxidase (anti-  TPO), anti-thyroglobulin (anti-Tg) or TSH receptor-  blocking antibodies. There may also be a goitre.  Hypothyroidism may also be associated with other  autoimmune diseases such as type 1 diabetes mellitus,  adrenal insufficiency and pernicious anaemia.   Rare causes of primary hypothyroidism are  exogenous goitrogens (substances that interfere with  thyroid iodine uptake and thus can result in a goitre)  and dyshormonogenesis, a term that includes inherited  deficiencies of any of the enzymes involved in thyroid  hormone synthesis, which may present in childhood.  Although the biochemical and clinical features differ, the  end result is hypothyroidism. In most cases, prolonged  TSH stimulation, due to reduced negative feedback,  causes goitre. The most common form is due to failure  to incorporate iodine into tyrosine. The perchlorate  discharge test may be useful to diagnose iodination and  trapping defects, although it is rarely used.   Secondary hypothyroidism is due to low  concentrations of TSH from the anterior pituitary  or to hypothalamic TRH deficiency; this is much  less common than primary hypothyroidism. In  long-standing secondary hypothyroidism, the  thyroid gland may atrophy irreversibly. The essential  biochemical difference between primary and secondary  hypothyroidism is in the plasma TSH concentration,  which is high in the former and inappropriately low in  the latter   selenoenzyme catalyses the conversion of T 4 to reverse  tri-iodothyronine and the conversion of T to 3,3' -di-  iodothyronine, both of which are biologically inactive.   As primary hypothyroidism develops, TSH secretion  from the anterior pituitary gland increases as the  negative feedback (associated with the falling plasma  T 4 or fT concentration) decreases. Plasma T or fT 3  concentrations may be normal and thus not usually  useful in making the diagnosis.   Generally in primary hypothyroidism the plasma  TSH concentration is high, but it is low in secondary  hypothyroidism due to pituitary or hypothalamic  disease. Initially, the plasma T 4 or fT concentration  maybe within the population reference range, although  abnormally low for the individual. For this reason the  plasma TSH concentration is the most sensitive index  of early hypothyroidism. If the patient is very ill,  investigations should be deferred (see ‘Sick euthyroid’  below).   Pregnancy and neonatal hypothyroidism  In about 0.3 per cent of pregnancies the mother has  Hashimoto’s disease. Thyroid hormones are essential  for fetal development, hence the importance of treating  the mother with thyroxine.   Routine screening for congenital hypothyroidism  (Fig. 11.3) is discussed in Chapters 26 and 27.   This is usually with T , which can be titrated until the  plasma TSH is within the reference range. However,  this has recently been challenged, as plasma TSH  concentrations may not adequately reflect tissue  hypothyroidism, and it may be better to be guided by  plasma fT concentrations and clinical features. On rare  occasions, such as in hypothyroid comas, T, is given  instead, as its action is more immediate. The response  to T 4 therapy can be checked every 2-3 months until  the patient is stable, after which 6- to 12-month blood  checks may be useful.   Thyroxine should be used with caution in patients  with ischaemic heart disease for fear of worsening angina  pectoris, and low doses initially plus [3-blockers may be  indicated. Thyroid- stimulating hormone assays are of  little value in monitoring secondary hypothyroidism;  fT is better. Thyroxine therapy may precipitate an  Addisonian crisis in patients with concomitant adrenal  insufficiency. Overtreatment with T 4 can evoke atrial    Figure 11.3 Congenital hypothyroidism. The head is broad,  the eyes wide apart, the tongue protrudes from the mouth  and all movements and responses are slow and sluggish.  Reproduced with kind permission from Browse NL,   Black J, Burnand KG and Thomas WEG (eds). Browse’s  Introduction to the Symptoms and Signs of Surgical  Disease, 4th edition. London: Hodder Arnold, 2005.   fibrillation and osteoporosis; in such cases, plasma TSH  concentrations are often low or suppressed.   If a patient is non-compliant with treatment and only  takes T 4 near to the time of thyroid function testing, a  high plasma TSH may be observed with high plasma  fT, concentrations. This is because there is insufficient  T 4 to normalize plasma TSH, and yet the high plasma  fT reflects the recent taking of T .   Subclinical (compensated hypothyroidism) is the state  in which plasma TSH concentration is raised but the  total or fT, concentration still falls within the reference  range. In individuals over the age of 60 years, the  prevalence maybe as high as 10 per cent. Some of these  patients have positive thyroid antibodies, for example  anti-TPO or anti-Tg, and each year about 2-5 per cent  of thyroid antibody-positive patients go on to develop  hypothyroidism.   Some patients may be asymptomatic, whereas  others have symptoms suggestive of hypothyroidism.  Thyroxine therapy may be indicated particularly in  pregnancy, when the patient is symptomatic, or with  positive thyroid antibodies and plasma TSH more than  lOmU/L. It can be associated with increased risk of  cardiovascular disease.   In generalized thyroid hormone resistance, the plasma  total T, and fT, concentrations are elevated, with  normal or slightly raised TSH concentration. Some  patients appear euthyroid, but others may present with  hypothyroid symptoms, and the defect maybe inherited  as an autosomal dominant trait in some patients. The  defect is thought to be due to a defect in T 4 and/or T .  receptors and may be associated with other end-organ  resistance states.   Laboratory investigation of suspected hypothyroidism  A careful history (including drugs) should be taken and  an examination performed, checking for a goitre.   • The plasma TSH and total T 4 or fT concentrations  should be measured.   • Slightly elevated plasma TSH and normal fT  concentrations suggest compensatedhypothyroidism.  Measuring circulating thyroid antibodies may be  useful, that is, anti-TPO. Tests should be repeated  after 3-6 months as some patients may develop full-  blown hypothyroidism.   • Raised plasma TSH and low fT concentrations  suggest primary hypothyroidism. The thyroid  antibodies should be measured and, if positive, other  autoimmune diseases excluded.   • Low plasma TSH and low fT concentrations may  indicate that the hypothyroidism is caused by a  hypothalamic or pituitary disorder. A TRH test   A 49-year-old woman was investigated in the medical  out-patient department for tiredness. The following  test results were returned:   These results are suggestive of compensated  hypothyroidism, in which the plasma TSH  concentration is raised and the fT, concentration  still remains within the reference range. The patient  also had positive thyroid antibodies (anti-thyroid  peroxidase). A trial of thyroxine of 50 pg a day  brought her plasma TSH concentration to within the  reference range and improved her symptoms.   should be done, if indicated, and the pituitary gland  assessed (see Chapter 7).   • Raised plasma TSH and raised/normal plasma  fT concentrations in the presence of hypothyroid  symptoms may indicate thyroid hormone resistance.   Hyperthyroidism (thyrotoxicosis)  Hyperthyroidism causes sustained high plasma  concentrations of T 4 and T . There is often generalized  increase in the metabolic rate, evidenced clinically by,  for example, heat intolerance, a fine tremor, tachycardia  including atrial fibrillation, weight loss, tiredness,  anxiety, sweating and diarrhoea.   • Hypercalcaemia is occasionally found in patients  with severe thyrotoxicosis. There is an increased  turnover of bone cells, probably due to a direct  action of thyroid hormone.   Transient subacute thyroiditis (de Quervain’s)  Following treatment of hyperthyroidism  Post thyroidectomy  Post radioiodine treatment  External irradiation to neck region  Surgery or trauma to neck  Defects of thyroid hormone synthesis  Congenital absence of thyroid gland  Infiltrative disease of the thyroid, e.g. sarcoid,  haemochromatosis, fibrosis (Riedel’s struma)   This is the most common form of thyrotoxicosis and  occurs more often in females than in males. It may  be caused by relatively autonomous secretion from a  diffuse goitre and is characterized by:   • exophthalmos, due to lymphocytic infiltration and  swelling of retro-orbital tissues of the eyes (Fig. 1 1.4),   • sometimes localized thickening of the subcutaneous  tissue over the shin (pretibial myxoedema).   Graves’ disease is an autoimmune thyroid disease  characterized by a variety of circulating antibodies,  including anti-TPO, as well as being associated with  other autoimmune diseases such as type 1 diabetes  mellitus, adrenal insufficiency and pernicious anaemia.  Thyroid antibodies are detectable in some cases, such   Toxic multinodular goitre (Plummer's disease) or a  single functioning nodule (occasionally an adenoma)  Subacute thyroiditis  Some metastatic thyroid carcinomas   Administration of iodine to a subject with iodine-  deficiency goitre  Jod-Basedow syndrome   Thyroid-stimulating hormone secretion by tumours,  including pituitary tumours or those of trophoblastic  origin   Struma ovarii (thyroid tissue in an ovarian teratoma)  Excess human chorionic gonadotrophin, e.g. molar  pregnancy or choriocarcinoma  Pituitary resistance to thyroid hormone   as thyroid-stimulating immunoglobulin (TSI), which  is directed towards epitopes of the TSH receptor and  thus acts as a TSH receptor agonist. Nuclear medicine  tests may show a high radioactive uptake of iodine by  the thyroid gland.   This is a destructive thyroiditis resulting in the  release of preformed thyroid hormones. There  are three subtypes: granulomatous or painful,  lymphocytic or silent and painless, and post-partum.  This condition is associated with extremely elevated  thyroid hormones and no radioactive iodine uptake  by the thyroid gland. The clinical course progresses  through 6-8 weeks of thyrotoxicosis, 2-4 months  of hypothyroidism and a return to euthyroidism in  about 90 per cent of patients.   The painful or granulomatous variety is thought  to be a viral disease and is associated with human  leucocyte antigen (HLA)-Bw35. The lymphocytic  variety is autoimmune, as is post-partum thyroiditis.  The post-partum form occurs in about 5-8 per cent  of pregnant women in Europe and the USA, but in  20 per cent in Japan. Treatment is supportive, as in  many cases the condition is self-limiting.   A 29-year-old woman was seen in the thyroid clinic  because of exophthalmos and a goitre. She had the  following thyroid function test results:   The patient had biochemical results typical of  hyperthyroidism. In fact she had Graves’ disease  and was shown to have positive thyroid- stimulating  immunoglobulins and increased diffuse radiolabelled  iodine uptake by the thyroid gland.   Compare these results with those of another patient  (a 54-year-old woman) in the same clinic:   Here, the plasma fT concentration is within the  reference range, but the plasma fT concentration is  raised, with suppressed plasma TSH concentration,  suggesting T 3 thyrotoxicosis.   Toxic nodules, either single or multiple, in a nodular  goitre may secrete thyroid hormones autonomously.  The secretion of TSH is suppressed by negative  feedback, as in Graves’ disease. The nodules may  be detected by their uptake of radioactive iodine or  technetium, with suppression of uptake in the rest of the  thyroid tissue (‘hot nodules’). Toxic nodules are found  most commonly in older patients, who may present  with only one of the features of hyperactivity, usually  cardiovascular symptoms such as atrial fibrillation.  Toxic multinodular goitre is also called Plummer’s  disease.   Jod-Basedow syndrome is hyperthyroidism in patients  with excess iodide intake, for example from the diet  or from iodine-containing contrast medium. High  iodine intake may be assessed by urinary iodide assay.  Metastatic thyroid carcinoma can produce thyroid  hormones. In struma ovarii, ectopic thyroid tissue   is found in dermoid tumours or ovarian teratomas.  Patients with choriocarcinoma or molar hydatidiform  pregnancy have extremely high concentrations of  (3-human chorionic gonadotrophin that can activate  the TSH receptor. Rarely, the pituitary tumour releases  TSH, resulting in thyrotoxicosis.   Pathophysiology of hyperthyroidism  Plasma T, or fT, and T„ and fT, concentrations are  usually increased in hyperthyroidism. Much of the T 3 is  secreted directly by the thyroid gland, and the increase  in plasma T 3 concentrations is greater, and usually  evident earlier, than that of T . Rarely, only plasma  T 3 and fT 3 concentrations are elevated (T, toxicosis).  In both situations, TSH secretion is suppressed by  negative feedback, and plasma TSH concentrations are  either very low or undetectable.   The aetiology of hyperthyroidism must be fully  investigated and treatment started. Various forms of  treatment are available, the selection of which depends  on the cause, the clinical presentation and the age of  the patient. (3-blocker drugs such as propranolol, which  inhibit the peripheral conversion of T 4 to T , maybe used  initially. Additional treatment includes the use of such  drugs as carbimazole or propylthiouracil. Carbimazole  inhibits the synthesis of T 3 and T 4 ; propylthiouracil  additionally inhibits T 4 to T 3 conversion. Some  clinicians use block-and-replace regimens: carbimazole  is used to ‘block’ thyroid secretion, and simultaneous  exogenous T 4 maintains and replaces T 4 concentrations.  It is important to remember that carbimazole can  have the potentially lethal side effect of bone marrow  suppression, and patients should be warned about  infections such as sore throats and about the need to  have their full blood count monitored.   Radioactive iodine can be used in resistant or  relapsing cases; surgery is rarely indicated, but may  have a place if there is a large toxic goitre that is exerting  pressure or if drug therapy fails but radioactive iodine  is contraindicated. Thyroid function must be checked  regularly, as some patients may become hypothyroid or  may relapse after radioiodine or surgery.   The progress of a patient being treated for  hyperthyroidism is usually monitored by estimating  plasma TSH, fT 4 and fT concentrations, and trying to  restore these to normal (although TSH concentration  may be slow to normalize). Overtreatment may  induce hypothyroidism, with a rise in plasma TSH   concentrations and low plasma T 4 /fT 4 and T 3 /fT,  concentrations. In some patients with severe prolonged  hyperthyroidism, such a rise in plasma TSH may be  delayed because of the effects of prolonged feedback  suppression of T 4 on the pituitary.   Subclinical hyperthyroidism may occur with a low or  suppressed TSH concentration but normal (usually  high-normal) plasma fT and fT concentrations. The  condition may progress to full-blown hyperthyroidism  with suppressed plasma TSH and raised plasma fT 4 and  fT concentrations. Subclinical hyperthyroidism may  be associated with atrial fibrillation, decreased bone  mineral density and other features of hyperthyroidism.  Plasma TSI may be raised.   Laboratory investigation of suspected hyperthyroidism  A careful history (including drugs) should be taken  and examination performed, checking for a goitre.  The plasma TSH, fT and fT concentrations should be  measured.   • The plasma fT and fT, concentrations are clearly  high and the TSH concentration is suppressed in  clinically thyrotoxic patients.   • In the face of suppressed plasma TSH, a clearly  elevated plasma fT concentration confirms the  diagnosis of hyperthyroidism. Remember that in T 3  thyrotoxicosis the plasma fT may be normal.   • If the plasma fT concentration is raised and the  TSH concentration is normal, this is suggestive of  biochemical euthyroid hyperthyroxaemia (see below  for causes).   • Measurement of thyroid antibodies is useful,  particularly if the concentration of TSIs is raised,  which supports a diagnosis of Graves’ disease.   • The rare TSH-secreting pituitary tumours need  pituitary assessment (see Chapter 7). a-subunit  concentrations may be useful, as they are usually  raised in such circumstances.   • In difficult cases, determination of plasma SHBG  concentration can help decide whether the patient is  hyperthyroid, as it is lowered in hypothyroidism and  raised in hyperthyroidism.   • Radioiodine uptake studies of the thyroid can  be useful to distinguish some of the causes of  hyperthyroidism (see Box 11.2).   • The TRH test is sometimes useful in the diagnosis of  unclear cases.   If plasma T 4 concentrations fall, enlargement of the  thyroid gland (goitre) maybe caused by TSH stimulation  resulting in cellular hyperplasia. Thyroxine synthesis  may be impaired by iodide deficiency, caused by drugs  such as para-aminosalicylic acid, or possibly by partial  deficiency of the enzymes involved in T 4 synthesis.  Under the influence of prolonged stimulation by TSH,  the number of thyroid cells increases and plasma  thyroid hormone concentrations are maintained at the  expense of the development of a goitre.   Inflammation of the thyroid gland (thyroiditis),  whether acute or subacute, may produce marked but  temporary aberrations of thyroid function tests (see  above). Ultrasound scanning can be useful in the  diagnosis of goitre, as can radiolabelled uptake studies  to see if there are hot (T 4 -producing) or cold (non-  producing) nodule(s).   Any severe illness may be associated with low plasma  total or fT concentrations and may make the  interpretation of thyroid function tests extremely  difficult. Plasma TSH concentrations may be normal or  slightly high or low. The TSH response to TRH may also  be impaired. There may be impaired conversion of T 4  to T with low plasma T concentrations. Consequently,  the assessment of thyroid function is best deferred until  the patient has recovered from the illness.   This is defined as a condition in which either the plasma  total or fT concentration is abnormally raised without  clinical evidence of thyroid disease. These changes may  be transient or persistent, with high, normal or low total  or fT concentrations. Heterophilic antibodies to fT  and/or fT 3 should be excluded [these can sometimes be  removed by the laboratory by treating the sample with  polyethylene glycol (PEG), which can precipitate these  antibodies], as they can interfere with some assays.   • Physiological conditions resulting in raised plasma  TBG concentration, for example pregnancy.  Concentrations oftotalT 4 andT 3 are both elevated, but  there are usually normal fT and fT concentrations.   - familial dysalbuminaemic hyperthyroxinaemia  (FDH) due to an abnormal form of albumin.   - oestrogens raise TBG concentration, as do  5-fluorouracil, heroin and methadone,   - amiodarone blocks conversion of T to T,,  resulting in an elevation of T 4 and reverse T.  concentrations,   - heparin, due to fatty acid release, inhibits fT  binding to TBG,   • Some patients with certain illnesses, for example  hyperemesis gravidarum, have low total and fT  concentrations due to reduced peripheral conversion  of T, to T because 5-deiodinase is inhibited. This  results in elevated total T and fT, concentrations.   Some hepatic disorders, including acute hepatitis,  result in raised concentrations of TBG and T, and  fT . In up to 10 per cent of cases of acute psychosis,  total and fT, concentrations are raised. The exact  mechanism is unknown, but it may be due to central  activation of the hypothalamic-pituitary axis.   Amiodarone and thyroid function  Amiodarone is sometimes used to treat certain cardiac  arrhythmias. This drug can evoke hypothyroidism,  partly because it interrupts the conversion of T 4  to T . However, it contains iodine and can also  evoke thyrotoxicosis by the Jod-Basedow or type 1  phenomenon. Conversely, it may elicit disruptive  thyroiditis and thyrotoxicosis with raised interleukin-6  concentration (type 2 phenomenon). The drug has a  long half-life (40-100 days) and thus takes a long time  to clear from the body (see Chapter 25).   • A first-line test for thyroid function (as stated  above) is plasma TSH, although this can be difficult  to interpret in the absence of fT . (Sometimes fT  is also required, particularly if hyperthyroidism is  suspected - Table 11.2.)   • If the plasma TSH concentration is normal and the  patient is clinically euthyroid, look at plasma fT :   — If fT concentration is low, consider sick euthyroid/  non-thyroidal illness, certain drugs, such as  carbamazepine or phenytoin (see Table 11.1).   - If fT concentration is also normal, thyroid  function is likely to be normal.   A 45-year-old man was on the coronary care unit  the day after an acute myocardial infarction. One of  his doctors thought that he looked hypothyroid and  requested thyroid function tests, the results of which  were as follows:   On repeating the tests 3 months later at a follow-up  appointment in the medical out-patient department,  the following results were obtained:   The first set of results could indicate hypothyroidism  due to pituitary or hypothalamic defects (secondary  hypothyroidism), that is, low TSH and ‘normal’ fT  and fT concentrations. However, the normalization  of the results when the patient was not acutely ill  suggested sick euthyroidism or non-thyroidal illness.  Beware of requesting thyroid function tests in acutely  ill patients.   - If fT concentration is high, consider euthyroid  hyperthyroxinaemia, interfering assay  autoantibodies.   • If the plasma TSH concentration is low, look at  plasma fT :   — If fT, concentration is low, consider sick  euthyroid/non-thyroid illness, pituitary or  hypothalamic disease (secondary hypo-  thyroidism?), certain drugs (see Table 11.1).   - If fT, concentration is normal, consider sick  euthyroid/non-thyroid illness, subclinical  hyperthyroidism, particularly if clinically  hyperthyroid, certain drugs, such as  glucocorticoids and dopamine that may affect  TSH, fT 3 toxicosis (fT 3 concentration is raised).   - If fT concentration is high, consider  hyperthyroidism (see Box 11.2), drugs such  as amiodarone (see Table 11.1), iodine excess,  hyperemesis gravidarum, molar pregnancy,  activating TSH receptor mutations.   T 4 , thyroxine; T 3 , tri-iodothyronine; fT 4 , free thyroxine; fT 3 , free tri-iodothyronine; TBG, thyroxine-binding globulin; TSH, thyroid-stimulating  hormone.   • If the plasma TSH concentration is high, look at  plasma fT 4 :   - If fT concentration is low, consider primary  hypothyroidism (see Box 11.1).   thyroid replacement for hypothyroidism, drugs  such as metoclopramide or domperidone, or  sick euthyroid.   - If fT concentration is high, consider  generalized thyroid hormone resistance, TSH-  secreting tumour, interfering assay antibodies.   • Thyroid-stimulating hormone from the anterior  pituitary acts upon the thyroid gland to release  two iodine-containing hormones, T 4 and T . The  latter is more active and can also be produced from  T 4 peripherally. These hormones are essential for  normal growth and development and increase  basal metabolic rate.   • Both T 4 and T 3 are bound to proteins, including  TBG, albumin and transthyretin. Only the free or  unbound form is physiologically active, although  this fraction constitutes less than 1 per cent of the  total hormone concentration.   Thyroid disease is relatively common.  Hypothyroidism can be primary or secondary  and manifest as weight gain, tiredness and  constipation - to name but a few of its clinical  features. Hyperthyroidism causes thyrotoxicosis  associated with weight loss, sweating and  sometimes thyroid eye disease. Both can present  with a goitre, although some patients with  a goitre may be euthyroid (normal thyroid  function tests).   This chapter discusses carbohydrate metabolism and its  abnormalities, with emphasis on diabetes mellitus and  hypoglycaemia. In the next decade it is predicted that  there will be about 250 million people worldwide with  type 2 diabetes mellitus.   The main monosaccharide hexoses are reducing sugars.  Naturally occurring polysaccharides are long-chain  carbohydrates composed of glucose subunits (Table  12.1):   • Starch, found in plants, is a mixture of amylose  (straight chains) and amylopectin (branched  chains).   • Glycogen, found in animal tissue, is a highly branched  polysaccharide.   The main function of glucose is as a major tissue energy  source. The simplified pathways of glycolysis and the  Krebs cycle [tricarboxylic acid (TCA) cycle] are shown  in Figures 12.1 and 12.2. The brain is highly dependent  upon the extracellular glucose concentration for its  energy supply; indeed, hypoglycaemia is likely to impair  cerebral function or even lead to irreversible neuronal  damage. This is because the brain cannot:   • metabolize substrates other than glucose and ketones  - plasma ketone concentrations are usually very low  and ketones are of little importance as an energy  source under physiological conditions,   • extract enough glucose from the extracellular fluid  (ECF) at low concentrations for its metabolic needs,  because entry into brain cells is not facilitated by  insulin.   Normally the plasma glucose concentration remains  between about 4mmol/T and lOmmol/L, despite the  intermittent load entering the body from the diet. The  maintenance of plasma glucose concentrations below  about lOmmol/L minimizes loss from the body as well  as providing the optimal supply to the tissues. Renal  tubular cells reabsorb almost all the glucose filtered  by the glomeruli, and urinary glucose concentration  is normally too low to be detected by the usual tests,    Figure 12.2 Simplification of the tricarboxylic acid (Krebs)  cycle. CoA, coenzyme A; TPP, thiamine pyrophosphate.  Reproduced with kind permission from Candlish JK and  Crook M. Notes on Clinical Biochemistry. Singapore:   even after a carbohydrate meal. Significant glycosuria  usually occurs only if the plasma glucose concentration  exceeds about lOmmol/L - the renal threshold.   During normal metabolism, little glucose is lost  unchanged from the body. Maintenance of plasma  glucose concentrations within the relatively narrow  range of 4-10 mmol/L, despite the widely varying input  from the diet, depends on the balance between the  glucose entering cells from the ECF and that leaving  them into this compartment.   Hormones concerned with glucose homeostasis  Some of the more important effects of hormones on  glucose homeostasis are summarized in Table 12.2.   Insulin is the most important hormone controlling  plasma glucose concentrations. A plasma glucose  concentration of greater than about 5 mmol/L acting via  the glucose transporter 2 stimulates insulin release from  the pancreas (3-cell. These cells produce proinsulin, which  consists of the 51-amino-acid polypeptide insulin and a  linking peptide (C-peptide, Fig. 12.3). Splitting of the  peptide bonds by prohormone convertases releases via  intermediates (mostly 32-33 split proinsulin) equimolar  amounts of insulin and C-peptide into the ECF.   Insulin binds to specific cell surface receptors on  muscle and adipose tissue, thus enhancing the rate  of glucose entry into these cells. Insulin-induced  activation of enzymes stimulates glucose incorporation  into glycogen (glycogenesis) in liver and muscle (Fig  12.4). Insulin also inhibits the production of glucose  (gluconeogenesis) from fats and amino acids, partly  by inhibiting fat and protein breakdown (lipolysis and  proteolysis).   The transport of glucose into liver cells is insulin  independent but, by reducing the intracellular glucose  concentration, insulin does indirectly promote the  passive diffusion of glucose into them. Insulin also  directly increases the transport of amino acids,  potassium and phosphate into cells, especially muscle;  these processes are independent of glucose transport.  In the longer term, insulin regulates growth and  development and the expression of certain genes.   Glucagon is a single-chain polypeptide synthesized  by the a-cells of the pancreatic islets. Its secretion is  stimulated by hypoglycaemia. Glucagon enhances  hepatic glycogenolysis (glycogen breakdown) and  gluconeogenesis.    Figure 12.3 Structure of proinsulin, indicating the  cleavage sites at which insulin and C-peptide are  produced.   +, stimulates; inhibits; IGF-1, insulin-like growth factor 1; NEFA, non-esterified fatty acid.   This peptide hormone is released from the D cells of  the pancreas and inhibits insulin and growth hormone  release.   When plasma insulin concentrations are low, for  example during fasting, the hyperglycaemic actions  of hormones, such as growth hormone (GH),  glucocorticoids, adrenaline (epinephrine) and  glucagon, become apparent, even if there is no increase  in secretion rates. Secretion of these so-called counter-  regulatory hormones may increase during stress and  in patients with acromegaly (GH, see Chapter 6),  Cushing’s syndrome (glucocorticoids, see Chapter 8) or   in phaeochromocytoma (adrenaline and noradrenaline  see Chapter 24) and thus oppose the normal action of  insulin.   The liver is the most important organ maintaining a  constant glucose supply for other tissues, including the  brain. It is also of importance in controlling the post-  prandial plasma glucose concentration.   Portal venous blood leaving the absorptive area of the  intestinal wall reaches the liver first, and consequently  the hepatic cells are in a key position to buffer the  hyperglycaemic effect of a high-carbohydrate meal  (Fig. 12.5).    Figure 12.4 Structure of glycogen. Open circles depict  glucose moieties in a-1,4 linkage and the black  circles those in a-1,6 linkages at branch points. R  indicates the reducing end group. The outer branches  terminate in non-reducing end groups. Reproduced  with permission from Nyhan WL and Barshop BA. Atlas  of Inherited Metabolic Diseases, 3rd edition. London:  Hodder Arnold, 2012.   The entry of glucose into liver and cerebral cells is  not directly affected by insulin, but depends on the  extracellular glucose concentration. The conversion of  glucose to glucose-6-phosphate (G6P), the first step in  glucose metabolism in all cells, is catalysed in the liver  by the enzyme glucokinase, which has a low affinity for  glucose compared with that of hexokinase, which is found  in most other tissues. Glucokinase activity is induced by  insulin. Therefore, hepatic cells extract proportionally  less glucose during fasting, when concentrations in  portal venous plasma are low, than after carbohydrate  ingestion. This helps to maintain a fasting supply of  glucose to vulnerable tissues such as the brain.   The liver cells can store some of the excess glucose as  glycogen. The rate of glycogen synthesis (glycogenesis)  from G6P may be increased by insulin secreted by  the (3-cells of the pancreas in response to systemic  hyperglycaemia. The liver can convert some of the excess  glucose to fatty acids, which are ultimately transported  as triglyceride in very low-density lipoprotein (VLDT)  and stored in adipose tissue.   Under normal aerobic conditions, the liver can  synthesize glucose by gluconeogenesis using the  metabolic products from other tissues, such as glycerol,   lactate or the carbon chains resulting from deamination  of certain amino acids (mainly alanine) (Table 12.3). The  liver contains the enzyme glucose-6-phosphatase, which,  by hydrolysing G6P derived from either glycogenolysis  or gluconeogenesis, releases glucose and helps to  maintain extracellular fasting concentrations. Hepatic  glycogenolysis is stimulated by the hormone glucagon,  secreted by the a-cells of the pancreas in response  to a fall in the plasma glucose concentration, and by  catecholamines such as adrenaline or noradrenaline.   During fasting, the liver converts fatty acids,  released from adipose tissue as a consequence of low  insulin activity, to ketones. The carbon chains of some  amino acids may also be converted to ketones (Table  12.3). Ketones can be used by other tissues, including  the brain, as an energy source when plasma glucose  concentrations are low.   The renal cortex is the only other tissue capable of  gluconeogenesis, and of converting G6P to glucose. The  gluconeogenic capacity of the kidney is particularly  important in hydrogen ion homeostasis and during  prolonged fasting.   Other tissues, such as muscle, can store glycogen but,  because they do not contain glucose-6-phosphatase,  they cannot release glucose from cells and so can only  use it locally; this glycogen plays no part in maintaining  the plasma glucose concentration.   The liver modifies the potential hyperglycaemic effect  of a high- carbohydrate meal by extracting relatively  more glucose than in the fasting state from the portal  plasma. However, some glucose does pass through  the liver and the rise in the systemic concentration   Table 12.3 Metabolism of the carbon skeleton of some  amino acids to either carbohydrate (glycogenic) or fat  (ketogenic)   Figure 12.5 Post-prandial metabolism of glucose. CoA, coenzyme A; G6P, glucose-6-phosphate; Glycerol-3-P,  glycerol-3-phosphate; Triose-P, triose phosphate or glyceraldehyde 3-phosphate; VLDL, very low-density lipoprotein.   stimulates the (3-cells of the pancreas to secrete insulin.  Insulin may further enhance hepatic and muscle  glycogenesis. More importantly, entry of glucose into  adipose tissue and muscle cells, unlike that into liver and  brain, is stimulated by insulin and, under physiological  conditions, the plasma glucose concentration falls to  near fasting levels. Conversion of intracellular glucose  to G6P in adipose and muscle cells is catalysed by the  enzyme hexokinase, which, because its affinity for  glucose is greater than that of hepatic glucokinase,  ensures that glucose enters the metabolic pathways in  these tissues at lower extracellular concentrations than  those in the liver. The relatively high insulin activity  after a meal also inhibits the breakdown of triglyceride  (lipolysis) and protein (proteolysis). If there is relative  or absolute insulin deficiency, as in diabetes mellitus,   these actions are impaired. Both muscle and adipose  tissue store the excess post-prandial glucose, but the  mode of storage and the function of the two types of  cell are very different, as will be shown later.   Adipose tissue triglyceride is the most important long-  term energy store in the body. Greatly increased use  of fat stores, for example during prolonged fasting,  is associated with ketosis. Adipose tissue cells, acting  in conjunction with the liver, convert excess glucose  to triglyceride and store it in this form rather than  as glycogen. The components are both derived from  glucose, fatty acids from the glucose entering hepatic  cells and glycerol from that entering adipose tissue cells.   In the liver, triglycerides are formed from glycerol-  3 -phosphate (from triose phosphate or glyceraldehyde-  3 -phosphate) and fatty acids [from acetyl coenzyme A  (CoA)]. The triglycerides are transported to adipose  tissue cells incorporated in VLDL, where they are  hydrolysed by lipoprotein lipase. The released fatty  acids (of hepatic origin) are re-esterified within these  cells with glycerol-3-phosphate, derived from glucose,  which has entered this tissue under the influence of  insulin. The resultant triglyceride is stored and is far  more energy dense than glycogen (see Chapter 13).   During fasting, when exogenous glucose is  unavailable and the plasma insulin concentration  is therefore low, endogenous triglycerides are  reconverted to free non-esterified fatty acids (NEFAs)  and glycerol by lipolysis (Fig. 12.6). Both are  transported to the liver in plasma, the NEFA being  protein bound, predominantly to albumin. Glycerol  enters the hepatic gluconeogenic pathway at the  triose phosphate stage; the glucose synthesized can be  released from these cells, thus minimizing the fall in  glucose concentrations.   Most tissues, other than the brain, can oxidize fatty  acids to acetyl CoA, which can then be used in the TCA  cycle as an energy source. When the rate of synthesis  exceeds its use, the hepatic cells produce acetoacetic acid  by enzymatic condensation of two molecules of acetyl  CoA; acetoacetic acid can be reduced to [3-hydroxybutyric  acid and decarboxylated to acetone. These ketones can  be used as an energy source by brain and other tissues at  a time when glucose is in relatively short supply.   Ketosis occurs when fat stores are the main energy  source and may result from fasting or from reduced  nutrient absorption, for example due to vomiting. Mild  ketosis may occur after as little as 12 h of fasting. After  short fasts, metabolic acidosis is not usually detectable,  but, after longer periods, more hydrogen ions may  be produced than can be dealt with by homeostatic  buffering mechanisms, depleting the plasma bicarbonate  concentration, which therefore falls (see Chapter 4).   The plasma glucose concentration is maintained  principally by hepatic gluconeogenesis, but during  prolonged starvation, such as that in anorexia nervosa or  during childhood, ketotic hypoglycaemia may occur. The    Figure 12.6 Intermediary metabolism during fasting: ketosis. CoA, coenzyme A; FA, fatty acid; G6P,  glucose-6-phosphate; NEFA, non-esterified fatty acid.   Figure 12.5 Post-prandial metabolism of glucose. CoA, coenzyme A; G6P, glucose-6-phosphate; Glycerol-3-P,  glycerol-3-phosphate; Triose-P, triose phosphate or glyceraldehyde 3-phosphate; VLDL, very low-density lipoprotein.   stimulates the (3-cells of the pancreas to secrete insulin.  Insulin may further enhance hepatic and muscle  glycogenesis. More importantly, entry of glucose into  adipose tissue and muscle cells, unlike that into liver and  brain, is stimulated by insulin and, under physiological  conditions, the plasma glucose concentration falls to  near fasting levels. Conversion of intracellular glucose  to G6P in adipose and muscle cells is catalysed by the  enzyme hexokinase, which, because its affinity for  glucose is greater than that of hepatic glucokinase,  ensures that glucose enters the metabolic pathways in  these tissues at lower extracellular concentrations than  those in the liver. The relatively high insulin activity  after a meal also inhibits the breakdown of triglyceride  (lipolysis) and protein (proteolysis). If there is relative  or absolute insulin deficiency, as in diabetes mellitus,   these actions are impaired. Both muscle and adipose  tissue store the excess post-prandial glucose, but the  mode of storage and the function of the two types of  cell are very different, as will be shown later.   Adipose tissue triglyceride is the most important long-  term energy store in the body. Greatly increased use  of fat stores, for example during prolonged fasting,  is associated with ketosis. Adipose tissue cells, acting  in conjunction with the liver, convert excess glucose  to triglyceride and store it in this form rather than  as glycogen. The components are both derived from  glucose, fatty acids from the glucose entering hepatic  cells and glycerol from that entering adipose tissue cells.   During gluconeogenesis, hydrogen ions are reused.  Under aerobic conditions, the liver consumes much  more lactate than it produces.   The physiological accumulation of lactic acid during  muscular contraction is a temporary phenomenon and  rapidly disappears at rest, when slowing of glycolysis  allows aerobic processes to ‘catch up’.   Lactic acid, produced by anaerobic glycolysis, may either  be oxidized to C0 2 and water in the TCA cycle or be  reconverted to glucose by gluconeogenesis in the liver.  Both the TCA cycle and gluconeogenesis need oxygen;  anaerobic glycolysis is a non-oxygen-requiring pathway.  Pathological accumulation of lactate may occur because:   • production is increased by an increased rate of  anaerobic glycolysis,   • use is decreased by impairment of the TCA cycle or  impairment of gluconeogenesis.   Tissue hypoxia (Fig. 12.8) due to the poor tissue  perfusion of the ‘shock’ syndrome is usually the most  common cause of lactic acidosis. Hypoxia increases  plasma lactate concentrations because:   • the TCA cycle cannot function anaerobically and  oxidation of pyruvate and lactate to C0 2 and water  is impaired,   • anaerobic glycolysis is stimulated because the  falling adenosine triphosphate (ATP) levels cannot  be regenerated by the TCA cycle under anaerobic  conditions.   The combination of impaired gluconeogenesis and  increased anaerobic glycolysis converts the liver from  an organ that consumes lactate and H + to one that  generates large amounts of lactic acid. Severe hypoxia,  for example following a cardiac arrest, causes marked  lactic acidosis. If diabetic ketoacidosis is associated with  significant volume depletion, this hypoxic syndrome  may aggravate the acidosis. (See Chapter 4 for a further  discussion of lactic acidosis.)   The glycolytic pathway as well as the TCA cycle are  summarized in Figures 12.1 and 12.2.   • severe stress (usually a transient effect) such as  trauma, myocardial infarction or cerebrovascular  accidents,   Diabetes mellitus is caused by an absolute or relative  insulin deficiency. It has been defined by the World  Health Organization (WHO), on the basis of  laboratory findings, as a fasting venous plasma glucose  concentration of 7.0 mmol/L or more (on more than one  occasion or once in the presence of diabetes symptoms)  or a random venous plasma glucose concentration  of 11.1 mmol/L or more. Sometimes an oral glucose  tolerance test (OGTT) maybe required to establish the  diagnosis in equivocal cases. The interpretation of this  test is shown below, but, briefly, diabetes mellitus can be  diagnosed if the venous plasma glucose concentration  is 7.0 mmol/L or more (fasting) and/or 11.1 mmol/L  or more 2h after the oral ingestion of the equivalent  of 75 g of anhydrous glucose. Diabetes mellitus can be  classified into the following categories.   Previously called insulin-dependent diabetes mellitus,  this is the term used to describe the condition in patients  for whom insulin therapy is essential because they are  prone to develop ketoacidosis. It usually presents during  childhood or adolescence. Most of these cases are due to  immune-mediated processes and may be associated with  other autoimmune disorders such as Addison’s disease,  vitiligo and Hashimoto’s thyroiditis. It has been suggested  that many cases follow a viral infection that has damaged  the (3-cells of the pancreatic islets. Individuals most  at risk are those with human leucocyte antigen (HLA)  types DR3 and DR4 of the major histocompatibility  complex. Autoantibodies to islet cells, insulin, tyrosine  phosphatases IA-2 and IA-2(3 and glutamic decarboxylase  (GAD) are found in about 90 per cent of cases. There  is a form of type 1 diabetes called idiopathic diabetes  mellitus that is not autoimmune mediated but is strongly  inherited and more common in black and Asian people.  The insulin requirement of affected people can fluctuate  widely and the cause is unknown. There is also LADA  (latent autoimmune diabetes of adults), sometimes  called slow-onset type 1 diabetes.   Previously called non-insulin-dependent diabetes  mellitus, this is the most common variety worldwide  (about 90 per cent of all diabetes mellitus cases) . Patients  are much less likely to develop ketoacidosis than those  with type 1 diabetes, although insulin may sometimes  be needed. Onset is most usual during adult life; there  is a familial tendency and an association with obesity.   There is a spectrum of disorders ranging from mainly  insulin resistance with relative insulin deficiency to a  predominantly secretory defect with insulin resistance.   A variety of inherited disorders may be responsible for  the syndrome, either by reducing insulin secretion or by  causing relative insulin deficiency because of resistance  to its action or of insulin receptor defects, despite high  plasma insulin concentrations.   - MODY 1: mutation of the hepatocyte nuclear  factor ( HNF4A ) gene,   Some cases are thought to be point mutations in  mitochondrial deoxyribonucleic acid (DNA) associated  with diabetes mellitus and deafness and are usually  autosomal dominant.   • Type A insulin resistance (insulin receptor defect),  for example leprechaunism, lipoatrophy and  Rabson-Mendenhall syndrome.   • Relative insulin deficiency, due to excessive GH  (acromegaly), phaeochromocytoma, glucocorticoid  secretion (Cushing’s syndrome).   In the UK, about 4-5 per cent of pregnancies are  complicated by gestational diabetes mellitus (GDM).  It is associated with increased fetal abnormalities,  for example high birthweight, cardiac defects and  polyhydramnios. In addition, birth complications,  maternal hypertension and the need for caesarean  section may occur. If maternal diet/lifestyle factors fail  to restore glucose levels, insulin is usually required to  try to reduce the risk of these complications.   Women at high risk for GDM include those who  have had GDM before, have previously given birth to a  high -birthweight baby, are obese, have a family history of  diabetes mellitus and/or are from high-risk ethnic groups,  for example black or South Asian. These women should  be screened at the earliest opportunity and, if normal,  retested at about 24-28 weeks, as glucose tolerance  progressively deteriorates throughout pregnancy. In some  units 50 g oral glucose is used and the blood glucose is  sampled at 1 h - plasma glucose of more than or equal to  7.8mmol/L being diagnostic (O’Sullivan’s screening test  for gestational diabetes). If fasting venous plasma glucose  is 7.0mmol/L or more and/or the random measurement  gives a concentration of ll.lmmol/L or more (some  doctors prefer to use a lower cut-off of about 9.0mmol/L  in pregnancy), the woman has GDM. In equivocal cases,  an OGTT is indicated. Six weeks post partum, the woman  should be reclassified with a repeat OGTT.   The WHO definition of impaired glucose tolerance  (IGT) is a fasting venous plasma glucose concentration  of less than 7.0mmol/L and a plasma glucose  concentration between 7.8mmol/L and ll.lmmol/L  2h after an OGTT. Some patients with IGT develop  diabetes mellitus later and may require an annual OGTT  to monitor for this. However, because of the increased  risk of vascular complications, secondary causes of IGT  should be sought, dietary advice given, if necessary, and  the patient followed up. In pregnancy IGT is treated as  GDM because of the risks to the fetus.   Impaired fasting glucose (IFG), like IGT, refers to a  metabolic stage intermediate between normal glucose   homeostasis and diabetes mellitus. The definition is  that the fasting venous plasma glucose is 6.1mmol/L  or more but less than 7.0mmol/L, and less than  7.8mmol/L 2h after an OGTT.   A strong family history of diabetes mellitus may suggest  that an individual is at risk of developing diabetes  mellitus (particularly type 2), as may a family history  of GDM, IGT or IFG. Those with predisposing HLA  types and autoimmune disease may be susceptible  to developing type 1 diabetes. Type 2 diabetes is  more common in certain racial groups, such as Afro-  Caribbeans, South Asians and Pacific Islanders. One of  the reasons why type 2 diabetes is on the increase is the  increasing tendency to obesity and central adiposity in  urbanized and more sedentary populations consuming  high-calorie diets.   The thrifty phenotype (Barker-Hales) hypothesis  proposes that nutritional deficiency in fetal and early  infancy associated with low birthweight increases  the risk of developing type 2 diabetes and insulin  resistance.   It has been recognized that certain coronary heart disease  risk factors occur together. There is an aggregation of  lipid and non-lipid risk factors of metabolic origin.  A particular cluster is known as the metabolic syndrome,  syndrome X or Reaven’s syndrome and is closely linked  to insulin resistance. One definition is the presence of  three or more of the following features:   Plasma levels of insulin would be expected to be  raised, that is, hyperinsulinaemia. Other associated  features may include polycystic ovary syndrome, fatty  liver, raised fibrinogen and plasminogen activator  inhibitor 1 concentrations, renal sodium retention,  hyperuricaemia and dense low-density lipoprotein  (LDL) particles (see Chapter 13).   Metabolic features of diabetes mellitus  Patients with type 1 diabetes tend to be diagnosed before  the age of 40 years, are usually lean and have experienced  weight loss at the time of presentation. They may  present with diabetic ketoacidosis. Conversely, patients  with type 2 diabetes often present later, usually after the  age of 40 years, and are often overweight or obese. The  presentation can be insidious and they may have had  diabetes years before diagnosis.   If plasma glucose concentration exceeds about  lOmmol/L, glycosuria would be expected. High urinary  glucose concentrations produce an osmotic diuresis and  therefore polyuria. Cerebral cellular dehydration due to  hyperosmolality, secondary to hyperglycaemia, causes  thirst (polydipsia). A prolonged osmotic diuresis may  cause excessive urinary electrolyte loss. These ‘classic’  symptoms are suggestive of diabetes mellitus.   Diabetic patients on insulin may show the  following conditions. The ‘dawn’ phenomenon is the  physiological response of the elevation of blood glucose  concentration in the early morning prior to breakfast  due to nocturnal spikes in GH concentration and a rise  in plasma cortisol concentration that increase hepatic  gluconeogenesis. Conversely, in some diabetic patients  nocturnal hypoglycaemia may evoke a rebound  counter-regulatory hyperglycaemia called the Somogyi  phenomenon. Patient blood glucose checking at 02.00-  04.00 h, or continuous glucose monitoring if available,  may distinguish these conditions, as the Somogyi  phenomenon reveals hypoglycaemia. It is sometimes  possible to ameliorate these conditions by giving  intermediate-acting insulin before bedtime.   These may be secondary to insulin deficiency. Lipolysis  is enhanced and plasma NEFA concentrations rise.  In the liver, NEFAs are converted to acetyl CoA and  ketones, or are re-esterified to form endogenous  triglycerides and incorporated into VLDLs; the latter  accumulate in plasma because lipoprotein lipase, which  is necessary for VLDL catabolism, requires insulin for  optimal activity. High-density lipoprotein cholesterol  concentration tends to be low in type 2 diabetes. If  insulin deficiency is very severe, there may also be  chylomicronaemia. The rate of cholesterol synthesis is  also increased, with an associated increase in plasma  LDL concentrations. Consequently, patients with  diabetes may show high plasma triglyceride, raised  cholesterol and low HDL cholesterol concentrations.   Long-term effects of diabetes mellitus  Vascular disease is a common complication of diabetes  mellitus. Macrovascular disease due to abnormalities of  large vessels may present as coronary artery, cerebrovascular  or peripheral vascular insufficiency. The condition is  probably related to alterations in lipid metabolism and  associated hypertension. The most common cause of death  is cardiovascular disease, including myocardial infarction.   Microvascular disease due to abnormalities of small  blood vessels particularly affects the retina (diabetic  retinopathy) and the kidney (nephropathy); both may  be related to inadequate glucose control. Diabetes is  one of the most common causes of patients requiring  renal dialysis. Microvascular disease of the kidney is  associated with proteinuria.   Kidney disease is associated with several  abnormalities, including proteinuria and progressive  renal failure. Diffuse nodular glomerulosclerosis  (Kimmelstiel-Wilson lesions) may cause the nephrotic  syndrome. The presence of small amounts of albumin  in the urine (microalbuminuria) is associated with an  increased risk of developing progressive renal disease,  which may sometimes be prevented by more stringent  plasma glucose and blood pressure control. The renal  complications may be partly due to the increased  glycation of structural proteins in the arterial walls  supplying the glomerular basement membrane; similar  vascular changes in the retina may account for the high  incidence of diabetic retinopathy. Glycation of protein  in the lens may cause cataracts.   Infections are also more common in diabetic  patients, for example urinary tract or chest infections,  cellulitis and Candida. Diabetic neuropathy can occur,  which can be peripheral symmetric sensory, peripheral  painful, acute mononeuropathies or autonomic. It  has been suggested that sorbitol is implicated in the  aetiology of diabetic neuropathy through the action of  aldolase reductase. Erectile dysfunction is also relatively  common and in some cases may be partly neurologically  mediated.   Diabetic ulcers, for example of the feet, can lead to  gangrene and amputation. The ulcers can be ischaemic,  neuropathic or infective. The joints can also be affected,  for example Charcot’s joints. Other features of diabetes  mellitus are skin disorders, such as necrobiosis lipoidica,  and abscesses.   The management of diabetes mellitus is considered  briefly, although consulting a specialist text is   recommended if further information is required. Insulin  requirements vary in patients with type 1 diabetes. For  example, the dose may need to be increased during  any illness or during pregnancy and reduced if there is  increased activity or meals are missed.   In patients with type 2 diabetes, plasma glucose  concentrations may be controlled by diet, associated  with weight reduction, and increased physical activity,  but insulin may be required during periods of stress  or pregnancy. In this group insulin secretion can be  stimulated by the sulphonylurea drugs, such as gliclazide,  glipizide, glibenclamide or glimepiride. Biguanides,  usually metformin, can also be used and are particularly  useful in obese patients. Metformin decreases intestinal  glucose absorption and hepatic gluconeogenesis as well  as increasing tissue insulin sensitivity. Metformin can  inhibit oxidative phosphorylation, which can, under  certain circumstances, lead to lactic acid accumulation.  Acarbose delays post-prandial absorption of glucose by  inhibiting a.-glucosidase.   Other oral agents are the thiazolidinediones or  ‘glitazones’, for example rosiglitazone and pioglitazone,  which activate y-peroxisome proliferator-activated  receptors and which can reduce insulin resistance  by a number of metabolic pathways, some of which  involve increasing the transcription of nuclear proteins  that control free fatty acid and tissue glucose uptake.  Repaglinide is a meglitinide that increases insulin release  from pancreatic (3-cells and enhances tissue insulin  sensitivity. The incretins are gastrointestinal hormones  that increase insulin release from the pancreas after  eating, for example glucagon-like peptide (GLP-1)  and gastric inhibitory peptide (GIP). They are rapidly  inactivated by the enzyme dipeptidyl peptidase-4 (DPP-  4). Incretin mimetics such as exenatide or liraglutide  or DPP-4 inhibitors such as sitagliptin, vildagliptin or  saxagliptin are being used in type 2 diabetes mellitus..   It is now recognized that diabetes mellitus is not  just a glucose disorder. It is important also to optimize  abnormal plasma lipids (see Chapter 13) and correct  hypertension, particularly if there is microalbuminuria  or proteinuria (see Chapter 19).   Glycosuria can be defined as a concentration of  urinary glucose detectable using relatively insensitive,  but specific, screening tests. These tests often depend  on the action of an enzyme, such as glucose oxidase,  incorporated into a diagnostic strip. Usually, the   proximal tubular cells reabsorb most of the glucose  in the glomerular filtrate. Glycosuria, as defined  above, occurs only when the plasma, and therefore  glomerular filtrate, concentrations exceed the tubular  reabsorptive capacity. This may be because the plasma  and glomerular filtrate concentrations are more than  about lOmmol/L, and therefore the normal tubular  reabsorptive capacity is significantly exceeded. Very  rarely, if the glomerular filtration rate is much reduced,  there may be no glycosuria despite plasma glucose  concentrations more than lOmmol/L. A diagnosis of  diabetes mellitus should never be made on the basis of  glycosuria.   Blood glucose concentrations may be measured using  glucose testing reagent strips. The colour change of  the strip can be assessed visually or by using a portable  glucose meter and the reaction often involves an enzyme  determination of glucose, for example glucose oxidase.  Meters should ideally be overseen by laboratory staff  expert in point of care testing (see Chapter 30) .Although  the measurement of blood glucose concentrations  involves the discomfort of several skin punctures,  many motivated patients are able to adjust their insulin  dose more accurately based on these results than on  those obtained by testing their urine. This method of  testing is also useful in the detection of hypoglycaemia.  For patients who do not like blood testing, urinary  glucose testing can be used, but of course cannot detect  hypoglycaemia and is dependent on the renal glucose  threshold.   Glycated haemoglobin (HbA lc ) is formed by non-  enzymatic glycation of haemoglobin and is dependent  on the mean plasma glucose concentrations and on the  lifespan of the red cell; falsely low values may be found  in patients with haemolytic disease. Measurement of  blood HbA k may not reveal potentially dangerous  short-term swings and nor does HbA lc detect  hypoglycaemic episodes and thus plasma glucose  estimations may also be useful.   This was expressed as a percentage of total blood  haemoglobin concentration and gives a retrospective  assessment of the mean plasma glucose concentration  during the preceding 6-8 weeks. The higher the  glycated haemoglobin, the poorer the mean diabetic or  glycaemic control.   Glycated haemoglobin used to be expressed in  percentage units but now is expressed as mmol/mol   and conversion between the units is by the following  equation: IFCC-HbA k (mmol/mol) = [DCCT-HbA k   - 2.15] x 10.929. HbA k tests are certified by the  National Glycohemoglobin Standardization Program  (NGSP) to standardize them against the results of  the 1993 Diabetes Control and Complications Trial  (DCCT) but now are expressed as IFCC (International  Federation of Clinical Chemistry) units. Intervention  trials for type 1 and type 2 diabetes have shown that  trying to optimize glycaemic control, as judged by HbA k ,  to about 7 per cent (or above 53 mmol/mol) reduces  the risk of microvascular diabetic complications.   The measurement of plasma fructosamine concentrations  may be used to assess glucose control over a shorter  time course than that of HbA lc (about 2-4 weeks), but  the assay has methodological limitations. Fructosamine  reflects glucose bound to plasma proteins, predominantly  albumin, which has a plasma half-life of about 20 days  but is problematic in patients with hypoalbuminaemia,  for example due to severe proteinuria. This assay may  sometimes be useful in pregnancy and also if haemoglobin  variants, for example HbS or HbC, exist that may interfere  with certain HbA lc assays.   Monitoring of blood ketones may have a place  in the home management of type 1 diabetes. A  |3-hydroxybutyrate below 0.60mmol/L is normal,  whereas values between 0.60mmol/L and l.Ommol/L  may necessitate more insulin, and concentrations  greater than l.Ommol/L a warning to seek medical  advice.   One of the earliest signs of diabetic renal dysfunction  is the development of small amounts of albumin in the  urine, called microalbuminuria. Untreated, this can  progress to overt albuminuria or proteinuria (more  than 300mg/day), impaired renal function and finally  end-stage renal failure.   Microalbuminuria is defined as a urinary albumin  excretion of 30-300mg/day or 20-200 pg/min. An  albumin concentration less than 30 mg/ day or less than  20 pg/min is defined as normoalbuminuria. A random  urine sample or timed overnight collection can be  useful to assess urinary albumin excretion, although  the standard test is the urinary albumin to creatinine  ratio (ACR), which avoids a timed urine collection.  This should normally be less than 2.5g/mol in males   and less than 3.5g/mol in females. An abnormal result  should be confirmed in two out of three urine samples  in the absence of other causes of proteinuria (see  Chapter 19). Apart from being predictive of diabetic  renal complications, urinary albumin excretion is also  associated with increased vascular permeability and  enhanced risk of cardiovascular disease.   Optimization of glycaemic control can slow the  progression of microalbuminuria, as can treating  hypertension. Some recommend a target blood  pressure lower than 140/80 mmHg in type 2 diabetes, or  135/75 mmHg or lower if microalbuminuria is present.  The blood pressure targets are usually more aggressive  in type 1 diabetes, partly as the lifetime risk of overt  nephropathy is greater. Angiotensin-converting  enzyme (ACE) inhibitor therapy, such as lisinopril  in type 1 diabetic patients with microalbuminuria,  can result in a decline in the albumin excretion rate;  similar findings have been shown with enalapril in  type 2 diabetes. This action of ACE inhibitors is only  partially dependent on their blood pressure-lowering  ability, and therefore they presumably also have other  important renal protective actions. The angiotensin II  receptor antagonists (ARAs), for example irbesartan  and losartan, have also been shown to have renal  protective actions.   Acute metabolic complications of diabetes mellitus  Patients with diabetes mellitus may develop various  metabolic complications that require emergency treat-  ment, including coma, and these include the following.   This is probably the most common cause of coma seen  in diabetic patients. Hypoglycaemia is most commonly  caused by accidental overadministration of insulin or  sulphonylureas or meglitinides. Precipitating causes  include too high a dose of insulin or hypoglycaemic  drug; conversely, the patient may have missed a meal or  taken excessive exercise after the usual dose of insulin  or oral hypoglycaemic drugs.   Hypoglycaemia is particularly dangerous, and  some patients lack awareness of this; that is to say,  they lose warning signs such as sweating, dizziness  and headaches. Driving is a major hazard under such  circumstances. Patients should monitor their own  blood glucose closely, carry glucose preparations  to abort severe hypoglycaemia and avoid high-risk  activities during which hypoglycaemic attacks could  be dangerous.   A 34-year-old woman with known type 1 diabetes  mellitus was admitted to hospital following a ‘black  out’ while driving. She had recently increased her  insulin dose because she felt unwell with ‘flu’ but  unwisely had missed two meals during the day. The  results of some of her biochemistry tests were as  follows:   The blood glucose shows hypoglycaemia, secondary  to the patient having increased her insulin dose  despite having missed meals. Hypoglycaemia can  present with neurological impairment, including  impaired memory, loss of consciousness and coma.  This can be treated in the emergency situation by  giving glucose intravenously to avoid irreversible  neurological damage. It is important for patients on  insulin to monitor their own blood glucose closely,  particularly if they wish to drive.   Diabetic ketoacidosis may be precipitated by infection,  acute myocardial infarction or vomiting. The patient  who reasons ‘no food, therefore no insulin’ could  mistakenly withhold insulin. In the absence of insulin,  there is increased lipid and protein breakdown,  enhanced hepatic gluconeogenesis and impaired  glucose entry into cells.   Plasma glucose concentrations are usually in the  range 20-40 mmol/L, but may be considerably higher,   A 24-year-old woman presented to the casualty  department in a coma. The relevant biochemical  results were as follows:   The patient was shown to have type 1 diabetes  mellitus and had presented in diabetic ketoacidosis,  with hyperglycaemia, hyponatraemia, hyperkalaemia  and a metabolic acidosis.   although euglycaemic diabetic ketoacidosis has been  described when plasma glucose concentrations are only  slightly elevated.   Hyperglycaemia causes glycosuria and hence an  osmotic diuresis. Water and electrolyte loss due to  vomiting, which is common in this syndrome, increases  fluid depletion. There may be haemoconcentration and  reduction of the glomerular filtration rate enough to  cause uraemia due to renal circulatory insufficiency.  The extracellular hyperosmolality causes a shift of water  out of the cellular compartment and severe cellular  dehydration occurs. Loss of water from cerebral cells is  probably the reason for the confusion and coma. Thus  there is both cellular and extracellular volume depletion.   The rate of lipolysis is increased because of decreased  insulin activity; more free fatty acids are produced than  can be metabolized by peripheral tissues. The free fatty  acids are either converted to ketones by the liver or,  of less immediate clinical importance, incorporated  as endogenous triglycerides into VLDL, sometimes  causing severe hypertriglyceridaemia (see Chapter 13).   Hydrogen ions, produced with ketones other than  acetone, are buffered by plasma bicarbonate. However,  when their rate of production exceeds the rate of  bicarbonate generation, the plasma bicarbonate falls.  Hydrogen ion secretion causes a fall in urinary pH.  The deep, sighing respiration (Kussmaul’s respiration)   and the odour of acetone on the breath are classic  features of diabetic ketoacidosis.   Plasma potassium concentrations may be raised,  secondarily to the metabolic acidosis, before treatment  is started. This is due to failure of glucose entry into  cells in the absence of insulin and because of the low  glomerular filtration rate. Despite hyperkalaemia,  there is a total body deficit due to increased urinary  potassium loss in the presence of an osmotic diuresis.  During treatment, plasma potassium concentrations  may fall as potassium re-enters cells, sometimes causing  severe hypokalaemia unless potassium is prescribed.   Plasma sodium concentrations may be low  (hyponatraemia) or low-normal at presentation, partly  because of the osmotic effect of the high extracellular  glucose concentration, which draws water from the cells and  dilutes the sodium. In the presence of a very high plasma  glucose concentration, a normal or raised plasma sodium  concentration is suggestive of significant water depletion.   If there is severe hyperlipidaemia, the possibility of  pseudohyponatraemia must be considered (see Chapter  2). When insulin is given, gluconeogenesis is inhibited,  glucose enters cells and sodium-free water follows along  the osmotic gradient. If plasma sodium concentrations  rise rapidly, the patient may remain confused or even  comatose as long as the plasma osmolality remains  significantly raised, despite a satisfactory fall in plasma  glucose concentration. This may also occur if isosmolar  or stronger saline solutions are given inappropriately.   Hyperphosphataemia followed by hypophosphataemia  as plasma phosphate concentrations parallel those of  potassium may persist for several days after recovery from  diabetic coma. Similarly, hypermagnesaemia can result,  partly because of the acidosis.   Plasma and urinary amylase activities may be  markedly elevated and, even in the presence of abdominal  pain mimicking an ‘acute abdomen’, do not necessarily  indicate acute pancreatitis. In some patients the amylase  is of salivary rather than pancreatic origin. Some plasma  creatinine assays cross-react with ketones, resulting in a  spurious plasma creatinine elevation. Sometimes severe  hypertriglyceridaemia and chylomicronaemia result,  due to reduced lipoprotein lipase activity in the face  of insulin deficiency. A summary of the usual clinical  and biochemical findings in a patient presenting with  diabetic ketoacidosis is shown in Table 12.4.   In diabetic ketoacidosis there is always plasma  hyperosmolality due to the hyperglycaemia, and many   Table 12.4 Clinical and biochemical findings in a  patient presenting with diabetic ketoacidosis    of the symptoms, including those of confusion and  coma, are related to it. However, the term ‘hyperosmolal’  coma or ‘pre-coma’ is usually confined to a condition  in which there is marked hyperglycaemia but no  detectable ketoacidosis. The reason for these different  presentations is not clear. It has been suggested that  insulin activity is sufficient to suppress lipolysis but  insufficient to suppress hepatic gluconeogenesis or to  facilitate glucose transport into cells.   Hyperosmolal non-ketotic (HONK) coma now  may be referred to as hyperosmolar hyperglycaemic  state (HHS) and may be of sudden onset. It is  more common in older patients. Plasma glucose  concentrations may exceed 50mmol/L. The effects of  glycosuria are as described above, but hypernatraemia  due to predominant water loss is more commonly  found than in ketoacidosis and aggravates the plasma  hyperosmolality. Cerebral cellular dehydration, which  contributes to the coma, may also cause hyperventilation,  and a respiratory alkalosis, although sometimes plasma  lactic acid may rise, evoking a metabolic acidosis and  thus a mixed acid-base disturbance may occur. There  may also be an increased risk of thrombosis.   Lactic acidosis can cause a high anion gap metabolic  acidosis and coma. It may be due to the use of  metformin in certain situations, such as high doses in   the very elderly, those with renal, liver or cardiac failure  or those dehydrated or undergoing imaging tests with  contrast media (see Chapter 4).   In addition to the comas described above, a patient  with diabetes mellitus may present with other comas:   • Cerebrovascular accidents are relatively common in  diabetic patients because of the increased incidence  of vascular disease.   • Diabetic patients can, of course, have any other  coma, for example drug overdose.   • Diabetic patients are also more at risk of diabetic  nephropathy and renal failure and thus uraemic coma.   The assessment of a diabetic patient presenting in  coma or pre-coma is outlined in Table 12.5.   Only the outline of treatment will be discussed. For  details of management, the reader should consult a  textbook of medical emergencies.   Hypoglycaemic coma needs prompt glucose replacement  to avoid irreversible brain damage, for example 50 mL  of 20 per cent glucose intravenously. If intravenous  access is not an option, glucagon lmg can be given  intramuscularly. Once the patient is awake, glucose-  containing drinks can be given.   Repletion of fluid and electrolytes should be vigorous.  A 0.9 per cent normal saline solution should be  administered, usually 1 L initially and then 1 L over the   next hour and then 2h and repeated at 4h. Monitoring  central venous pressure may be useful to assess fluid  replacement. Dextrose-saline may be used when the  plasma glucose concentration is less than 15mmol/L.   If the plasma glucose concentration is more than  20mmol/T, 10 U soluble insulin should be given. A  sliding insulin scale should be instigated. Insulin is  given either by continuous intravenous infusion or  by intermittent intramuscular injections, as soon as  the plasma glucose and potassium concentrations  are known. Once the patient is eating, subcutaneous  insulin can be given instead.   If the metabolic acidosis is very severe (pH less than  7.0), bicarbonate may be infused, but only until the  blood pH rises to between about 7.15 and 7.20. It is  unnecessary and often dangerous to correct the plasma  bicarbonate concentration completely; it rapidly returns  to normal following adequate fluid and insulin therapy.  Remember that 8.4 per cent sodium bicarbonate is  very hyperosmolar and may cause hypernatraemia and  aggravate hyperosmolality. A rapid rise in the blood  pH may aggravate the hypokalaemia associated with  treatment.   The plasma potassium concentration should be  measured before insulin is given. It is almost always  raised at presentation due to the metabolic acidosis and  reduced glomerular filtration rate, although total body  potassium may be decreased. The plasma potassium  concentration may fall rapidly once treatment is  started, and therefore it should be monitored frequently  and potassium given as soon as it starts to fall. Usually  20mmol/L potassium is given to each litre bag apart  from the first litre and provided there is no oliguria or  hyperkalaemia. Diabetic ketoacidosis is severe if blood   Table 12.5 Clinical and biochemical features of a diabetic patient presenting in coma   ketones are greater than 6mmol/L and the treatment  aim is for these to be less than 0.30 mmol/L.   Urinary volume should be monitored; if it fails to  rise despite adequate rehydration, further fluid and  potassium should be given only if clinically indicated,  and then with care. The risk of deep vein thrombosis is  increased, in part due to dehydration, and thus heparin  5000 U every 8 h subcutaneously can be given.   Clinical conditions such as infection that may have  precipitated the coma should be sought and treated.  Frequent monitoring of plasma glucose, potassium and  sodium concentrations is essential to assess progress and  to detect developing hypoglycaemia, hypokalaemia or  hypernatraemia. Acid-base balance should also be assessed.   A 7 7 -year- old man with known type 2 diabetes  mellitus presented to the casualty department feeling  drowsy. His home blood glucose monitoring had  recently averaged about 25 mmol/L and a recent  glycated haemoglobin (HbA k ) result obtained by his  general practitioner was 12 per cent (108 mmol/mol).  The following blood results were returned in hospital:   The patient was found to be in a hyperosmolal non-  ketotic (HONK) diabetic coma. Note the severe  hyperglycaemia, hypernatraemia and high plasma  osmolality and presentation in an elderly patient.  HONK coma is associated with type 2 diabetes  mellitus. Ketoacidosis is usually absent, as there has  been no conversion to ketone metabolism. This is  more common in the elderly, and severe dehydration  is present and there is an increased risk of thrombotic  events and focal neurological signs. Treatment is with  careful intravenous rehydration, insulin and heparin.   The treatment of HONK coma is similar to that of  ketoacidosis. A sudden reduction of extracellular  osmolality may be harmful, and it is important to  give small doses of insulin to reduce plasma glucose  concentrations slowly, for example 1 U/h. These patients  are often very sensitive to the action of insulin. Hypo-  osmolal solutions are often used to correct volume  depletion, but these too should be given slowly. Heparin  is usually given, as there is an increased risk of venous  thrombosis.   A diabetic patient maybe in coma due to hyperglycaemia,  hypoglycaemia or any of the causes shown in Tables  12.4 and 12.5. After a thorough clinical assessment,  proceed as follows:   • Notify the laboratory that specimens are being taken  and ensure that they are delivered promptly. This  minimizes delays.   • Determination of plasma lactate will help diagnose a  lactic acidosis (see Chapter 4).   • A rapid assessment of blood glucose concentration  may be obtained using a point-of-care (POCT)  device, but results may be dangerously wrong so  these should always be checked against the results  obtained from the laboratory (see Chapter 30).   • If severe hypoglycaemia is suspected on clinical  grounds or because of the results obtained using  reagent strips, glucose should be given immediately  while waiting for the laboratory results. It is  less dangerous to give glucose to a patient with  hyperglycaemia than to give insulin to a patient with  hypoglycaemia.   • The results of point-of-care testing (see Chapter 30)  must be interpreted with caution.   • Also look for precipitating causes such as acute  myocardial infarction or infection.   In most cases a diagnosis can be established from either  fasting or random blood glucose determinations. In  equivocal cases an OGTT may be required.   Blood for plasma glucose estimation should be taken if  a patient presents with symptoms of diabetes mellitus  or glycosuria or if it is desirable to exclude the diagnosis,  for example because of a strong family history.   Diabetes mellitus is confirmed if one of the following  is present:   • a fasting venous plasma concentration of 7.0 mmol/L  or more on two occasions or once with symptoms,   • a random venous plasma concentration of  11.1 mmol/L or more on two occasions or once with  symptoms.   Diabetes mellitus is unlikely if the fasting venous  plasma glucose concentration is less than 5.5 mmol/L  on two occasions. Samples taken at random times after  meals are less reliable for excluding than for confirming  the diagnosis.   The indications for performing an OGTT to diagnose  diabetes mellitus may include:   • fasting venous plasma glucose concentration  between 5.5 mmol/L and less than 7.0 mmol/L  - this is debatable as the WHO recommends an  OGTT only if fasting plasma glucose is greater than   • a high index of clinical suspicion of diabetes mellitus,  such as a patient at high risk of gestational diabetes  with equivocal blood glucose results.   The OGTT is sometimes also useful in the diagnosis  of acromegaly (see Chapter 7).   It has been suggested that an HbA R of greater than  6.5 per cent is diagnostic of diabetes mellitus, but  this is not universally agreed as other factors such as  haemoglobin variants and abnormal erythrocyte  lifespan may affect HbA lc levels.   Before starting this test, contact your laboratory: local  details may vary.   The patient should be resting and should not smoke  during the test.   The patient fasts overnight (for at least 10 h but  not more than 16 h). Water, but no other beverage, is  allowed.   A venous sample is withdrawn for plasma glucose  estimation. If the glucose concentration is measured  in whole blood, the results will be approximately   A solution containing 75 g of anhydrous glucose  in 300 mL of water is hyperosmolar, and not only  may cause nausea and occasionally vomiting and  diarrhoea, but also, because of delayed absorption,  may affect the results of the test. It is therefore more  usual to give a solution of a mixture of glucose and  its oligosaccharides, because fewer molecules per unit  volume have less osmotic effect than the equivalent  amount of monosaccharide; the oligosaccharides are  all hydrolysed at the brush border, and the glucose  immediately enters the cells.   A solution that contains the equivalent of 75 g of  anhydrous glucose is: 113mL of Polycal made up to  approximately 300 mL with water.   This solution should be drunk slowly over a few  minutes. Further blood is taken 2h after the ingestion  of glucose.   Note that in the investigation of acromegaly, sampling  is half-hourly over the 2-h period (see Chapter 7).   Interpretation of the OGTT is shown in Table  12.6. There is controversy as to how best to interpret  the OGTT in pregnancy because of the differences in  maternal glucose metabolism, as stated earlier.   • Previous diet No special restrictions are necessary if  the patient has been on a normal diet for 3-4 days.  However, if the test is performed after a period of  carbohydrate restriction, for example as part of a  weight-reducing diet, this may cause abnormal glucose  tolerance, probably because metabolism is adjusted to  the ‘fasted state’ and so favours gluconeogenesis.   • Time of day Most OGTTs are performed in the  morning and the reference values quoted are for this  time of day. There is evidence that tests performed  in the afternoon yield higher plasma glucose  concentrations and that the accepted ‘reference   Table 12.6 Interpretation of the oral glucose tolerance test (glucose mmol/L); venous plasma preferred   values’ may not be applicable. This may be due to a  circadian variation in islet cell responsiveness.   • Drug Steroids, oral contraceptives and thiazide  diuretics may impair glucose tolerance.   By definition, hypoglycaemia is present if the plasma  glucose concentration is less than 2.5 mmol/L in a  specimen collected into a tube containing an inhibitor  of glycolysis, for example fluoride oxalate. Blood  cells continue to metabolize glucose in vitro, and low  concentrations found in a specimen collected without  such an inhibitor can be dangerously misleading  (pseudohypoglycaemia).   Symptoms of hypoglycaemia may develop at higher  concentrations if there has been a rapid fall from a  previously raised value, when adrenaline secretion is  stimulated and may cause sweating, tachycardia and  agitation. As discussed earlier, cerebral metabolism  depends on an adequate supply of glucose from ECF,  and the symptoms of hypoglycaemia may resemble  those of cerebral hypoxia (neuroglycopenia). Faintness,  dizziness or lethargy may progress rapidly to coma and,  if untreated, permanent cerebral damage or death may  occur. Existing cerebral or cerebrovascular disease may  aggravate the clinical picture. Whipple’s triad is defined  as hypoglycaemia, neuroglycopenic symptoms, and  relief of these symptoms on raising the blood glucose.   Hypoglycaemia is a disease manifestation and  not a diagnosis. There is no completely satisfactory  classification of its causes. However, one useful approach  is to divide hypoglycaemia into (inappropriate)  hyperinsulinaemia, (appropriate) hypoinsulinaemia  and reactive hypoglycaemia (Box 12.1).   Although carcinomas (especially of the liver) and  sarcomas have been reported to cause hypoglycaemia,  this occurs most commonly in association with  retroperitoneal tumours of mesenchymal origin,  but also with lymphomas, haemangiopericytomas,  liver carcinoma and leukaemia. Pleural spindle cell  tumours can be associated with a paraneoplastic   Inappropriately high insulin concentrations due to:  Pancreatic tumour - insulinoma  Hyperplasia of the pancreatic islet cells  Insulin receptor antibodies  Autoimmune insulin syndrome  Exogenous insulin  Sulphonylureas, meglitinides   Severe liver disease  End-stage renal disease  Severe congestive cardiac failure  Malaria (particularly if taking quinine)   Insulin-like growth factor (IGF)-2-secreting tumours,  e.g. liver, adrenal, breast,  mesenchymal, haemangiopericytomas  Leukaemias, lymphomas, myeloma  Widespread metastases  Reactive hypoglycaemia  Idiopathic   Von Gierke’s disease (type 1 glycogen storage disease)  Drugs, e.g. salicylates, quinine, haloperidol,  pentamidine, sulphonamides   hypoglycaemia, sometimes called Doege-Potter  syndrome. Hypoglycaemia may be the presenting  feature. The mechanism is not always clear, but may  sometimes be due to the secretion of insulin-like  growth factor 2 (IGF-2) or abnormal glycosylated big  IGF-2. The IGF-2 suppresses GH and IGF-1. Tumours  secreting IGF-2 are characterized by an increased  plasma total IGF-2:IGF-1 ratio and low plasma insulin  concentration.   Hypoglycaemia may occur in hypothyroidism,  pituitary or adrenal insufficiency. However, it is rarely  the presenting manifestation of these conditions.   The functional reserve of the liver is so great that, despite  its central role in the maintenance of plasma glucose  concentrations, hypoglycaemia is a rare complication  of liver disease. It may complicate very severe hepatitis,  hypoxic liver disease associated with congestive cardiac  failure or liver necrosis if the whole liver is affected.  Plasma IGF-1 concentration maybe low.   Renal failure can result in hypoglycaemia as the kidney,  like the liver, is a gluconeogenic organ.   Insulin or other drugs are probably the most common  causes. It is most important to take a careful drug  history. Unless the facts are deliberately concealed by the  patient, the offending drug should be easily identifiable.  Hypoglycaemia in a diabetic patient may be caused  by accidental insulin overdosage, by changing insulin  requirements, or by failure to eat after insulin has been  given. Self-administration for suicidal purposes or to  gain attention is not unknown, and homicidal use is a  remote possibility. Sulphonylureas or meglitinides may  also induce hypoglycaemia, especially in the elderly.   Hypoglycaemia due to exogenous insulin suppresses  insulin and C-peptide secretion. Measurement  of plasma C-peptide concentrations may help to  differentiate exogenous insulin administration, when  C-peptide secretion is inhibited, from endogenous  insulin secretion, when plasma C-peptide is raised,  whether it is from an insulinoma or following pancreatic  stimulation by sulphonylurea drugs.   An insulinoma is usually a small, histologically  benign primary tumour of the islet cells of the pancreas.   A 45-year-old woman was being investigated in the  endocrine unit because of hypoglycaemic episodes,  which manifested as sweating and dizzy attacks and  which were relieved by sweet drinks. Her renal, liver  and thyroid functions were all normal. Some of her  fasting biochemical results were as follows:   This patient has raised plasma insulin concentrations  in the presence of fasting hypoglycaemia. She was  subsequently shown to have an insulinoma. Note the  raised plasma insulin and C-peptide concentrations  in the presence of hypoglycaemia, suggesting the  presence of endogenous insulin secretion (exogenous  insulin administration would not be expected to be  associated with raised C-peptide concentrations).  Her symptoms and their relief by glucose-containing  drinks are classic indicators of hypoglycaemic  episodes. Insulinomas can be associated with  multiple endocrine neoplasia (MEN) syndrome,  which should be excluded.   It may present at any age. Multiple tumours may occur  and may be part of the syndrome of multiple endocrine  neoplasia (MEN). As with other functioning endocrine  tumours, hormone secretion is inappropriate and  usually excessive. C-peptide and proinsulin are released  in parallel with insulin, and plasma concentrations  are therefore inappropriately high in the presence  of hypoglycaemia. Some insulinomas secrete just  proinsulin. Attacks of hypoglycaemia occur typically at  night and before breakfast, associated with hunger, and  may be precipitated by strenuous exercise. Personality  or behavioural changes may be the first feature; some  patients present initially to psychiatrists.   Insulin antibodies can form in response to exogenous  insulin, probably less so for human insulin than for  animal types. Sometimes insulin antibodies form despite  the patient never having been exposed to exogenous  insulin - autoimmune insulin syndrome (AIS).  Insulin receptor antibodies may cause hypoglycaemia,  although they sometimes lead to insulin resistance and  hyperglycaemia.   Some people develop symptomatic hypoglycaemia  between 2 and 4h after a meal or a glucose load. Loss  of consciousness is very rare. Similar symptoms may  follow a gastrectomy or bariatric gastric banding, when  rapid passage of glucose into the intestine, and rapid  absorption, may stimulate excessive insulin secretion  (‘late dumping syndrome’). Reactive hypoglycaemia is  uncommon.   Hypoglycaemia may develop between 2 and 10 h  after the ingestion of large amounts of alcohol. It is  found most often in undernourished subjects and  chronic alcoholics but may occur in young subjects  when they first drink alcohol. Hypoglycaemia is  probably caused by the suppression of gluconeogenesis  during the metabolism of alcohol. Differentiation of  hypoglycaemia from alcoholic stupor maybe impossible  unless the plasma glucose concentration is estimated. It  may be necessary to infuse glucose frequently during  treatment, until glycogen stores are repleted and plasma  glucose concentrations are stable.   See Chapter 26 for a discussion of hypoglycaemia in  neonates and children.   Investigation of adult hypoglycaemia  Some of the causes of hypoglycaemia are shown in Box  12.1 and can be divided into hyperinsulinaemic and  hypoinsulinaemic groups. The following scheme may  be useful in investigating hypoglycaemia. It is important  to exclude pseudohypoglycaemia due to in vitro  glucose metabolism, for example an old blood sample  or one not collected into fluoride oxalate anticoagulant.  Sometimes a cause may be evident from the medical  and drug histories and clinical examination.   One of the most important tests in a patient with  proven hypoglycaemia is to measure the plasma insulin  and C-peptide concentrations when the plasma glucose  concentration is low. Plasma for these assays should  be separated from cells immediately and the plasma  stored at -20°C until hypoglycaemia has been proven.  These tests should differentiate exogenous insulin  administration and endogenous insulin production,  for example an insulinoma, from other causes of  hypoglycaemia.   Raised plasma insulin concentrations and  suppressed plasma concentrations of C-peptide suggest  exogenous insulin administration (hyperinsulinaemic  hypoglycaemia). Conversely, a high plasma insulin   and high C-peptide can be seen in sulphonylurea or  meglitinide administration, and a urine or plasma drug  screen is thus important.   Autoantibodies positive to the insulin receptor or  insulin may also evoke hypoglycaemia, such as AIS. If a  sulphonylurea drug screen and an insulin autoantibody  screen are negative, raised plasma insulin and C-peptide  concentrations are suggestive of an insulinoma.   Some tumours release proinsulin, which can also be  assayed. Imaging will also be necessary, such as magnetic  resonance imaging or computerized tomography  scanning, to localize the tumour and to help exclude  MEN syndrome (see Chapter 24). If both the plasma  insulin and C-peptide concentrations are suppressed,  the hypoglycaemia (hypoinsulinaemic hypoglycaemia)  can then be divided into non-ketotic and ketotic forms.  Hypoglycaemia with low plasma ketone concentrations,  that is, (3-hydroxybutyrate less than 600pmol/L, is  suggestive of increased insulin or IGF-1 activity such  as may occur in non-islet cell tumour hypoglycaemia  (NICTH). In NICTH, there is often increased IGF-2  secretion. Some spindle cell tumours, for example of  the thorax, may release a variant ‘big-IGF-2’ resulting  in hypoglycaemia (Doege-Potter syndrome).   hypoketonaemia can also be seen in hepatic failure or  renal disease. Therefore liver and renal function shouldbe  checked. Conversely, hypoinsulinaemic hypoglycaemia  with high plasma ketones, that is, (3-hydroxybutyrate  more than 600 pmol/F, can be found in hypopituitarism  (see Chapter 7) when the plasma GH concentration is  usually low. In addition, consider adrenal insufficiency  (see Chapter 8) and hypothyroidism (see Chapter 11),  in which plasma GH is usually high. High alcohol intake  can also present with hypoinsulinaemic hypoglycaemia  and raised ketone concentrations.   However, more commonly, the patient has been  referred for investigation of previously documented  hypoglycaemia or with a history strongly suggestive  of hypoglycaemic attacks. It is also essential to exclude  adrenal insufficiency, especially in diabetes mellitus  patients with unexplained hypoglycaemia or reduced  insulin requirements. If no cause is identified at this  point, it may be possible to induce hypoglycaemia by  provocation tests, although these are not without the  risk of severe hypoglycaemic episodes.   glucose concentration less than 2.5mmol/F during  an overnight (18-h) fast when assayed on three  separate occasions.   • Exercise test Exercise may be used in the induction of  insulin-induced hypoglycaemia. Blood is collected at  10-min intervals during 30 min of intense exercise,  for example treadmill, and 30 min after stopping.  Plasma insulin and C-peptide (and proinsulin, if  necessary) are assayed and will be inappropriately  high in endogenous hyperinsulinaemia and  suppressed appropriately in hypoinsulinaemic  hypoglycaemia.   • Prolonged fast The prolonged fast, of up to 4 h, may  be reserved for cases where there is a high index  of suspicion of hypoglycaemia that has not been  provoked spontaneously or by the above tests. It  should be noted that, during the prolonged fast,  the patient must be under close supervision as  potentially dangerous.   Blood should be taken every 6 h for plasma glucose  and insulin estimations and, if symptoms occur, should  be assayed for glucose immediately. The test can be  stopped if hypoglycaemia is demonstrated. The patient  should be fed after the test. If prolonged fasting does not  induce hypoglycaemia, endogenous hyperinsulinism  is unlikely to be the cause of the symptoms. A few  normal individuals may show plasma glucose levels  less than 2.5mmol/E during the test, but they do not  exhibit neuroglycopenic symptoms or Whipple’s triad.  A plasma insulin to C-peptide ratio of less than 1 has  about 89 per cent sensitivity and 100 per cent specificity  for insulinoma.   The insulin provocation test is now rarely used (as  it can evoke dangerous hypoglycaemia) - in it, insulin  administration fails to suppress plasma insulin and  C-peptide in cases of insulinoma. Reactive hypoglycaemia  should not be diagnosed by a prolonged 75 g OGTT,  as normal individuals may show false-positive results.  However, the mixed meal, based on the sort of foods that  evoke the attacks, can be used to investigate post-prandial  neuroglycopenic symptoms. Capillary blood samples are  taken prior to and every half-hour for 6 h after mixed-meal  ingestion. Reactive hypoglycaemia is a possible diagnosis  if the patient develops neuroglycopenic symptoms and  the capillary plasma glucose concentration is 3.0mmol/F  or less. Note that venous plasma should ideally not be  used, as false-positive results may result because post-  prandial glucose concentrations can be l-2mmol/E  lower than in capillary blood samples.   Treatment of hypoglycaemia  Milder cases can be managed with oral glucose-containing  preparations. Severe hypoglycaemia should be treated  by urgent intravenous administration of 10-20 mL of  at least 10 per cent (and in adults 20 per cent) glucose  solution. Some patients may need to be maintained on a  glucose infusion until the cause has been established and  treated. Intramuscular glucagon 1 mg can also be used if  intravenous access is a problem, but this should not be  given in cases of insulinoma. An insulinoma should be  removed surgically. If this is contraindicated for clinical  reasons, diazoxide may maintain normoglycaemia.   Laboratory tests  Estimation of plasma or blood glucose  Glucose is measuredin the laboratory by specific enzymatic  methods. The supply of glucose to cells depends on  extracellular (plasma) concentrations. The measurement  of plasma glucose concentrations is preferable to that of  whole blood, which is now rarely used. Blood cells, with  very low glucose concentrations, ‘dilute’ glucose, giving  results 10-15 per cent lower than in plasma, the actual  amount depending on the haematocrit. Capillary blood  from a finger prick is used for home glucose monitoring  and the results for such samples may fall between those of  venous whole blood and venous plasma.   Unless plasma is separated from the blood cells  within about an hour of collection, the whole blood  sample must be mixed with an inhibitor of glycolysis,  such as fluoride. This helps prevent an in vitro fall in the  plasma glucose concentration as glycolysis continues,  which may result in pseudohypoglycaemia.   Enzyme reagent strips specific for glucose, such as  those that use a glucose oxidase method, best detect  glycosuria. The directions for use are supplied with the  strips. Glycosuria maybe due to:   • renal glycosuria, which may be inherited as an  autosomal dominant trait or in Fanconi’s syndrome,   • if the urine contains large amounts of ascorbic acid  after the ingestion of therapeutic doses,   • after the injection of tetracyclines, which contain  ascorbic acid as a preservative.   False-positive results may occur if the urine container  is contaminated with detergent.   Reducing substances in the urine can be detected  using copper-containing reagents such as those  incorporated in Clinitest tablets. These are rarely  used now in the management of diabetes mellitus;  however, in the neonatal period, the finding of other  reducing substances may suggest an inborn error of  metabolism, such as galactosaemia (see Chapter 27).  It is therefore important to use this test, rather than  one that is specific for glucose, in screening for such  conditions.   Non-glucose-reducing substances are identified by  chromatography and specific tests. The significance of  a Clinitest-positive result varies with the substances,  which are as follows.   • Glucuronates are relatively common urinary  reducing substances. Numerous drugs, such as  salicylates and their metabolites, are excreted in  the urine after conjugation with glucuronate in the  liver.   • Fructose may appear in the urine after very large oral  doses of sucrose, or after excessive fruit ingestion, but  usually fructosuria is due to one of two rare inborn  errors of metabolism, both transmitted as autosomal  recessive disorders:   - essential fructosuria is usually a benign  condition (hepatic fructokinase deficiency),   - hereditary fructose intolerance is characterized  by hypoglycaemia that may lead to death in  infancy.   - alimentary pentosuria, after excessive ingestion  of fruits such as cherries and grapes - the  pentoses are arabinose and xylose,   - essential pentosuria, a rare recessive disorder  due to L-xylulose reductase deficiency,  characterized by the excretion of xylose - it is  usually benign.   • Homogentisic acid appears in the urine in the rare  inborn error alkaptonuria. It is usually recognizable  because it forms a blackish precipitate in urine on  standing. Urea and creatinine may give weak positive  results at high concentrations.   Most simple urine tests for ketones are more sensitive for  detecting acetoacetate than acetone; (3-hydroxybutyrate  does not always react in these tests. Occasional colour  reactions resembling, but not identical to, that of  acetoacetate may be given by phthalein compounds  when used as a laxative.   Ketostix and Acetest are strips and tablets,  respectively, impregnated with ammonium sulphate  and sodium nitroprusside.   Remember that raised concentrations of urinary  ketones can occur not only in diabetic ketoacidosis but  also in alcoholic ketoacidosis or starvation, and in some  urinary tract infections.   • Diabetes mellitus is a common medical condition,  and an understanding of its biochemistry aids its  medical management. Type 1 diabetes mellitus is  associated with insulin deficiency and may present  with weight loss and urinary ketones in young  individuals. There is a relationship with autoimmune  disease. Treatment is with insulin. Conversely,  type 2 diabetes mellitus is usually associated with  insulin resistance, increased body weight and later  age presentation. There may be a family history  of diabetes mellitus. Treatment involves diet and  biguanides, sulphonylureas, glitazones or incretins,  although insulin may sometimes be needed.   • Biochemical tests have a major role in the  management of diabetes mellitus and in monitoring   its complications, such as in the control of blood  glucose, HbA k , plasma lipids and urinary ACR.  Diabetes mellitus can present with various comas,  including hypoglycaemia, diabetic ketoacidosis  (type 1), HONK and lactic acidosis.  Hypoglycaemia can present with neurological  impairment and coma. A useful classification is to  divide hypoglycaemia into that with high plasma  insulin and that with low insulin levels. The causes  of hyperinsulinaemic hypoglycaemia include  insulinomas and following insulin administration.  The causes of hypoinsulinaemic hypoglycaemia  include severe hepatic disease, adrenal insufficiency,  pituitary failure and non-pancreatic tumours  producing insulin-like substances.   Lipids play a critical role in almost all aspects of  biological life - they are structural components in  cells and are involved in metabolic and hormonal  pathways. The importance of having a knowledge of  lipid disorders cannot be overstated, not least because  they are common in clinical practice and, in some cases  associated with atherosclerosis such as coronary heart  disease, one of the biggest killers in urbanized societies.   Lipids are defined as organic compounds that are  poorly soluble in water but miscible in organic solvents.  Lipidology is the study of abnormal lipid metabolism.  An understanding of the pathophysiology of plasma  lipid metabolism is usefully based on the concept of  lipoproteins, the form in which lipids circulate in plasma.   The chemical structures of the four main forms of lipid  present in plasma are illustrated in Figure 13.1.   These are straight-chain carbon compounds of varying  lengths. They may be saturated, containing no double  bonds, monounsaturated, with one double bond, or  polyunsaturated, with more than one double bond  (Table 13.1).   Fatty acids can esterify with glycerol to form  triglycerides or be non-esterified (NEFAs) or free.  Plasma NEFAs liberated from adipose tissue by lipase  activity are transported to the liver and muscle mainly  bound to albumin. The NEFAs provide a significant  proportion of the energy requirements of the body.  Summary diagrams of fatty acid synthesis and oxidation  are shown in Figures 13.2 and 13.3.   Triglycerides are transported from the intestine to  various tissues, including the liver and adipose tissue, as  lipoproteins. Following hydrolysis, fatty acids are taken  up, re-esterified and stored as triglycerides. Plasma   Figure 13.1 Lipid structures. P, phosphate; N, nitrogenous base; R, fatty acid.   Table 13.1 Some of the major fatty acids found in the  plasma   triglyceride concentrations rise after a meal, unlike that  of plasma cholesterol.   Phospholipids are complex lipids, similar in structure  to triglycerides but containing phosphate and a  nitrogenous base in place of one of the fatty acids. They  fulfil an important structural role in cell membranes,  and the phosphate group confers solubility on non-  polar lipids and cholesterol in lipoproteins.   A family of nuclear receptors that are activated by  fatty acids - called peroxisome proliferator-activated  receptors (PPARs) - has been described and implicated  in insulin resistance and dyslipidaemia. The PPARs  can be subdivided into a- PPARs, which are activated  by fibrate drugs, and y-PPARs, which are activated by  thiazolidinedione drugs, for example pioglitazone or  rosiglitazone.   Cholesterol is a steroid alcohol found exclusively in  animals and present in virtually all cells and body fluids.  It is a precursor of numerous physiologically important  steroids, including bile acids and steroid hormones. A  summary of the cholesterol synthetic pathways is shown  in Figure 13.4. The rate-limiting enzyme is 3-hydroxy-  3-methylglutaryl coenzyme A reductase (HMG-CoA  reductase), which is controlled by negative feedback by  the intracellular concentration. About two-thirds of the  plasma cholesterol is esterified with fatty acids to form  cholesterol esters.   Because lipids are relatively insoluble in aqueous media,  they are transported in body fluids as, often spherical,    Figure 13.2 Summary of fatty acid synthesis and  adipose tissue substrates. Reproduced with kind  permission from Candlish JK and Crook M. Notes on  Clinical Biochemistry. Singapore: World Scientific  Publishing, 1993.   Figure 13.3 Summary of fatty acid oxidation. CoA,  coenzyme A. Reproduced with kind permission  from Candlish JK and Crook M. Notes on Clinical  Biochemistry. Singapore: World Scientific  Publishing, 1993.   Figure 13.4 Summary of pathways of cholesterol  synthesis. CoA, coenzyme A. Reproduced with kind  permission from Candlish JK and Crook M. Notes on  Clinical Biochemistry. Singapore: World Scientific  Publishing, 1993.   soluble protein complexes called lipoproteins. Lipids  can be derived from food (exogenous) or synthesized  in the body (endogenous). The water-soluble (polar)  groups of proteins, phospholipids and free cholesterol  face outwards and surround an inner insoluble (non-  polar) core of triglyceride and cholesterol esters.   Lipoproteins are classified by their buoyant density,  which inversely reflects their size. The greater the lipid  to protein ratio, the larger their size and the lower the  density. Lipoproteins can be classified into five main  groups (Table 13.2). The first three are triglyceride   rich and, because of their large size, they scatter light,  which can give plasma a turbid appearance (lipaemic)  if present in high concentrations:   • Chylomicrons are the largest and least dense  lipoproteins and transport exogenous lipid from the  intestine to all cells.   • Very low-density lipoproteins (VLDLs) transport  endogenous lipid from the liver to cells.   • Intermediate-density lipoproteins (IDLs), which  are transient and formed during the conversion of  VLDL to low-density lipoprotein (LDL), are not  normally present in plasma.   The other two lipoprotein classes contain mainly  cholesterol and are smaller in size:   • Low-density lipoproteins are formed from VLDLs  and carry cholesterol to cells.   • High-density lipoproteins (HDLs) are the most  dense lipoproteins and are involved in the transport  of cholesterol from cells back to the liver (reverse  cholesterol transport). These lipoproteins can be  further divided by density into HDL 2 and HDL 3 .   If a lipaemic plasma sample, for example after a  meal, is left overnight at 4°C, the larger and less dense  chylomicrons form a creamy layer on the surface. The  smaller and denser VLDL and IDL particles do not rise,  and the sample may appear diffusely turbid. The LDL  and HDL particles do not contribute to this turbidity  because they are small and do not scatter light. Fasting  plasma from normal individuals contains only VLDL,  LDL and HDL particles.   In some cases of hyperlipidaemia, the lipoprotein  patterns have been classified (Fredrickson’s  classification) according to their electrophoretic  mobility. Four principal bands are formed, based on  their relative positions, by protein electrophoresis,  namely a (HDL), pre-|3 (VLDL), |3 (LDL) and  chylomicrons (Table 13.3).   Cho, cholesterol; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; PL, phospholipid; Pro,  protein; Tg, triglyceride; VLDL, very low-density lipoprotein.   Intermediate-density lipoproteins in excess may  produce a broad (3-band. Some individuals with  hyperlipidaemia may show varying electrophoretic  patterns at different times.   Ultracentrifugation (separation based upon particle  buoyant density) or electrophoretic techniques  are rarely used in routine clinical practice as these  may require completed apparatus and experienced  operators. Instead, the lipoprotein composition  of plasma may be inferred from standard clinical  laboratory lipid assays. As fasting plasma does not  normally contain chylomicrons, the triglyceride  content reflects VLDL. Furthermore, generally about  70 per cent of plasma cholesterol is incorporated as  LDL and 20 per cent as HDL. The latter particles,  because of their high density, can be quantified  by precipitation techniques that can assay their  cholesterol content by subtraction, although direct  HDL assays are now often used.   The Friedewald equation enables plasma LDL  cholesterol concentration to be calculated and is often  used in clinical laboratories:   This equation makes certain assumptions, namely  that the patient is fasting and the plasma triglyceride  concentration does not exceed 4.5mmol/L (otherwise  chylomicrons make the equation inaccurate).   There has been recent interest in the subdivision  of LDL particles into small dense LDL, and LDL 3 ,  which appear to be more atherogenic and more easily  oxidized than the larger LDL particles. Additionally,   another lipoprotein called lipoprotein (a), or Lp(a),  has been found. This is similar in lipid composition  to LDL but has a higher protein content. One of its  proteins, called apolipoprotein (a), shows homology to  plasminogen and may disrupt fibrinolysis, thus evoking  a thrombotic tendency. The plasma concentration of  Lp(a) is normally less than 0.30 g/L and it is thought to  be an independent cardiovascular risk factor.   The proteins associated with lipoproteins are called  apolipoproteins (apo). ApoA (mainly apoA t and apoA 2 )  is the major group associated with HDL particles. The  apoB series (apoB 100 ) is predominantly found with  LDL particles and is the ligand for the LDL receptor.  Low-density lipoprotein has one molecule of apoB 100  per particle. Some reports have suggested that the  plasma apoA 3 to apoB ratio may be a useful measure of  cardiovascular risk (increased if the ratio is less than 1)  and it is not significantly influenced by the fasting status  of the patient. The apoC series is particularly important  in triglyceride metabolism and, with the apoE series,  freely interchanges between various lipoproteins. Some  of the functions of these apolipoproteins are described  in Table 13.4.   Lipoprotein-associated phospholipase A 2 [also  called platelet-activating factor acetylhydrolase (PAF-  AH)] is present mainly on LDL and to a lesser degree  HDL. It is produced by inflammatory cells and is  involved in atherosclerosis formation and levels are  associated with increased risk of coronary artery  disease and stroke.   HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein;  LCAT, lecithin-cholesterol acyltransferase; LDL, low-density  lipoprotein; VLDL, very low-density lipoprotein.   Figure 13.4 Summary of pathways of cholesterol  synthesis. CoA, coenzyme A. Reproduced with kind  permission from Candlish JK and Crook M. Notes on  Clinical Biochemistry. Singapore: World Scientific  Publishing, 1993.   soluble protein complexes called lipoproteins. Lipids  can be derived from food (exogenous) or synthesized  in the body (endogenous). The water-soluble (polar)  groups of proteins, phospholipids and free cholesterol  face outwards and surround an inner insoluble (non-  polar) core of triglyceride and cholesterol esters.   Lipoproteins are classified by their buoyant density,  which inversely reflects their size. The greater the lipid  to protein ratio, the larger their size and the lower the  density. Lipoproteins can be classified into five main  groups (Table 13.2). The first three are triglyceride   rich and, because of their large size, they scatter light,  which can give plasma a turbid appearance (lipaemic)  if present in high concentrations:   • Chylomicrons are the largest and least dense  lipoproteins and transport exogenous lipid from the  intestine to all cells.   • Very low-density lipoproteins (VLDLs) transport  endogenous lipid from the liver to cells.   • Intermediate-density lipoproteins (IDLs), which  are transient and formed during the conversion of  VLDL to low-density lipoprotein (LDL), are not  normally present in plasma.   The other two lipoprotein classes contain mainly  cholesterol and are smaller in size:   • Low-density lipoproteins are formed from VLDLs  and carry cholesterol to cells.   • High-density lipoproteins (HDLs) are the most  dense lipoproteins and are involved in the transport  of cholesterol from cells back to the liver (reverse  cholesterol transport). These lipoproteins can be  further divided by density into HDL 2 and HDL 3 .   If a lipaemic plasma sample, for example after a  meal, is left overnight at 4°C, the larger and less dense  chylomicrons form a creamy layer on the surface. The  smaller and denser VLDL and IDL particles do not rise,  and the sample may appear diffusely turbid. The LDL  and HDL particles do not contribute to this turbidity  because they are small and do not scatter light. Fasting  plasma from normal individuals contains only VLDL,  LDL and HDL particles.   In some cases of hyperlipidaemia, the lipoprotein  patterns have been classified (Fredrickson’s  classification) according to their electrophoretic  mobility. Four principal bands are formed, based on  their relative positions, by protein electrophoresis,  namely a (HDL), pre-|3 (VLDL), |3 (LDL) and  chylomicrons (Table 13.3).   Cho, cholesterol; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; PL, phospholipid; Pro,  protein; Tg, triglyceride; VLDL, very low-density lipoprotein.    Figure 13.6 Endogenous lipid pathways. HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein;  LDL, low-density lipoprotein; NEFA, non-esterified (free) fatty acid; VLDL, very low-density lipoprotein.   from chylomicron remnants via the exogenous pathway  or synthesized locally. These lipids are transported from  the liver as VLDL.   Very low-density lipoprotein is a large triglyceride-  rich particle consisting also of apoB 100 , apoC and apoE.  Following hepatic secretion, it incorporates additional  apoC from HDL particles within the circulation. Like  chylomicrons, VLDL is hydrolysed by lipoprotein  lipase in the peripheral tissues, albeit more slowly. The  resulting VLDL remnant or IDL contains cholesterol  and triglyceride as well as apoB and apoE and is rapidly  taken up by the liver or converted by the action of  hepatic lipase to LDL by losing apoE and triglyceride.   Low-density lipoprotein is a small cholesterol-rich  lipoprotein containing only apoB. It represents about  70 per cent of the total plasma cholesterol concentration.  It can be taken up by most cells, although mainly the  liver by the LDL or B/E receptor which recognizes and   binds apoB 100 . Within the cell, the LDL particles are  broken down by lysosomes, releasing cholesterol. This  cholesterol can be incorporated into cell membranes or  in specific tissues such as the adrenal cortex or gonads  and utilized in steroid synthesis.   Most cells are able to synthesize cholesterol, but, to  avoid intracellular accumulation, there is a feedback  control system reducing the rate of synthesis of the  LDL receptors. Although most of the plasma LDL is  removed by LDL receptors, if the plasma cholesterol  concentration is excessive, LDL particles, by virtue  of their small size, can infiltrate tissues by passive  diffusion and can even cause damage, as in atheroma  formation within arterial walls. An alternative route of  removal of LDL is via the reticuloendothelial system,  collectively termed the scavenger cell pathway, which  recognizes only chemically modified LDL, for example  oxidized LDL.   • it is responsible for most of the endogenous  cholesterol synthesis,   Cholesterol is synthesized via a series of enzymatic  steps, with HMG-CoA reductase being the rate-limiting  enzyme (Fig. 13.4). Suppression of this enzyme may  occur if cholesterol synthesis is excessive. Involved in  these processes is a family of transcription-regulating  proteins called sterol regulatory element-binding  proteins. Intracellular cholesterol accumulation also  reduces the number of hepatic LDL receptors, and  therefore LDL entry into cells declines and the plasma  concentration rises. However, if the dietary intake of  cholesterol is excessive, intracellular accumulation can  still occur. About 30-60 per cent of the dietary intake  of cholesterol (of 1-2 mmol) is absorbed, this amount  being increased if the diet is rich in saturated fat. High   saturated fat intake can also suppress LDL receptor  activity and is a bigger driver of cholesterol metabolism  than dietary cholesterol. The richest dietary sources of  cholesterol are egg yolks, dairy products and red meat.   Net loss of body cholesterol can occur by bile  excretion. Some bile salts are reabsorbed from the  intestinal lumen and return to the liver via the  enterohepatic circulation. Interruption of this process  results in enhanced conversion of cholesterol to bile  salts, reduced hepatic cholesterol stores and increased  LDL receptors (Fig. 13.7). The rate of LDL receptor  synthesis is also increased by thyroxine and oestrogens  and decreases with age.   The transport of cholesterol from non-hepatic cells  to the liver involves HDL particles, in a process called  reverse cholesterol transport (Fig. 13.8). The HDL is  synthesized in both hepatic and intestinal cells and  secreted from them as small, nascent HDL particles  rich in free cholesterol, phospholipids, apoA and    Figure 13.7 The low-density lipoprotein (LDL) receptor. ACAT, acyl coenzyme A acyl transferase; CoA, coenzyme  A; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A.   Figure 13.8 Reverse high-density lipoprotein  (HDL) cholesterol transport. Al, apoAl; E, apoE.  Reproduced with kind permission from Candlish  JK and Crook M. Notes on Clinical Biochemistry.  Singapore: World Scientific Publishing, 1993.   apoE. This cholesterol acquisition is stimulated by  adenosine triphosphate-binding cassette protein 1  (ABC1). If the plasma concentration of VLDL or  chylomicrons is low, apoC is also carried in HDL, but  as the plasma concentrations of these lipoproteins rise,  these particles take up apoC from HDL. In addition,  HDL can be formed from the surface coat of VLDL  and chylomicrons. Various factors control the rate of  HDL synthesis, including oestrogens, thus explaining  why plasma concentrations are higher in menstruating  women than in menopausal women or men.   The enzyme lecithin-cholesterol acyltransferase  (LCAT) is present on HDL and catalyses the  esterification of free cholesterol and is activated by  apoA , the predominant apolipoprotein of HDL.  Some HDL particles also contain apoA,. Most of this  esterified cholesterol is transferred to LDL, VLDL  and chylomicron remnants and thus ultimately  reaches the liver. Some may be stored within the core  of the HDL particle and taken directly to the liver.  Cholesterol ester transfer protein (CETP) is involved  in these processes.   The HDL particles can be divided into pre-(3 (or  precursor) HDL, HDL, and HDL r The HDL 2 , which is  a precursor of smaller HDL particles, interconverts as a  result of the acquisition of cholesterol by HDL, through  the actions of LCAT and hepatic lipase.   High-density lipoprotein also contains other  enzymes, including paroxanase, which may have an   antioxidant role. Removal of HDL may occur by  endocytosis, although there may be specific receptors  such as the murine class B type I scavenger receptor  (SR-BI) in liver and steroidogenic tissue, for example  adrenal glands, ovaries and testes. Thus HDL-derived  cholesterol can be ‘off-loaded’ in the liver and secreted  in bile or taken up and utilized for steroid synthesis.   In hypertriglyceridaemia there is increased VLDL  concentration and, under the action of hepatic lipase,  the HDL becomes overloaded with triglyceride; they  reduce in size, losing apoA , and the concentration of  HDL cholesterol falls. Thus, in hypertriglyceridaemia  one often sees an inverse relationship between plasma  triglyceride and HDL cholesterol concentrations.   cardioprotective not only because of the reverse  cholesterol transport system, which helps to remove  cholesterol from the peripheral tissues, but also  because of the mechanisms that include increased  atherosclerotic plaque stability, protection of LDL from  oxidation, and maintaining the integrity of the vascular  endothelium.   A plasma HDL cholesterol concentration of less  than l.Ommol/L confers increased cardiovascular risk  and can be raised by various lifestyle changes, such as  smoking cessation, regular exercise and weight loss.  The fibrate drugs or nicotinic acid are sometimes used  if these measures fail (see later). A low HDL cholesterol  concentration is associated with diabetes mellitus type  2, obesity and the metabolic syndrome (see Chapter  12). Concentration of plasma non-HDL cholesterol  (total cholesterol - HDL cholesterol) may be a better  indicator of cardiovascular risk than that of LDL  cholesterol.   The study of hyperlipidaemias is of considerable  importance, mainly because of the involvement of lipids  in cardiovascular disease. Lredrickson, Levy and Lees first  defined the hyperlipidaemias in a classification system  based on which plasma lipoprotein concentrations  were increased (Table 13.3). Although this so-called  Lredrickson’s classification helped to put lipidology on  the clinical map, it was not a diagnostic classification.  It gives little clue as to the aetiology of the disorder;  indeed, all of the phenotypes can be either primary  or secondary. Lurthermore, the Lredrickson type can  change as a result of dietary or drug intervention.  Nowadays, a more descriptive classification is used for  the primary hyperlipidaemias, as follows.   This can be due to familial lipoprotein lipase deficiency,  an autosomal recessive disorder affecting about 1 in  1 000 000 people. The gene for lipoprotein lipase is found  on chromosome 8, and genetic studies have shown  insertions or deletions within the gene. Lipoprotein  lipase is involved in the exogenous lipoprotein pathway  by hydrolysing chylomicrons to form chylomicron  remnants, and also in the endogenous pathway by  converting VLDL to IDL particles.   Presentation as a child with abdominal pain (often  with acute pancreatitis) is typical. There is probably no  increased risk of coronary artery disease. Gross elevation  of plasma triglycerides due to the accumulation  of uncleared chylomicron particles occurs (Fig.  13.9). Lipid stigmata include eruptive xanthomata,  hepatosplenomegaly and lipaemia retinalis (Fig. 13.10).   Other variants of the chylomicron syndrome  include circulating inhibitors of lipoprotein lipase  and deficiency of its physiological activator apoC,.  Apolipoprotein C 2 deficiency is also inherited as an  autosomal recessive condition affecting about 1 in  1000 000 people. The gene for apoC 2 is located on  chromosome 19 and mutations resulting in low plasma  concentrations have been found.    Figure 13.9 Whole blood of a patient with lipoprotein  lipase deficiency. Note chylomicron creamy  appearance. Reproduced with kind permission  from Nyhan WL and Barshop BA. Atlas of Inherited  Metabolic Diseases, 3rd edition. London: Hodder  Arnold, 2012.    Figure 13.10 Lipaemia retinalis in a patient with  lipoprotein lipase deficiency. Reproduced with kind  permission from Nyhan WL and Barshop BA. Atlas of  Inherited Metabolic Diseases, 3rd edition. London:  Hodder Arnold, 2012.   Treatment of the chylomicron syndrome involves  a low- fat diet, aiming for less than 20 g of fat a day, if  possible, although compliance on such a diet may be  difficult. Some clinicians supplement the diet with  medium-chain triglycerides and also give 1 per cent of  the total calorie intake as linoleic acid.   In cases of apoC 2 deficiency, fresh plasma may  temporarily restore plasma apoC, levels. To confirm  the diagnosis of familial lipoprotein lipase deficiency,  plasma lipoprotein lipase can be assayed after the  intravenous administration of heparin, which releases  the enzyme from endothelial sites. The assay is  complicated in that other plasma lipases (hepatic lipase  and phospholipase, for example) contribute to the  overall plasma lipase activity. Inhibition of lipoprotein  lipase can be performed using protamine, high saline  concentrations or specific antibodies and its overall  activity can be calculated by subtraction.   If apoC 2 deficiency is suspected, the plasma  concentrations of this activator can be assayed. Patients  may show a type I or type V Fredrickson’s phenotype.  Family members should be investigated.   Familial hypercholesterolaemia  This condition is usually inherited as an autosomal  dominant trait and was described by Goldstein and  Brown. The inheritance of one mutant gene that  encodes for the LDL receptor affects about 1 in every  500 people (more common in certain groups such  as Afrikaners and French Canadians), resulting in   impaired LDL catabolism and hypercholesterolaemia.  At least five types of mutation of the LDL receptor  have been described, resulting in reduced synthesis,  failure of transport of the synthesized receptor to the  Golgi complex within the cell, defective LDL binding or  inadequate expression or defective recycling of the LDL  receptor at the cell surface.   According to the Simon Broome register, definite  familial hypercholesterolaemia (FH) is defined as  a plasma cholesterol concentration of more than  7.5mmol/L in an adult (more than 6.7mmol/L in  children under 16 years) or a plasma LDL cholesterol  concentration of more than 4.9mmol/L in an adult  in the presence of tendon xanthoma. Possible FH is  defined as a plasma cholesterol concentration of more  than 7.5mmol/L in an adult (more than 6.7mmol/L in  children under 16 years) or a plasma LDL cholesterol  concentration of more than 4.9mmol/L in an adult,  plus a family history of either an elevated plasma  cholesterol concentration of more than 7.5mmol/L in  a first-degree or second-degree relative or myocardial  infarction below the age of 50 years in a first-degree   A 23-year-old woman had her plasma lipids checked  by her general practitioner because her father had  died of a myocardial infarction aged 44 years. Her  24-year-old brother had hyperlipidaemia. Her renal,  liver and thyroid function tests were normal, as was  her blood glucose.   On examination, she had tendon xanthomata on her  Achilles tendons and bilateral corneal arci.   Note the considerably raised plasma cholesterol  concentration. The absence of an obvious secondary  hyperlipidaemia, in conjunction with the family  history of a first- degree relative with premature  cardiovascular disease and hyperlipidaemia, suggests  a genetic hyperlipidaemia. The presence of tendon  xanthomata and premature corneal arci supports the  diagnosis of familial hypercholesterolaemia. This is  usually an autosomal dominant disorder and usually  a defect of the low-density lipoprotein (LDL) receptor.   relative or below the age of 60 years in a second-degree  relative.   Typically, patients manifest severe hypercholes-  terolaemia, with a relatively normal plasma triglyceride  concentration in conjunction with xanthomata, which  can affect the back of the hands, elbows, Achilles tendons  or the insertion of the patellar tendon into the pretibial  tuberosity (Fig. 13.11). Premature cardiovascular  disease is often observed, along with premature corneal  arci (Fig. 13.12).   Using the Fredrickson’s classification, this  condition has also been termed familial type Ha    Figure 13.1 1 Tendinous xanthomas in familial  hypercholesterolaemia. Reproduced with kind  permission from Nyhan WL and Barshop BA. Atlas of  Inherited Metabolic Diseases, 3rd edition. London:  Hodder Arnold, 2012.    Figure 13.12 Corneal arcus in familial  hypercholesterolaemia. Reproduced with kind  permission from Nyhan WL and Barshop BA. Atlas of  Inherited Metabolic Diseases, 3rd edition. London:  Hodder Arnold, 2012.   hyperlipoproteinaemia, although some patients may  show a type lib phenotype. Plasma HDL cholesterol  concentration can vary in different individuals,  although low concentrations may increase the  likelihood of cardiovascular disease. It has been shown  that in heterozygote FH there is more likely to be an  increased amount of plasma Lp(a) in those subjects  with cardiovascular disease.   The diagnosis of FH is usually obvious from the  markedly elevated plasma cholesterol concentration  and the presence of tendon xanthomata in the patient or  first-degree relation. The diagnosis may not be so clear  cut in patients without the lipid stigmata. A functional  assay of the LDL receptors has recently been described  using cultured lymphocytes, but this has not yet gained  wide routine acceptance, and deoxyribonucleic acid  (DNA) screening is now important. The response to  a lipid-lowering diet is often disappointing and the  treatment is usually with the HMG-CoA reductase  inhibitors, that is, the statins.   Homozygous FH can be very severe. There is a  considerable risk of coronary artery disease, aortic  stenosis and early fatal myocardial infarction before  the age of 20 years. Florid xanthoma occurs in  childhood including tendon, planar and cutaneous  types. Atheroma of the aortic root may manifest before  puberty, associated with coronary ostial stenosis.   In homozygous FH, treatment to lower the plasma  cholesterol concentration (which can be as high as  20 mmol/L or more) is essential in order to try to reduce  the likelihood of sudden death due to coronary artery  disease. Plasma exchange, LDL apheresis or heparin  extracorporeal LDL precipitation (HELP) can be used  in an attempt to remove the plasma LDL particles and  thus reduce the plasma cholesterol concentration.   There is a rare recessive form of FH and also a form  of FH associated with increased proprotein convertase  subtilisin/kexin type 9 (PCSK9) expression which  regulates LDL receptors.   Familial defective apoB 3500  This condition is due to a mutation in the apoB gene  resulting in a substitution of arginine at the 3500 amino  acid position for glutamine. Apolipoprotein B is the  ligand upon the LDL particle for the LDL receptor. It  may be indistinguishable clinically from FH and is also  associated with hypercholesterolaemia and premature  coronary artery disease. The treatment is similar to that  for heterozygote FH. The apoB gene is located upon  chromosome 2.   In familial combined hyperlipidaemia (FCH), the plasma  lipids may elevated, plasma cholesterol concentrations  often being between 6 mmol/L and 9 mmol/L and plasma  triglyceride between 2 mmol/L and 6 mmol/L. The  Fredrickson’s phenotypes seen in this condition include  Ila, lib and IV. Familial combined hyperlipidaemia may  be inherited as an autosomal dominant trait (although  others suggest that there may be co-segregation of more  than one gene). About 0.5 per cent of the European  population is affected, and there is an increased  incidence of coronary artery disease in family members.  The metabolic defect is unclear, although plasma apoB  is often elevated due to increased synthesis; LDL and  VLDL apoB concentration is increased. The synthesis of  VLDL triglyceride is increased in FCH and there may  also be a relationship with insulin resistance.   The diagnosis of FCH is suspected if there is a  family history of hyperlipidaemia, particularly if family  members show different lipoprotein phenotypes. There  is often a family history of cardiovascular disease.  However, the diagnosis can be difficult and it sometimes  needs to be distinguished from FH (xanthomata  are not usually present in FCH) and familial  hypertriglyceridaemia (the Ila and lib phenotypes are  not usually found in familial hypertriglyceridaemia,  although they are in FCH). Children with FCH usually  show hypertriglyceridaemia and not the type Ila  phenotype (unlike the situation found in FH). Unlike  familial hypertriglyceridaemia, plasma VLDL particles  are usually smaller in FCH. Dietary measures and, if  indicated, either a statin or a fibrate are sometimes used.   Familial hypertriglyceridaemia is often observed with low  HDL cholesterol concentration. The condition usually  develops after puberty and is rare in childhood. The exact  metabolic defect is unclear, although overproduction of  VLDL or a decrease in VLDL conversion to LDL is likely.  There may be an increased risk of cardiovascular disease.  Acute pancreatitis may also occur, and is more likely  when the concentration of plasma triglycerides is more  than 10 mmol/L. Some patients show hyperinsulinaemia  and insulin resistance. Dietary measures, and sometimes  lipid-lowering drugs such as the fibrates or C0-3 fatty  acids, are used to treat the condition.   This condition is also called familial dysbeta-  lipoproteinaemia or broad (3-hyperlipidaemia. The   underlying biochemical defect is one of a reduced  clearance of chylomicron and VLDL remnants. The  name broad (Thyperlipidaemia is sometimes used  because of the characteristic plasma lipoprotein  electrophoretic pattern that is often observed (the  broad |3-band that is seen being remnant particles).   An association with type Ill/broad |3-hyperlipidaemia  and homozygosity for apoE or variants of apoE 2 has  been described. Apolipoprotein E shows three common  alleles, E2, E3 and E4, coded for on chromosome 19,  which are important for the binding of remnant  particles to the remnant receptor.   The mechanism for the disorder seems to be that  apoE 2 -bearing particles have poor binding to the  apoB/E (remnant) receptor and thus are not effectively  cleared from the circulation.   It is becoming apparent that it is not just inheriting  the apoE, genotype that is important in developing  broad |3-hyperlipidaemia. The prevalence of the apoE,/  E 2 genotype is about 1 in 100 in the general population,  yet only about 1 in 5000-10000 individuals manifest   A 43-year-old man attended the vascular surgery out-  patient clinic for peripheral vascular disease. He was  a non-smoker but had undergone a coronary artery  bypass graft the year before. Some of his laboratory  results were as follows:   The diagnosis was type III hyperlipoproteinaemia  (familial dysbetalipoproteinaemia or broad  (3-hyperlipidaemia). Note the mixed hyperlipidaemia  (both cholesterol and triglyceride concentrations  raised) in an approximately 1:1 molar ratio and also  apoE genotype E 2 homozygote (usual is apoE 3 /E 3 ).  The type III hyperlipoproteinaemia is associated  with raised concentration of remnant lipoprotein  particles, which are particularly atherogenic. Note  also the characteristic lipid stigmata and premature  peripheral vascular and coronary heart disease.   type III hyperlipoproteinaemia. A concurrent increase  in plasma VLDL concentration also seems necessary  for the condition to be expressed, such as might occur  in diabetes mellitus, hypothyroidism or obesity. Some  patients may show either an autosomal recessive or a  dominant mode of inheritance of the condition.   The palmar striae (palmar xanthomata) are  considered pathognomonic for the disorder, but  tuberoeruptive xanthomata, typically on the elbows and  knees, xanthelasma and corneal arcus have also been  described in this condition. Peripheral vascular disease  is a typical feature of this hyperlipidaemic disorder, as  is premature coronary artery disease.   Plasma lipid determination frequently reveals  hypercholesterolaemia and hypertriglyceridaemia,  often in similar molar proportions with plasma  concentrations of around 9-10 mmol/L. Plasma HDL  cholesterol concentration is usually low. Plasma LDL  concentration may also be low due to the fact that there  is reduced conversion from IDL particles, although it  may also be normal or elevated.   Plasma lipoprotein electrophoresis can show the  classic type III picture with a broad |3-band composed  of remnant particles, although this is not always present.  An association of type III hyperlipoproteinaemia with  homozygosity for apoE 2 has been described, and thus  apoE phenotyping or genotyping by a specialized  laboratory can be useful, although some patients  with broad |3-hyperlipidaemia can show other apoE  phenotypes or variants.   Another investigation that can be useful in  establishing the diagnosis is ultracentrifugation to  separate the lipoprotein particles. The cholesterol of  the VLDL particles is then quantified and expressed  as a total of the plasma triglyceride concentration. In  molar terms, normal individuals show a ratio of less  than 0.30, while ratios more than 0.30 are more likely  in type III hyperlipoproteinaemia, particularly nearer  0.60.Treatment consists of dietary measures, correcting  the precipitating causes and either the statin or fibrate  drugs.   This is one of the most common causes of a raised  plasma cholesterol concentration. This condition is  the result of a complex interaction between multiple  environmental and genetic factors. In other words, it  is not due to a single gene abnormality, and it is likely  that it is the result of more than one metabolic defect.  There is usually either an increase in LDL production   or a decrease in LDL catabolism. The plasma lipid  phenotype is usually either Ha or lib Fredrickson’s  phenotype. The plasma cholesterol concentration  is usually either mildly or moderately elevated. An  important negative clinical finding is the absence of  tendon xanthomata, the presence of which would tend  to rule out the diagnosis. Usually less than 10 per cent of  first-degree relations have similar lipid abnormalities,  compared with FH or FCH in which about 50 per cent  of first-degree family members are affected. There may  also be a family history of premature coronary artery  disease. Individuals may have a high intake of dietary  fat and be overweight. Treatment involves dietary  intervention and sometimes the use of lipid-lowering  drugs such as the statins.   Hyperalphalipoproteinaemia  Hyperalphalipoproteinaemia results in elevated  plasma HDL cholesterol concentration and can be  inherited as an autosomal dominant condition or, in  some cases, may show polygenic features. The total  plasma cholesterol concentration can be elevated,  with normal LDL cholesterol concentration. There is  no increased prevalence of cardiovascular disease in  this condition; in fact, the contrary probably applies,  with some individuals showing longevity. Plasma HDL  concentration is thought to be cardioprotective, and  individuals displaying this should be reassured. Box  13.1 gives the causes of raised plasma HDL cholesterol  concentrations.   Secondary hyperlipidaemias  One should not forget that there are many secondary  causes of hyperlipidaemia. These may present  alone or sometimes concomitantly with a primary  hyperlipidaemia. Some of the causes of secondary  hyperlipidaemia are listed in Box 13.2. Secondary causes   of hyperlipidaemia include obesity, type 2 diabetes  mellitus, hypothyroidism, chronic kidney disease,  cholestasis and certain drugs. The reader should refer  to the other chapters in this book for details of the  relevant diseases.   Inherited disorders of low plasma HDL concentration  (hypoalphalipoproteinaemia) occur, and plasma HDL  cholesterol concentration should ideally be more than  l.Ommol/L. A number of such conditions have been  described (such as apoAj deficiency), many of which  are associated with premature cardiovascular disease. In  Tangier’s disease, individuals have very lowlevels of HDL,  large, yellow tonsils, hepatomegaly and accumulation  of cholesterol esters in the reticuloendothelial system.  There is a defect in the ABC1 gene involved in HDL  transport. The causes of a low plasma HDL cholesterol  concentration are shown in Box 13.3.   Defects of apoB metabolism have also been  described. In abetalipoproteinaemia or LDL deficiency  there is impaired chylomicron and VLDL synthesis.  This results in a failure of lipid transport from the  liver and intestine. Transport of fat-soluble vitamins is  impaired and steatorrhoea, progressive ataxia, retinitis   Diabetes mellitus and metabolic syndrome  Certain drugs, e.g. estrogens, (3-blockers (without  intrinsic sympathomimetic activity), thiazide diuretics,  acitretin, protease inhibitors, some neuroleptics and  glucocorticoids  Chronic kidney disease   Box 13.3 Causes of low plasma high-  density lipoprotein (HDL) cholesterol   Poorly controlled diabetes mellitus  Insulin resistance and metabolic syndrome  Chronic kidney disease   Certain drugs, e.g. testosterone, probucol, (3-blockers  (without intrinsic sympathomimetic activity),  progestagens, anabolic steroids, bexarotene   pigmentosa and acanthocytosis (abnormal erthyrocyte  shape) can result. In hypobetalipoproteinaemia, a less  severe syndrome occurs, sometimes due to a truncated  form of apoB.   In LCAT deficiency, the accumulation of free  unesterified cholesterol in the tissues results in corneal  opacities, renal damage, premature atherosclerosis and  haemolytic anaemia. The enzyme LCAT catalyses the  esterification of free cholesterol. Another condition  that is probably due to a defect of LCAT is fish-eye  disease, in which there may be low HDL cholesterol  concentrations and eye abnormalities.   The help of a dietitian is invariably useful in treating  dyslipidaemias. Low-saturated fat/reduced cholesterol  diets are instigated. Total fat intake should be less than  30 per cent of the total calorie intake, with an increase  in monounsaturated fat intake up to 20 per cent of  total calories. Dietary cholesterol intake should not  exceed about 200mg/day. Five daily portions of fruit  and vegetables are advisable. Ideally, patients should  aim to achieve their recommended body mass index.  Alcohol intake should be less than 14 units/week for  females and 21 units/week for males. Increased intake  of plant sterols and stanols may lead to competition  for cholesterol intestinal absorption, thereby reducing  the plasma cholesterol concentration. If diet and  lifestyle measures fail, drug therapy may be indicated  (Table 13.5).   Table 13.5 Some lipid-lowering drugs and their effects  on plasma lipoprotein fractions   The rate-limiting enzyme of cholesterol synthesis,  HMG-CoA reductase, is inhibited by HMG-CoA  reductase inhibitors, also known as the statins, which  can be used to treat hypercholesterolaemia. These  agents include lovastatin, simvastatin, pravastatin,  atorvastatin, fluvastatin and rosuvastatin. The HDL  cholesterol concentration is modestly increased and  triglyceride concentration reduced by varying degrees  by these agents. The side effects notably include myalgia,  myositis (and rarely rhabdomyolysis) and abnormal  liver function.   Bile salt sequestrants such as colestipol and  colestyramine bind bile salts in the intestinal lumen  and thus interrupt their reabsorption and reutilization.  The removal of bile acids stimulates hepatic cholesterol  synthesis, which in turn results in an increase in hepatic  LDL receptors, resulting in decreased plasma LDL  concentration. The side effects include gastrointestinal  symptoms, such as constipation. To avoid interference  with their absorption, these drugs should not be given  at the same time as other drugs. They lower plasma  cholesterol concentration by about 10-20 per cent;  although HDL cholesterol concentration is also  modestly raised, triglyceride concentrations can be  paradoxically increased. Another agent acting on the  gut is ezetimibe, which inhibits intestinal cholesterol  uptake specifically.   The fibrate drugs include gemfibrozil, bezafibrate,  fenofibrate and ciprofibrate. These drugs have good  triglyceride-lowering and HDL-raising abilities,  although LDL cholesterol concentration may not be  much changed. They are PPAR a-agonists and work  on a number of lipid pathways, including increasing  lipoprotein lipase activity, reducing apoC 3 and VLDL  synthesis and increasing apoA synthesis. The side   effects include myalgia, myositis and gastrointestinal  disturbance, and they should not be used in patients  with active gallstones or significant renal disease. They  can lower plasma alkaline phosphatase concentration,  which can be used to monitor drug compliance.   Nicotinic acid and its derivatives have been used  to reduce VLDL secretion and LDL concentration  and, interestingly, also Lp(a) levels. This drug is  good at raising HDL cholesterol. However, the side  effects include hepatic toxicity, hyperuricaemia,  impaired glucose tolerance and flushing, to -3 fatty  acids in the form of fish oils or flaxseed oil can lower  plasma triglyceride concentrations by reducing VLDL  synthesis. They also have an antiplatelet aggregatory  action. They can sometimes be used to treat severe  hypertriglyceridaemia. However, they show little, if any,  LDL-lowering activity. Their side effects may include  gastrointestinal upset or bruising. The plant sterols and  stands can also lower serum LDL-cholesterol, probably  by interrupting gut micelle formation, and hence  reduce cholesterol absorption.   Combination drug therapy is sometimes used, for  example statin/ ezetimibe or statin/nicotinic acid, but is  best instigated by an expert with close monitoring, as  dangerous side effects may be increased.   Coronary artery disease remains one of the major  causes of morbidity and mortality in the industrial  world. Traditionally, the major risk factors are  hyperlipidaemia, hypertension and smoking, to which  can be added diabetes mellitus, a family history of  premature coronary heart disease and obesity.   With primary (the prevention of the occurrence)  and secondary (the prevention of further occurrences)  coronary heart disease prevention in mind, the usual  strategy adopted is to try to reduce the modifiable  risk factors. Cardiovascular risk factors tend to cluster  together in individuals and interact in such a way that  the overall combined effect is greater than the combined  risk of individual factors (see Chapter 22).   Before collecting blood, consider whether the patient  is on lipid-lowering therapy, including lipid-containing  infusions. Also ensure that the patient fasts overnight  for around 12 h (if safe to do so) and is allowed only  water to drink, if required. Although plasma cholesterol   A 15-year-old woman presented to the surgical unit  with acute pancreatitis. Some of her laboratory  results were as follows:   On examination, she had eruptive xanthomata  on her arms and thighs and fundoscopy revealed  lipaemia retinalis.   This patient has grossly elevated lipid concentrations  with severe hypertriglyceridaemia. The blood  sample would be lipaemic and some plasma  sodium assays (indirect ion electrodes) may show  pseudohyponatraemia. She was found to have  lipoprotein lipase deficiency when this enzyme was  measured before and after heparin administration,  which releases the enzyme from capillaries into the  circulation. Lipoprotein lipase deficiency can result in  the chylomicron syndrome and eruptive xanthomata  may be present. Plasma amylase concentration is  normally elevated in acute pancreatitis but, due to the  gross lipaemia, the assay was unsatisfactory, giving  a spuriously low result. The latter is an important  practical point and a spot urinary amylase may be  preferable, or assay of plasma amylase after separation  from the lipid fraction, under such circumstances.   concentration is little affected by fasting, triglyceride  concentrations rise and HDL cholesterol concentration  decreases if not, and thus ideally fasting samples should  be requested. The patient should be on his or her usual  diet for a couple of weeks preceding the test.   Plasma lipids should not be assessed in patients who  are acutely ill, for example acute myocardial infarction,  as plasma cholesterol concentration may be decreased  due to the acute-phase response. Wait for about  3 months after the event, although if a sample is taken  within 12 h of an event, a ‘true’ result may be obtained.   Posture can alter plasma lipid concentrations: in the  upright position, plasma cholesterol concentration  can be 10 per cent higher than in the recumbent  position.   The blood sample should be taken to the laboratory  and assayed promptly. The usual fasting lipid profile  consists of plasma cholesterol, triglyceride and HDL  cholesterol concentrations.   When faced with a hyperlipidaemia, decide whether it  is primary or secondary. A family history, clinical features  and appropriate blood tests can be useful to help make  this decision. Lipid stigmata such as tendon xanthomata  or premature corneal arci may point to familial  hypercholesterolaemia, and tuberous xanthomata or  palmar striae to type III hyperlipoproteinaemia.   Blood glucose concentration is useful to help assess  for diabetes mellitus, liver function tests for liver disease  such as cholestasis, urinary protein and plasma albumin  concentrations for nephrotic syndrome and thyroid  function tests for hypothyroidism. It is also important  to determine alcohol consumption and medications  from the clinical history.   It is generally wise to retest patients’ lipids, a few  months apart, as it is recognized that the within-  individual variation of lipids can be significant, and  reliance cannot be placed on just one set of readings.   It is also useful to assess the patient for other  cardiovascular risk factors, including smoking habits,  diabetes mellitus, blood pressure, body weight and  family history of cardiovascular disease. These should  also be managed in their own right. Assessment should  also be made for possible atherosclerotic disease, for  example does the patient have evidence of coronary,  peripheral or carotid artery disease?   If the patient is known to have coronary artery  disease, aim for a plasma LDL cholesterol concentration  of about 2.0mmol/L. Statins are first-line drugs for  patients with raised LDL cholesterol concentration.   Alternatively, there may be a place for a fibrate drug if  fasting plasma triglyceride concentrations are raised by  more than 5 mmol/L, particularly if there is also a low  HDL cholesterol concentration and LDL cholesterol  concentration is not much raised. The ideal is to aim  for fasting plasma cholesterol concentration of about  4.0 mmol/L, triglyceride concentration less than  1.5 mmol/L and HDL cholesterol concentration more  than 1.0 mmol/L.   Severe hypertriglyceridaemia, particularly if the  plasma triglyceride concentration is more than  10 mmol/L, is a risk factor for acute pancreatitis.  Low-fat diets may help in conjunction with a fibrate  (sometimes CO-3 fatty acids are used), aiming ideally  for a fasting plasma triglyceride concentration lower  than about 1.5 mmol/L. Remember that severe  hypertriglyceridaemia can cause problems with  certain assays, for example falsely low plasma amylase  concentration or pseudohyponatraemia.   In terms of primary coronary heart disease  prevention, the use of a cardiovascular risk factor  assessment may be helpful to determine if a lipid-  lowering drug is indicated, for example Framingham  Study-based or QRISK cardiovascular risk calculators.  Close family members should be screened in cases of  genetic hyperlipidaemias such as FH.   Specialist lipid assays may help define the abnormality.  The apoE genotype is useful in the diagnosis of type  III hyperlipoproteinaemia, as many of these patients  are apoE 2 /E v Plasma lipoprotein lipase and apoC 2  (its activator) assays may be useful in chylomicron  syndrome, and LDL receptor DNA studies for familial  hypercholesterolaemia. Plasma apoA and apoB  concentrations and also Lp(a) may help define risk status.   • Lipids are essential for health, but raised plasma  cholesterol and triglyceride concentrations  are associated with an increased incidence of  cardiovascular disease.   • Conversely, plasma HDL cholesterol is  cardioprotective, partly because of its central role in  reverse cholesterol transport returning cholesterol  from the tissues to the liver.   • There are many cases of secondary hyperlipidaemias,  including obesity, alcohol excess, diabetes mellitus,   • The genetic hyperlipidaemias include FH, FCH  and type III hyperlipoproteinaemia. Familial  hypercholesterolaemia usually results from a defect  of the LDL receptor.   • The statins or HMG-CoA reductase inhibitors  lower plasma cholesterol concentration and are  associated with decreased cardiovascular disease.   This chapter gives an outline of certain nutritional  abnormalities and how they overlap with aspects of  chemical pathology. It is not, however, a substitute for a  nutrition textbook. The reader may also find Chapters  12 and 13 (on carbohydrate and lipid disorders,  respectively), Chapter 15 (on vitamin/ trace elements)  and Chapter 16 (on gastrointestinal function) relevant.  Nutrition is an important topic, as about 1 billion of the  world’s population are overweight yet, ironically, at the  same time approximately 1 billion are undernourished  or starving.   Adequate nutrition is essential for a variety of  reasons, including optimal cardiovascular function,  muscle strength, respiratory ventilation, protection  from infection, wound healing and psychological well-  being.   The principles of carbohydrate and lipid metabolism  and gastrointestinal digestion and absorption all  have important implications in the management of  nutrition, and of intravenous (parenteral) nutrition  in particular. These principles, including those of fluid  and electrolyte homeostasis, must be fully understood  in order to manage patients receiving parenteral  nutrition.   Daily energy loss as heat is about 120 kj (30kcal) per  kilogram of body weight in a normal adult. In addition,  there is a daily protein turnover of about 3 g/kg body  weight (about 0.5 g of nitrogen), of which about 0.15 g  of nitrogen/kg body weight is excreted ( 1 g of nitrogen  is derived from about 6.25 g of protein). These losses  are usually balanced by dietary intake of equivalent  amounts of energy, as carbohydrate, fat and protein.  Glucose provides 4kcal/g, and fat 9kcal/g.   Excess energy is stored as glycogen and triglyceride.  If expenditure exceeds intake, these energy stores are  drawn upon. In a well-nourished adult, enough energy  is stored as hepatic glycogen to last at least a day, and  therefore post-operative patients without complications  do not need intravenous feeding. Once this store has   been depleted, energy is derived from triglyceride,  and later from body tissue components such as the  proteins of cells, including those of muscle. This may  cause severe ketosis from the metabolism of fats and  ketogenic amino acids and increase nitrogen turnover  and loss. The daily energy and nitrogen requirements  are not constant, as we will now see.   During starvation the body tries to maintain blood  glucose levels in the acute phase and in the longer term  to preserve body protein mass.   Hepatic glycogen stores are largely consumed  within 24 h. The obligatory glucose requirements  of the brain, erythrocytes and other organs are met  by gluconeogenesis. During the first week or so of  acute starvation, up to 150 g of protein is utilized to  achieve this. Insulin levels decrease, resulting in amino  acid (mainly alanine) release from protein, glycogen  conversion to glucose, and adipocyte fatty acid release for  energy needs. Plasma insulin concentration is reduced,  but concentrations of glucagon, glucocorticoids,  catecholamines and growth hormone are raised. Thus,  blood glucose concentrations are generally maintained  despite starvation.   In later starvation, ketone bodies replace glucose as  the predominant brain fuel (ketoadaptive phase) and  a metabolic acidosis may result. The ketone bodies  also inhibit muscle protein degradation and the flow  of alanine into the circulation. There is thus a decrease  in urinary nitrogen excretion, a decline in hepatic  gluconeogenesis and increased brain oxidation of  ketone bodies while plasma amino acid concentrations  decrease. Plasma insulin is reduced, as are glucagon,  glucocorticoids, catecholamines and growth hormone.   Starvation results in initial rapid weight loss,  mostly due to protein breakdown and diuresis. The  latter is partly due to increased renal tubule urea load  and results in renal loss of calcium, phosphate and   potassium. Continuing starvation results in a slower  decline in body weight, as after the gluconeogenic  phase fat is catabolized. This is associated with water  conservation about 3-5 days later. The basal metabolic  rate (BMR) decreases and the kidney becomes the most  important gluconeogenic organ, using glutamine to  synthesize glucose.   Feeding converts the situation to an anabolic state.  The level of ketone bodies falls, along with a decline in  urinary nitrogen. Thus, positive nitrogen balance can  be achieved with fat gain.   In the face of trauma, including burns, the body strives  for wound healing and the evoked response is directly  related to the severity of the injury. Two phases have  been described.   • Early ebb or shock phase Here, in an attempt  to survive, there is a decrease in body energy  expenditure to conserve resources. Plasma insulin  concentration is reduced but concentrations of  glucagon, glucocorticoids, catecholamines and  growth hormone are raised. There is reduced oxygen  consumption and decreased glucose oxidation.   • Later flow or hypermetabolic phase Should blood  volume be restored and circulation satisfactory,  then the flow phase may take place. Concentrations  of hormones, such as glucagon, glucocorticoids,  catecholamines and growth hormone, increase, as  may that of insulin. Consequently, gluconeogenesis  (which may lead to hyperglycaemia) occurs, as does  proteolysis. Urinary nitrogen loss can range from  more than 25g/day in severe burns to 10 g/ day in  the case of an uncomplicated surgical procedure.  Accelerated muscle proteolysis results in a loss of  lean body mass. Furthermore, there is increased fat  catabolism, and lipolysis provides the predominant  non-protein source of energy in traumatized  patients.   Resting energy expenditure increases after trauma  or sepsis, by 20 per cent in long bone fracture to  100 per cent in severe burns and trauma. Most  adults require 1750-2500 kcal/day. Indeed, even in  undernourished patients, more than 2500 kcal/day is  rarely needed, as their BMR is lower. Only in severely  traumatized patients, such as those with major burns,  is 3000 kcal/day necessary. Most patients need 11-  16 g nitrogen/day; rarely is 20 g nitrogen/ day needed  (protein requirement = 6.25 x nitrogen requirement).   The diagnosis of undernutrition is mainly a clinical  one. In many cases, patient weight is suitable to assess  nutritional status, with a loss of 10 per cent or more  being suggestive of possible undernutrition. However,  this can be inaccurate if there are significant changes in  body water and does not provide adequate information  about body fat or protein stores.   A good dietary history is essential for helping to assess  nutritional status. Ask about food intake, including  type of food and frequency. A useful screening method  is the malnutrition universal screening tool (MUST).   Examination of the patient is also essential, including  weight and height. Body mass index (BMI) is a useful  indicator of nutrition, with the main exceptions being  patients with oedema or dehydration.   • Fat assessment Measurement of triceps skin-fold  thickness using suitable calipers may be useful but  is dependent on operator technique, the presence of  oedema and skin pliability.   • Skeletal muscle protein Arm muscle circumference  (AMC) can be calculated from the mid-arm  circumference and triceps skin-fold thickness by the  following equation:   - Operator technique may result in inaccuracies  in mid-arm circumference measurement.   - Hand- grip strength gives indirect evidence  of body protein status by looking at muscle  strength.   • Biochemical tests (these rarely have a major role  in assessing nutritional status) Measurement of  circulating visceral proteins has been used to assess  the impairment of hepatic protein production  indirectly. Various plasma proteins have been used:   - Albumin: this protein has a plasma half-life of  about 20 days but is a poor index of nutritional  status as it is influenced by degree of hydration,  loss from the body (e.g. gastrointestinal or  renal), hepatic well-being and intravascular/  extravascular movement (see Chapter 19), and  thus has little place as a nutritional marker.   - Transferrin: this has a shorter half-life, of about  9 days, and may be a better guide to nutritional  status, although it is dependent upon iron status.   - Other plasma proteins or peptides that  have been studied as potential markers of  nutritional status include retinol-binding  protein, insulin-like growth factor 1 (IGF-1)  and fibronectin, but these are rarely used. Pre-  albumin, now referred to as transthyretin, may  be a marker not so much of poor nutrition  but of adequate nutritional replacement when  plasma concentrations rise.   - Urinary creatinine-to-height ratio can be a  measure of lean body mass, but this test has the  problem of requiring accurate urine collection.   - 24-h urinary urea excretion (mmol/L) X 0.034  approximates to urinary nitrogen excretion.  This can be used as a guide to nitrogen, and  thus protein, requirements, that is, catabolic  status.   - Complex laboratory tests are available, such as  the assessment of total body fat by impedance  measurement, or total body nitrogen to assess  protein status. However, these are usually used  only in research settings and not in routine  clinical practice.   - Laboratory tests may also show fatty acid  deficiencies (see Chapter 13) and/or vitamin  and trace element abnormalities (see Chapter  15). Poor nutrition may cause impaired  immunological function with decreased  lymphocyte count and abnormalities of  delayed hypersensitivity.   Prognostic nutritional index  The prognostic nutritional index (PNI) has been devised  as a marker of nutritional status and incorporates a  number of the components discussed above including  plasma albumin and transferrin concentration, skin-  fold thickness and lymphocyte function.   Subjective global assessment  This looks at a number of subjective weightings on  key variables, including weight loss, gastrointestinal  disorders, functional capacity and physical signs of  deficiency. Weighting A is well nourished, B moderately  nourished and C poorly nourished.   This starts with reduced intake or nutrient loss.  The stores of nutrients are depleted, which leads  to biochemical and metabolic consequences and,  eventually, clinical symptoms and signs.   Kwashiorkor occurs in individuals with visceral  protein loss associated with impaired immunological  function, although other nutritional components  are satisfactory. Visceral proteins are decreased, but  body weight, mid-arm circumference and triceps  skin-fold thickness are relatively normal. Conversely,  marasmus is generalized undernutrition with  reduction in weight, mid-arm circumference and  skin-fold thickness, although visceral proteins are  relatively normal.   Do not forget that patients can be undernourished  while in the hospital ward, particularly the elderly and  those who are severely ill, post surgery or in intensive  care; alas, about 10-40 per cent of all adults in hospitals  or nursing homes may be undernourished.   Nutritional support or artificial nutrition  Nutritional support can take many forms. For example,  in coeliac disease (Chapter 16), a gluten-free diet is  important, and for lactose intolerance, specific dietary  restriction of lactose is required.   • when the patient has lost 10 per cent or more of  body weight and continues to lose weight because of  inadequate intake, or   • if the disease process is thought likely to result in  impaired nutritional intake for 10 days or more.   Undernutrition is associated with increased  mortality and morbidity and prolonged hospital stay  and should be remedied promptly. It may be possible  to encourage eating and, if necessary, enhance it with  supplements. However, sometimes patients are not  able to eat, and artificial nutrition becomes necessary.  The average daily adult requirements are shown in  Table 14.1.   Approximations based on the Harris-Benedict  equation give an estimate of the total energy  requirements. The daily BMR in kilocalories is:   where W = weight (kg), H = height (cm) and A = age  (years). The activity factor, for example, when confined   a AII units are expressed as per kilogram of body weight. It is  important to note that these values are extremely variable  depending upon individual circumstances.   to bed is 1.2. The injury factor after a minor operation  is 1.4 and with major sepsis is 1.6.   If the patient cannot take food orally, there are  two main alternatives depending on the presenting  indications: enteral or parenteral nutrition.   If the gastrointestinal tract is functioning, enteral  feeding is usually indicated. This is the most effective  and natural route, and is preferable because it is  more physiological and has fewer complications. The  indications for enteral nutrition may include:   • jejunostomy (perioperative placement may  be considered if likelihood of ongoing upper  gastrointestinal dysfunction).   The complications of enteral nutrition include tube  complications, poor gut absorption and biochemical  disturbances (these may be similar to those seen  in parenteral nutrition - see below). More specific  complications of enteral nutrition include aspiration  pneumonia and diarrhoea.   If possible, all patients should be fed orally or enterally,  both of which are simpler than parenteral feeding and  generally cause fewer complications that might consist  of infections, metabolic abnormalities and mechanical  problems for example. The basic rule is that if the gut  works, use it. However, if oral or enteral feeding is  contraindicated, for example when there is intestinal  obstruction or fistula, short bowel syndrome, ileus or  persistent vomiting, parenteral feeding maybe indicated.   Parenteral nutrition can be given through a  peripheral vein or through a central venous catheter  into a large vein. The amount of energy that can be  given into a peripheral vein is limited because glucose  and amino acid solutions are hyperosmolar and cause  irritation to small vessel walls, sometimes causing  thrombophlebitis. Thus, peripheral parenteral feed  osmolarity should usually be less than about 600 mmol/  kg. Hyperosmolar solutions infused through a central  venous catheter minimize the risk of thrombophlebitis,  which is more likely via a peripheral line.   The choice depends partly on the length of time for  which parenteral feeding is required, as the peripheral  route is used only for short periods, usually 1-2 weeks.  Some experts use a glyceryl trinitrate patch at the site of  insertion of peripheral catheters to facilitate insertion  and reduce thrombophlebitis. The insertion of central  venous catheters requires expertise and the involvement  of a multidisciplinary nutrition team working closely  with the clinical biochemistry laboratory to ensure  correct positioning of the catheter, and strict attention  must be paid to aseptic technique to reduce the risk  of line infection. The regimen depends on the clinical  condition of the patient and the volume that can safely  be infused.   The energy source is usually a combination of glucose-  and fat-containing fluids, for example Intralipid. Fat  is more dense in energy, has a lower osmolality and  generates less carbon dioxide. At least 50 per cent of the  energy requirements should be provided as glucose/  lipid. Giving energy as glucose and fat minimizes the  use of amino acids for gluconeogenesis and reduces  urinary nitrogen loss.   Glucose. Severe illness may cause insulin resistance  and glucose excess may evoke fatty liver and  hyperglycaemia.   Fat. The fat particles in emulsion form are similar  to chylomicrons. Provided that some carbohydrate is   given at a constant rate, there is no risk of significant  ketoacidosis. Sometimes in sepsis or insulin-resistant or  hyperlipidaemic patients intravenous lipid is not fully  cleared. Grossly lipaemic plasma may interfere with  some laboratory analyses, particularly that of plasma  sodium. If lipaemia persists, it indicates that the rate  of administration is faster than the rate at which the fat  can be metabolized, and the infusion should be slowed.  Rarely, fat overload can occur. The administration of fat  protects against essential fatty acid deficiency.   Nitrogen supplements containing amino acids are  needed to replace the daily nitrogen loss and to promote  tissue healing. The assessment of nitrogen requirements  may be difficult. A normal adult patient needs about  9g nitrogen/ day, but this increases as the catabolic rate  increases, for example up to 20 g nitrogen/ day in stress  and infection. During the catabolic phase, the body  cannot use administered nitrogen efficiently, and much  of it is excreted in the urine as urea or free amino acids.  About 840 kj (200kcal) of energy per gram of nitrogen  are needed by a normal adult to synthesize protein  from amino acids. This proportion decreases slightly as  the catabolic rate and nitrogen requirements increase.  Protein intake should be about 10-15 per cent of total  calorie requirement.   The essential amino acids are arginine, histidine,  isoleucine, leucine, threonine, lysine, methionine,  phenylalanine, tryptophan and valine. These should  constitute about 25 per cent of the total amino acids.  The essential amino acids tend to have more complex  structures such as aromatic rings or long side-chains;  they can be synthesized by bacteria and plants, but not by  humans. Arginine and histidine may become essential  only under certain circumstances. The following is a  useful mnemonic to remember these essential amino  acids: any help in learning these little molecules proves  truly valuable.   Glutamine, although not an essential amino acid, is  an important energy source for the cells of the immune  system and gut. There are data indicating that the  addition of glutamine to parenteral feeds may benefit  certain patients, such as those on intensive care.   Once tissue repair predominates, the use of amino  acid increases (anabolic phase) and urinary nitrogen  loss may fall suddenly; this indicates an increased, not  a decreased, need for nitrogen. Conversely, during the  catabolic phase, increasing nitrogen intake may result  in an increased urinary loss because it cannot be used.   This must be given with all parenteral feeding, in addition  to meeting the nitrogen and energy requirements.  Urinary trace metal loss is high during the catabolic  phase, but falls as anabolism becomes predominant.  If parenteral feeding has been very prolonged,  micronutrient deficiencies may become apparent.   Daily parenteral nutrition solutions are sometimes  prepared in 3-L bags that contain the appropriate  daily energy, glucose, lipid, nitrogen, vitamin and trace  element requirements in addition to electrolytes, calcium,  phosphate and magnesium. Intravenous feeding should  not be stopped suddenly; the patient should gradually be  weaned on to oral or enteral feeding.   Some clinical and metabolic complications associated  with long-term parenteral feeding are shown in Box  14.1. One of the most important complications is  infection of the central line, and careful nursing  attention is therefore essential. It is also important to  review the patient’s fluid balance. The biochemical  investigations that may be used to prevent the onset of  these complications are discussed below.   Box 14.1 Some clinical and metabolic  complications of long-term parenteral  nutrition   Hyperglycaemia/hypoglycaemia  Acid-base disturbances  Electrolyte disorders  Fluid overload  Hypophosphataemia  Metabolic bone disease  Liver disease  Biliary sludging   Essential vitamin and mineral deficiencies  Aluminium (sometimes by contamination) and  manganese toxicity   Init ially, measurement of the daily plasma  sodium, potassium, bicarbonate and urea/ creatinine  concentrations helps in the assessment of water and  electrolyte needs and renal function. Plasma glucose  concentrations must be monitored carefully, as patients  may develop stress-related glucose intolerance with  hyperglycaemia, consequent cell dehydration and  polyuria or rebound hypoglycaemia if glucose infusion  is stopped suddenly.   Plasma albumin, protein, calcium, phosphate and  magnesium concentrations should be measured to  detect possible metabolic complications. The full blood  count, including haemoglobin, white cells and platelets,  as well as clotting, should also be monitored.   A cholestatic type of liver disorder associated with  biliary sludging may develop in some patients receiving  intravenous nutrition; thus, liver function tests also  need to be monitored. The plasma alkaline phosphatase  activity increases, with a later rise in plasma transaminase  activities. Unless significant symptoms occur, such as  severe jaundice, this is not necessarily an indication  to stop parenteral feeding because liver dysfunction  usually resolves when the parenteral feeding is stopped.  Fatty liver may also occur with raised transaminases,  especially if overinfusion of glucose.   Plasma concentrations of trace elements zinc,  selenium, manganese and copper should be monitored  about monthly once the patient is clinically stable,  and similarly for ferritin, vitamin B [2 and folate. Some  patients may not clear the fat in the parenteral feed,  and this can be assessed by measuring the plasma  triglyceride concentration.   Estimation of 24-h urinary nitrogen output is very  rarely helpful but has been used to assess nitrogen  use and the amount that should be replaced. If urea  excretion increases quantitatively when nitrogen intake  is increased, this indicates that the nitrogen cannot be  used, and thus should not be increased. If nitrogen loss  falls while supplements are being given, this indicates  that anabolism is increasing, and supplements should  be increased until urinary excretion increases. Once the  patient has been established on long-term feeding, the  frequency of monitoring can be reduced.   This potentially lethal condition can be defined as  severe electrolyte and fluid shifts associated with  metabolic abnormalities in undernourished patients  undergoing refeeding, whether this is oral, enteral or  parenteral.   It can be associated with significant morbidity  and mortality. Clinical features include fluid balance  abnormalities, abnormal glucose metabolism,  hypophosphataemia (see Chapter 6), hypomagnesaemia  and hypokalaemia. In addition, thiamine deficiency  can occur. The conditions associated with refeeding  syndrome are shown in Box 14.2.   Prior to refeeding, electrolyte disorders should be  corrected and the circulatory volume carefully restored.  In practice, this may delay the administration of  nutrition but it can usually be accomplished within the  first 12-24h.   A 64-year-old man with an inoperable oesophageal  carcinoma had been unable to eat solid foods for  about 2 months. A day after he had been commenced  on total parenteral nutrition, the following blood  results were obtained.   The patient has biochemical features of refeeding  syndrome, with hypokalaemia, hypophosphataemia  and hypomagnesaemia. Considerable care is needed  when feeding patients after prolonged lack of food  to avoid these dangerous biochemical features. Other  associated complications may include thiamine  deficiency and fluid balance disturbance.   Vitamin and trace element deficiencies should also  be corrected and, specifically, thiamine should be  given before refeeding is instigated. Further thiamine  may be necessary until the patient is stabilized. Some  preparations containing thiamine have been associated  with anaphylaxis, and therefore facilities for treating  this should be readily at hand.   The calorie repletion should be slow, at approximately  20kcal/kg per day or, on average, 1 000 kcal/ day initially.  However, this may not meet the patient’s fluid, sodium,  potassium or vitamin requirements unless these are  specifically addressed. The usual protein requirement is  about 1.2-1.5g/kg per day, or about 0.17 g nitrogen/kg  per day. Gradual introduction of calories, particularly  over the first week of refeeding, may be prudent until  the patient is metabolically stable.   In this condition, which is more common in females,  there are psychological problems with body self-  image and self-inflicted starvation. In some respects  this condition can resemble hypopituitarism with  amenorrhoea resulting from decreased luteinizing  hormone and follicle-stimulating hormone release.  However, instead of the loss of axillary and pubic hair, as  in hypopituitarism, there is additional lanugo hair. There  maybe severe weight loss and elevated growth hormone  and cortisol concentrations. Severe hypokalaemia,  hypomagnesaemia and hypophosphataemia are  also seen, particularly if refeeding syndrome ensues.  Strangely, hypercholesterolaemia may also occur.   It may seem strange to talk about obesity in the same  chapter as undernutrition and starvation; however,  obesity could be considered a form of malnutrition.  About 20 per cent of the European population is obese,  with higher prevalence figures in other populations  such as African Americans and Pacific Islanders.   Although these definitions rely on BMI, an alternative  approach is to measure the waist circumference, with  88 cm for females and 102 cm for males being indicative  of central obesity in some patient groups (Table 14.2).   The reason for this epidemic of obesity is partly the  decrease in physical activity seen in sedentary societies  combined with the increased intake of calorie-dense  food such as saturated fat. However, there may also be  underlying genetic or molecular mechanisms, which  are reviewed briefly here.   adipocytes, is an afferent signal that relays the magnitude  of the fat stores to the central nervous system, primarily  the hypothalamus, and plasma levels correlate with  the adipose tissue mass. In the ob/ob mouse, there is  defective leptin production associated with obesity and  insulin resistance. Leptin administration causes weight  loss in ob/ob mice partly by reducing food intake. In  times of starvation, leptin levels decline. Adiponectin  is another adipose-released hormone that is thought to  sensitize tissues to insulin.   Neuropeptide Y is a potent stimulator of food  intake, the production of which is inhibited by leptin.  Pro-opiomelanocortin is also involved in obesity  and is cleaved to form adrenocorticotrophin and   A 49-year-old man presented to his general  practitioner wanting to lose weight. He had  osteoarthritis of his knees and sleep apnoea. His  blood pressure was raised, at 168/98 mmHg, and his  BMI was 40.4 kg/m 2 . His fasting blood glucose was  6.0mmol/L and plasma lipids showed cholesterol  6.3mmol/L, triglyceride 4.8mmol/L and high-  density lipoprotein (HDL) cholesterol 0.67 mmol/L.  The GP requested an oral glucose tolerance test  (OGTT), which had the following results:   2h after oral 75 g anhydrous glucose load - plasma  glucose: 9.4 mmol/L.   This patient shows grade 3 obesity, which is  associated with hypertension, mixed hyper-  lipidaemia, osteoarthritis, sleep apnoea and  impaired glucose tolerance. This is an increasing  public health problem.   a-melanocyte-stimulating hormone (MSH) (see  Chapter 7). The latter acts on the melanocorticortin-4  receptor (MC4-R) in the hypothalamus, which in  turn increases energy expenditure and reduces food  intake. Agouti protein, which is also expressed in hair  follicles, antagonizes the actions of MSH by blocking  MC4-R. Obese humans paradoxically have high levels  of leptin, presumably because of tissue resistance  to it. A minority of cases of obesity may be due to  mutations in leptin receptors. Also involved in obesity  is the (3-3-adrenoreceptor, which mediates adipose  metabolism by the sympathetic nervous system.   Obesity is associated with numerous medical  problems, including type 2 diabetes mellitus,  hyperlipidaemia, hypertension, coronary artery disease  and stroke. There are various treatment strategies for  obesity. Remember that energy balance and weight   gain are extremely tightly regulated and it only needs  an excess of lOOkcal/day, such as a chocolate biscuit, to  result in a 4-kg gain in a year.   Increased physical activity and reduced calorie  intake, sometimes combined with psychotherapy or  behavioural therapy, are the cornerstone of treatment,  although many people fail to achieve satisfactory  weight loss. Some new drug therapies are available,  but these should not be viewed as a universal panacea  for obesity as there are no short-cut easy answers and  they may have side effects. One such drug is orlistat,  a pancreatic lipase inhibitor that causes reduced small  intestine fat absorption. Other therapeutic possibilities  in the future may be neuropeptide Y antagonists, leptin  analogues and (3-3-adrenoreceptor agonists. Surgical  options such as gastric banding or Roux-en-Y gastric  bypass are being used and maybe cost-effective.   • Daily energy loss as heat is about 120 kj (30kcal)  per kilogram of body weight in a normal adult. In  addition, there is a daily protein turnover of about  3g/kg body weight (about 0.5 g of nitrogen), of  which about 0.15 g of nitrogen/kg body weight  is excreted ( 1 g of nitrogen is derived from about  6.25 g of protein). These losses are usually balanced  by dietary intake of equivalent amounts of energy,  as carbohydrate, fat and protein. Glucose provides  4kcal/g; fat provides 9kcal/g.   • During starvation, the body tries to maintain blood  glucose levels in the acute phase and in the longer  term to preserve body protein mass.   • Kwashiorkor occurs in individuals with visceral  protein loss associated with impaired immunological   function, although other nutritional components  are satisfactory. Visceral proteins are decreased,  but body weight, mid-arm circumference and  triceps skin-fold thickness are relatively normal.  Conversely, marasmus is generalized undernutrition  with reduction in weight, mid-arm circumference  and skin-fold thickness, although relatively normal  visceral proteins.   • Enteral or parenteral feeding may be necessary for  patients needing nutritional support.   • Although about 1 billion of the world’s population  are undernourished, approximately 1 billion are  overweight. Obesity is increasing, particularly in  Western urbanized societies.   This chapter looks at vitamins and trace elements, and  might usefully be read in conjunction with Chapter 14  (Nutrition). It also discusses certain elemental metals  that are important in disease states.   At one time, vitamins were thought to be amines and  hence the term ‘vitamines’ was coined for substances  that are essential for life but needed in only minute  amounts. Vitamins are now known to be organic  compounds, not necessarily amines, which are essential  for normal growth and development. They must be  included in the diet because the body either cannot  synthesize them at all or cannot do so in amounts  sufficient for its needs.   Trace elements are inorganic compounds that, like  vitamins, are essential for health and needed only  in small amounts, known as the reference nutrient  intake. A normal mixed diet should provide adequate  amounts of vitamins and trace elements, and thus  supplementation is not usually necessary.   Testing for vitamin and trace element deficiency  should be carried out as soon as the diagnosis is  suspected; the results of laboratory tests usually revert  rapidly to normal once the patient has resumed eating  a normal diet, for example after admission to hospital,  and it may then be impossible to confirm the original  diagnosis. Where the diagnosis is difficult, a trial of the  micronutrient may be the most reliable and simplest  method of assessment.   • inadequate intake: deficiencies are rarely seen in  affluent populations except in:   Some vitamins (notably A and D) are toxic if taken  in excess, and overdosage has recently become more  common, possibly because of the increased availability  of these compounds in over-the-counter preparations.   Vitamins are classified into two groups on the basis  of their solubilities: fat soluble and water soluble.  The distinction is of clinical importance because  steatorrhoea may be associated with a deficiency of fat-  soluble vitamins, with relatively little clinical evidence  of lack of most of the water-soluble vitamins except B p  and folate.   Each of these has more than one active chemical  form, but variations in structure are minimal and  in this chapter each vitamin is considered as a single  substance.   Precursors of vitamin A (the carotenes) are found in  the yellow and green parts of plants and are especially  abundant in carrots. The active vitamin is formed by  the hydrolysis of |3-carotene in the intestinal mucosa;   each molecule can produce two molecules of vitamin  A, which are absorbed as retinol esters and stored in  the liver. Retinol is transported to tissues bound to the  (/-globulin retinol-binding protein (RBP).   Vitamin A is stored in animal tissues, particularly the  liver, and is also present in milk products and eggs.   Rhodopsin (visual purple), the retinal pigment  that is necessary for vision in poor light (scotopic  vision), consists of a protein (opsin) combined with  vitamin A. Rhodopsin decomposes in bright light. It  is partly regenerated in the dark, but, because this is  not quantitatively complete, vitamin A is needed to  maintain retinol levels. Vitamin A is also essential for  normal mucopolysaccharide synthesis and mucus  secretion.   is associated with poor vision in dim light,  especially when the eyes have recently been  exposed to bright light.   • Owing to deficient mucus secretion leading to drying  and squamous metaplasia of ectodermal tissue:   - Skin secretion is diminished and there may  be hyperkeratosis of hair follicles. Dry, horny  papules (follicular hyperkeratosis) are found  mainly on the extensor surfaces of the thighs  and forearms. Squamous metaplasia of the  bronchial epithelium has also been reported  and may be associated with a tendency to  develop chest infections.   - The conjunctiva and cornea become dry and  wrinkled, with squamous metaplasia of the  epithelium and keratinization of the tissue  (xerosis conjunctivae and xerophthalmia).  Bitot’s spots are elevated white patches,  composed of keratin debris, found in the  conjunctivae. Prolonged deficiency leads to  keratomalacia, with ulceration and infection  and consequent scarring of the cornea, causing  blindness.   - Poor bone growth in the skull, leading to  cranial nerve compression.   - Anaemia, which responds to vitamin A but not  to iron therapy.   Hepatic stores of vitamin A are large and therefore  clinical signs develop only after many months, or even  years, of dietary deficiency. Such prolonged deficiency  is very rare in affluent communities. In steatorrhoea,  clinical evidence of vitamin A is rare, although plasma  concentrations may be low. Deficiency is relatively  common in poor countries, especially in children, and  can cause blindness.   The diagnosis is usually made on the basis of clinical  criteria; very low plasma vitamin A concentrations  usually confirm deficiency. In conditions such as non-  cirrhotic liver disease, in which plasma concentrations  of RBP are low, concentrations of vitamin A may be  decreased despite normal liver stores. In cirrhosis of the  liver, the stores may be very low. Laboratory tests for  the diagnosis of vitamin A deficiency consist of testing  for plasma retinol concentration. Retinol-binding  protein is also low in vitamin A deficiency, but this may  also occur in protein deficiency and the acute-phase  response.   Vitamin A deficiency can be treated with retinyl  palmitate. High doses of vitamin A should be given to  treat xerophthalmia and advanced skin lesions. ‘Night  blindness’ and early retinal and corneal changes often  respond rapidly to treatment, although corneal scarring  may be irreversible.   Vitamin A in large doses is toxic. Acute intoxication  has been reported in Arctic regions as a result of eating  polar bear liver, which has very high vitamin A content.  More commonly, overdosage is due to the excessive use  of vitamin preparations.   In acute poisoning, symptoms include nausea and  vomiting, abdominal pain, drowsiness and headache.  Pregnant women are usually advised not to eat liver,  which is a storage organ for many vitamins, to avoid  the risk of fetal damage.   Chronic hypervitaminosis A is associated with  fatigue, insomnia, bone pain, loss of hair, desquamation  and discoloration of the skin, hepatomegaly,  headaches, abdominal pain, bone and joint pain,  benign intracranial hypertension, osteoporosis and  weakness.   Additionally, a very high intake of carrots or orange  juice can lead to carotenaemia, which can mimic  jaundice except that plasma bilirubin is normal.   The metabolism and functions of vitamin D, the effects  and treatment of its deficiency and hypocalcaemia  are discussed in Chapter 6, as are disorders of bone,  including rickets and osteomalacia.   Overdosage may cause hypercalcaemia. In chronic  overdosage, stores of cholecalciferol are large and  therefore hypercalcaemia may persist, or even progress,  for several weeks after ingestion of the vitamin is  stopped.   Vitamin E acts as an antioxidant, and deficiency can  have many clinical sequelae.   The common causes of vitamin E deficiency are poor  intake and fat malabsorption, for example cystic fibrosis.  Low plasma concentrations may also be seen in the rare  hypobetalipoproteinaemia or abetalipoproteinaemia,  in which there are low concentrations of the low-  density lipoprotein (LDL) that is involved in carrying  some of the fat-soluble vitamins (see lipid metabolism,  Chapter 13). The clinical features of vitamin E  deficiency include increased haemolysis, a possibly  increased risk of atherosclerosis and, in low-birthweight  babies, retrolental fibroplasias and intraventricular  haemorrhages.   Laboratory tests for deficiency consist of  measurement of plasma vitamin E levels (or perhaps  better expressed as vitamin E to LDL cholesterol ratio);  (/-tocopherol is the most active form. Sometimes  increased erythrocyte haemolysis can be measured in  the laboratory as an index of vitamin E deficiency.   It has been proposed that supplementation of dietary  antioxidants such as vitamins A, C and E may protect  against atherosclerosis by reducing LDL oxidation.  However, the results of some intervention trials have  been disappointing, having failed to show any significant  reduction in cardiovascular events for those on  antioxidant supplementation. Vitamin E excess is rare.   Vitamin K is needed for the synthesis of prothrombin  and coagulation factors VII, IX and X in the liver, and  deficiency is accompanied by a bleeding tendency with  a prolonged prothrombin time. Clinically, vitamin  K is sometimes given to reverse the actions of the  anticoagulant warfarin in patients in whom it is causing  bleeding problems, as evidenced by a prolonged   prothrombin time or international normalized ratio  (INR) . Vitamin K is also involved in bone mineralization.   Vitamin K can be synthesized by bacteria in the  ileum, from where it can be absorbed, and thus dietary  deficiency is unlikely. However, deficiency may occur:   • in patients with steatorrhoea - the vitamin, whether  taken in the diet or produced by intestinal bacteria,  cannot be absorbed normally,   • after the administration of some broad-spectrum  antibiotics, which may alter the intestinal bacterial  flora and so reduce the synthesis of vitamin K,  especially in children.   If vitamin K deficiency occurs, it can be corrected by  parenteral administration.   In the newborn infant, plasma vitamin K  concentrations are lower than in adults because  very little can be transported across the placenta, the  neonatal gut is only gradually colonized by bacteria  capable of synthesizing vitamin K and protein  synthesis has not yet reached full adult capacity,  particularly in premature infants. Deficiency may be  severe enough to cause haemorrhagic disease of the  newborn infant, a condition that may present within  2-3 days of birth.   Laboratory tests for vitamin K deficiency are usually  indirect, involving measurement of the prothrombin  time. Although one can measure blood vitamin K  or its metabolites or PIVKA (proteins induced by  vitamin K absence) in specialized laboratories, these  are rarely indicated. Overdose of vitamin K is rare and  may sometimes cause haemolytic anaemia. Vitamin  K treatment may perturb warfarin requirements of  patients being anticoagulated.   Thiamine, folate, vitamin B 12 and ascorbate are  actively absorbed from the intestinal tract and the rest  diffuse passively through the intestinal mucosal wall.   Most of these vitamins act as enzyme cofactors and  many are synthesized by colonic bacteria. As the  absorption of water-soluble vitamins from the large  intestine is poor, probably most of those synthesized  within the colon are unavailable to the body.   Clinical deficiency is rare in affluent communities.  When deficiency does occur, it is usually multiple,  involving most of the B group, and is associated  with protein undernutrition; for this reason it may  be difficult to decide which signs and symptoms are  specific for an individual vitamin and which are part  of a general undernutrition syndrome (Fig. 15.1). With  the exception of vitamin B |2 , assay of these vitamins  is rarely indicated and treatment is usually given  empirically if a deficiency state is suspected.   Humans cannot synthesize thiamine. It is found in  many dietary components; wheat germ, oatmeal and   yeast are particularly rich sources. Adequate amounts  are present in a normal diet and deficiency is most  common in alcoholics and in patients with anorexia  nervosa.   Thiamine is a component of thiamine pyrophosphate,  which is an essential cofactor for decarboxylation  of 2-oxoacids; one such reaction is the conversion of  pyruvate to acetyl coenzyme A (see Chapter 12). In  thiamine deficiency, pyruvate cannot be metabolized  and accumulates in the blood. Thiamine pyrophosphate  is also an essential cofactor for transketolase in the  pentose-phosphate pathway.   Deficiency is usually due to excess ethanol intake with  high carbohydrate but poor vitamin intake, although  it can also be seen in intensive-care patients with high  carbohydrate intake. One of the commonest causes  worldwide is a diet high in un-enriched white flour  or rice. The level of thiaminase, which breaks down  thiamine, is high in raw fish.   Deficiency of thiamine causes beri beri, in which  anorexia, emaciation, neurological lesions (motor and  sensory polyneuropathy, amnesia and encephalopathy  known as Wernicke-Korsakoff syndrome) and  cardiac arrhythmias may occur. This form is called  ‘dry’ beri beri (shoshin) and is associated with low  cardiac output. In ‘wet’ beri beri there is peripheral  oedema, sometimes associated with cardiac failure.  Thiamine deficiency can be seen as part of the  refeeding syndrome (see Chapter 14). Beri beri may  be aggravated by a high-carbohydrate diet, possibly  because this leads to an increased rate of glycolysis  and therefore of pyruvate production.   Figure 15.1 Some biochemical interrelations of the B  vitamins. A, substrate (e.g. pyruvate); AH 2 , reduced  substrate (e.g. lactate); CoA, coenzyme A; Fp,  flavoprotein; NAD, nicotinamide adenine dinucleotide;  TPP, thiamine pyrophosphate.   One test for thiamine deficiency is the estimation of  erythrocyte transketolase activity, with and without  added thiamine pyrophosphate. Reduced activity, if  due to thiamine deficiency, becomes normal after the  addition of the cofactor. This test is rarely indicated  and of little use once a normal diet, or vitamin  supplementation, has been started because plasma  concentrations are rapidly corrected. Other tests may  be useful, including the measurement of both blood  and urinary thiamine concentrations, and a raised  blood pyruvate concentration is suggestive as is a lactic  acidosis (see Chapter 4).   Riboflavin is found in large amounts in yeasts and  germinating plants such as peas and beans, and in smaller  amounts in fish, poultry and meat, especially offal.   Riboflavin is present in many naturally occurring  flavoproteins, in most of which it is incorporated in  the form of flavine mononucleotide (FMN) and flavine  adenine dinucleotide (FAD). Both FMN and FAD are  reversible electron carriers in biological oxidation  systems, which are, in turn, oxidized by cytochromes.   The causes of riboflavin deficiency include poor intake,  malabsorption, alcoholism and increased metabolic rate  in severe illness. Riboflavin deficiency (ariboflavinosis)  causes a rough, scaly skin, especially on the face,  cheilosis (red, swollen, cracked lips), angular stomatitis  and similar lesions at the mucocutaneous junctions  of the anus and vagina (orogenital syndrome), and a  swollen, tender, red tongue that is described as magenta  coloured. Congestion of conjunctival blood vessels may  be visible if the eye is examined with a slit lamp.   Riboflavin acts as a cofactor for glutathione reductase.  The finding of a low erythrocyte activity of this enzyme,  which increases by about 30 per cent after the addition  of FAD, suggests riboflavin deficiency. A low urinary  riboflavin concentration may also be a useful marker  of deficiency. The treatment for deficiency is to give  riboflavin. Riboflavin excess is very rare.   Nicotinamide can be formed in the body from nicotinic  acid. Both substances are plentiful in animal and plant  foods, although much of that in plants is bound in  an unabsorbable form. Some nicotinic acid can also  be synthesized in humans from tryptophan. Probably  both dietary and endogenous sources are necessary to  provide enough nicotinamide for normal metabolism.   Nicotinamide is the active constituent of nicotinamide  adenine dinucleotide (NAD + ) and its phosphate  (NADP + ), which are important cofactors in oxidation-  reduction reactions. Reduced NAD + and NADP + are,   in turn, re-oxidized by flavoproteins, and the functions  of riboflavin and nicotinamide are closely linked. The  NAD + and NADP + and their reduced forms are essential  for glycolysis and oxidative phosphorylation, and for  many synthetic processes.   It may be difficult to distinguish between the clinical  features due to coexistent deficiencies, such as of  pyridoxine, and those specifically due to nicotinamide.  However, nicotinamide deficiency is probably the most  important factor precipitating the clinical syndrome  of pellagra. The mnemonic ‘three Ds’ - dementia,  dermatitis, diarrhoea - may help in remembering the  symptoms.   • Dementia, with delusions, may be preceded by  irritability and depression.   • Dermatitis is a sunburn-like erythema, especially  severe in areas exposed to the sun, which may  progress to pigmentation and thickening of the skin;  ‘pellagra’ literally means ‘rough skin’.   • Diarrhoea is due to widespread inflammation of the  mucosal membranes of the gastrointestinal tract;  anorexia may aggravate weight loss.   Dietary deficiency of nicotinamide, like that of the  other B vitamins, is rare in affluent communities.   Hartnup’s disease is due to a rare inborn error of  metabolism involving the renal, intestinal and other  cellular transport mechanisms for the monoamino-  monocarboxylic amino acids, including tryptophan  (see Chapter 27). Subjects with the disorder may  present with a pellagra-like rash that can be cured by  giving nicotinamide daily. If the endogenous supply  of tryptophan is reduced, dietary nicotinic acid is  probably insufficient to supply the body’s needs over  long periods of time; under these circumstances,  only a slight reduction of intake may precipitate  pellagra. A similar clinical picture has been reported  as a rare complication of the carcinoid syndrome,  when tryptophan is diverted to the synthesis of large  amounts of 5-hydroxytryptamine (see Chapter 24).   Nicotinic acid (but not nicotinamide) may reduce  the hepatic secretion of very low-density lipoprotein  (VLDL) and therefore plasma concentrations of VLDL  and LDL (see Chapter 13).   If a normal diet has not been started, the diagnosis can  sometimes be made by measuring urinary V- methyl -  nicotinamide concentration, which is low in deficiency.  Treatment is usually with nicotinamide.   Pyridoxal phosphate, formed in the liver from  pyridoxine, pyridoxal and pyridoxamine, is a cofactor  mainly for the transaminases, and for decarboxylation  of amino acids.   Pyridoxine (pyridoxol), its aldehyde (pyridoxal) and  amine (pyridoxamine) are widely distributed in food;  dietary deficiency is very rare. The antituberculous drug  isoniazid (isonicotinic hydrazide) and possibly also  levodopa have been reported to produce the picture of  pyridoxine deficiency, probably by competing with it in  metabolic pathways. Other causes of deficiency include  alcoholism, malabsorption and inborn errors.   Deficiency may cause roughening of the skin, peripheral  neuropathy and a sore tongue. A rare hypochromic  microcytic anaemia, with increased iron stores  (sideroblastic anaemia), responds to large doses of  pyridoxine even when there is no evidence of vitamin  deficiency (‘pyridoxine-responsive’ anaemia).   Pyridoxal phosphate is needed for the conversion  of tryptophan to nicotinic acid, and this pathway is  impaired in pyridoxine deficiency. Xanthurenic acid is  the excretion product of 3-hydroxykynurenic acid, the  metabolite before the ‘block’; in pyridoxine deficiency,  it is found in abnormally high amounts in the urine  after an oral tryptophan load. The urinary metabolite  of pyridoxal phosphate, 4-pyridoxic acid, may also  be measured. Increase in the activity of erythrocyte  aspartate transaminase after the addition of pyridoxal  phosphate may be measured. The more severe the  pyridoxine deficiency, the greater the increase in enzyme  activity after addition of the vitamin. Excess pyridoxine  can occur with overdose regularly greater than 10 mg /  day and may lead to a peripheral neuropathy.   It is not clear whether lack of these two vitamins  produces a clinical deficiency syndrome.   Biotin is produced by intestinal bacteria. It is  also present in eggs, but large amounts of raw egg  white in experimental diets have caused loss of hair  and dermatitis that are thought to be due to biotin  deficiency. Probably the protein avidin, present in  egg white, combines with biotin and prevents its  absorption. Biotin is a cofactor in carboxylation  reactions.   Pantothenate is a component of coenzyme A, which  is essential for fat and carbohydrate metabolism. The  vitamin is widely distributed in foodstuffs.   Both folate and vitamin B 12 are essential for the normal  maturation of erythrocytes; deficiency of either causes  macrocytosis or megaloblastic anaemia. Their effects are  so closely inter-related that they are usually considered  together. A fuller discussion of the haematological  diagnosis and treatment of megaloblastic anaemia,  such as a raised mean corpuscular volume (MCV),  will be found in haematology textbooks. Serum and  erythrocyte folate may be low in folate deficiency.  Serum vitamin B 12 is usually reduced in deficiency   A 67-year-old woman saw her general practitioner  because of tiredness and paraesthesiae in her feet as  well as anaemia. On examination, there was evidence  of a sensory peripheral neuropathy. A number of  blood tests were requested, and the abnormal results  are shown below.   A full blood count showed mean corpuscular  volume (MCV) of 102 fL (82-98); concentrations of  haemoglobin, white cells and platelets were all low  (pancytopenia).   The diagnosis is vitamin B 12 deficiency resulting  in pernicious anaemia. Note the raised MCV, as  macrocytosis, and pancytopenia, is a feature of vitamin  B 12 deficiency. Symptoms suggestive of a neuropathy  may occur. Here, there is an abnormality of vitamin  B 12 intestinal absorption usually with positive parietal  cell and/or intrinsic factor antibodies.   states, as may holotranscobalamin, whereas plasma  homocysteine and methylmalonic acid may be elevated.   Folate is present in green vegetables and some  meats. It is easily destroyed during cooking, and dietary  deficiency may occasionally occur. Folate is absorbed  throughout the small intestine and, in contrast to most  of the other B vitamins (except B p ), clinical deficiency  is relatively common in intestinal malabsorption  syndromes, especially in the ‘contaminated bowel’  syndrome. In these conditions, and during pregnancy  and lactation, low red-cell folate concentrations may be  associated with megaloblastic anaemia or macrocytosis.  Low maternal folate intake is also associated with neural  tube defects in the fetus. The active form of the vitamin  is tetrahydrofolate, which is essential for the transfer  of one-carbon units; it is particularly important in  purine and pyrimidine, and therefore deoxyribonucleic  acid (DNA) and ribonucleic acid (RNA), synthesis.  Methotrexate, a cytotoxic analogue of folate, competes  with it for metabolism, and therefore inhibits DNA  synthesis.   The vitamin B p group includes several cobalamins,  found in animal products but not in green vegetables.  Dietary deficiency is rare. The cobalamins are  transported in plasma by a specific carrier protein,  transcobalamin II. Deoxyadenosyl-cobalamin and  methylcobalamin have, like folate, coenzyme activity in  nucleic acid synthesis. Hydroxycobalamin is the form  most commonly used in treatment, and both it and  cyanocobalamin are converted to cofactor forms in the  body. All forms are absorbed mainly in the terminal  ileum, combined with intrinsic factor derived from the  gastric parietal cells. In pernicious anaemia, antibodies  to gastric parietal cells and/or to intrinsic factor cause  malabsorption of vitamin B p . The Schilling test has  been used to diagnose pernicious anaemia.   A small dose of radiolabelled vitamin B p is given orally  and its excretion is measured in the urine. A flushing  dose of non-radiolabelled vitamin is given parenterally  at the same time as, or just after, the labelled dose to  ensure quantitative urinary excretion. Haematological  tests, such as the examination of blood and bone  marrow films, should have been completed before the  vitamin B p is given.   Interpretation. If malabsorption of the vitamin  is due to pernicious anaemia, administration of the  labelled vitamin with intrinsic factor restores normal  absorption; if it is due to intestinal disease, for example   ileal resection or bacterial overgrowth, malabsorption  persists. Intrinsic factor and parietal antibodies should  be tested, as these maybe positive in pernicious anaemia  (see Chapter 16).   Deficiency of vitamin B p , like that of folate, causes  megaloblastic anaemia. However, unlike that of folate,  it can cause subacute combined degeneration of the  spinal cord. Although the megaloblastic anaemia of  vitamin B p deficiency can be reversed by folate, this  treatment should never be given in pernicious anaemia  because it does not improve, and may even aggravate,  the neurological lesions.   It has recently become established that homocysteine,  a sulphur- containing amino acid, is an independent  cardiovascular risk factor. Both folate and vitamin  B p regulate the enzyme methylenetetrahydrofolate  reductase and methionine synthase, respectively, while  vitamin B fi is a cofactor of the enzyme cystathionine  synthase (Fig. 15.2). Supplementation with folate and /  or vitamin B p may lower homocysteine plasma levels  (see also Chapter 22).   The known biological functions and the clinical  syndromes associated with deficiencies of the B vitamins  are summarized in Table 15.1. Generally, deficiencies of  this group cause lesions of skin, mucous membranes  and the nervous system.   Table 15.1 The biochemical functions and clinical deficiency syndromes associated with the B vitamins   FAD, flavine adenine dinucleotide; FMN, flavine mononucleotide; NAD, nicotinamide adenine dinucleotide;  NADP, nicotinamide adenine dinucleotide phosphate.   Ascorbate is found in fruit, particularly citrus fruits, and  vegetables. It is added to other foods as a preservative. It  cannot by synthesized by humans.   Ascorbate can be reversibly oxidized in biological  systems to dehydroascorbate and, although its functions  in humans are not clear, it probably acts as a hydrogen  carrier and antioxidant as well as being involved in  collagen synthesis.   Deficiency of ascorbate causes scurvy, which was  common on long sea voyages before the 18 th century  when fresh fruit and vegetables were unavailable.  Dehydroascorbate is easily and irreversibly oxidized  and loses its biological activity in the presence of  oxygen; this reaction is catalysed by heat. Deficiency  occurs most commonly in the elderly, especially those  who do not eat fresh fruit and vegetables. It can also  occur in iron overload.   Many of the signs and symptoms of scurvy are related to  deficient collagen formation and include the following.   • Fragility of vascular walls causing a bleeding  tendency, often with a positive Hess test; petechiae   and ecchymoses; swollen, tender, spongy, bleeding  gums; and, sometimes, haematuria, epistaxis and  retinal haemorrhages. In infants, subperiosteal  bleeding and haemarthroses are extremely painful  and may lead to permanent joint deformities.   • Deficiency of bone matrix causing osteoporosis and  poor healing of fractures. In children, bone formation  is impaired at the epidiaphyseal junctions, which look  ‘frayed’ radiologically and can mimic non-accidental  injury. The enzyme procollagen hydroxylase is  mediated by vitamin C and is important for the  intracellular matrix. In children there are tender  limbs due to poor osteoid, costochondral bleeding  (scorbutic rosary) and ground-glass bone X-ray  appearance.   • Anaemia, possibly due to impaired erythropoiesis  and poor iron absorption. The anaemia is aggravated  by bleeding.   These signs and symptoms are often cured by the  administration of ascorbate.   Laboratory confirmation of the clinical diagnosis of  ascorbate deficiency can only be made before therapy  has started. Once ascorbate has been given, it is difficult  to prove that deficiency was previously present.  Although the assay of leucocyte ascorbate was thought  to be more reliable than that of plasma in confirming   the diagnosis, levels probably alter in parallel; plasma  assay is technically more satisfactory, but chemical  estimations are rarely indicated. There is also an oral  vitamin C loading test that may show a low urinary  excretion of vitamin C in deficiency states. Treatment  of deficiency is with oral vitamin C.   Excess vitamin C is rare, but may increase oxalate levels  and the likelihood of renal oxalate calculi.   Inorganic micronutrients, or trace metals, are essential  for normal health and, by definition, make up less than  0.01 per cent of the body’s dry weight.   Zinc is a cofactor for certain enzymes, for example  polymerases, carbonic dehydratase and alkaline  phosphatase. Zinc deficiency may result in a number of  clinical states, including growth retardation, alopecia,  dermatitis, diarrhoea, poor wound healing, infertility  and increased risk of infections.   The causes of zinc deficiency are acrodermatitis  enteropathica (a rare autosomal recessive condition  associated with dermatitis, diarrhoea and alopecia  and a defect of the SLC39A4 gene, which encodes for  a membrane zinc-transporting protein) poor intake  (e.g. parenteral nutrition or malabsorption), increased  utilization and high levels of dietary phytates. Useful  laboratory tests for deficiency include the measurement  of plasma or urinary zinc levels. The plasma zinc-carrying  protein metallothionine may also prove useful. Note that  plasma zinc levels may be low during an acute-phase  response, as the zinc is also partly bound to albumin, which  is a negative acute-phase reactant. Zinc toxicity is rare,  usually occurring after inappropriate administration, and  may result in pulmonary oedema, jaundice and oliguria  as well as hypocupraemia (low plasma copper).   Copper functions as an enzyme cofactor, for example  for cytochromes. Copper deficiency may cause cardiac  arrhythmias, neutropenia, hypochromic anaemia  and, in children, bony problems such as subperiosteal  haematomas.   The causes of deficiency include poor intake, for  example long-term parenteral nutrition. Menke’s  disease is an inborn error of copper transport resulting  in low plasma copper concentrations and abnormal  hair that has a characteristic ‘kink’ appearance.   Laboratory tests for deficiency include the  measurement of plasma copper. Copper is carried on  the protein caeruloplasmin, the level of which may be  increased due to an acute-phase response, oestrogens  or pregnancy. Copper deficiency can be treated with  certain copper salts, but care is needed in case there is  an effect on zinc and iron absorption.   Copper excess can lead to toxicity, causing renal  problems, fits, haemolysis and hypotension. Wilson’s  disease, in which copper excess and decreased  caeruloplasmin occur, is an inborn error of metabolism.   Some plasma copper is loosely bound to albumin, but  most is incorporated in the protein caeruloplasmin.  Copper is mainly excreted in bile. There are two defects  of copper metabolism in Wilson’s disease:   • deficiency of caeruloplasmin results in low plasma  copper concentrations. Most is in a loosely bound  form and is therefore deposited in tissues; more  than normal is filtered at the glomeruli and urinary  copper excretion is increased.   A 21 -year-old man was referred to the neurology  out-patient department because of dysarthria and leg  weakness. A number of investigations were organized  and some of the pertinent results are as follows.  Urine analysis showed amino aciduria.   Albumin 44g/L (35-45)  y-Glutamyl transferase 234 U/L (<55)  Caeruloplasmin 0.08 g/L (0.2-0. 6)   The patient was confirmed by liver biopsy and  genetic studies to have Wilson’s disease presenting  with neurological sequelae, abnormal liver function  tests and renal tubular damage leading to amino  aciduria. The concentration of copper-binding  protein caeruloplasmin is low. High urinary free  copper concentration would be expected.   Excessive deposition of copper in the eyes, basal  ganglia of the brain, liver and renal tubules produces:   • Kayser-Fleischer rings at the edges of the cornea due  to deposition of copper in Des 9 emet’s membranes  (Fig 15.3),   • renal tubular damage with any or all of the associated  biochemical features, including amino aciduria  (Fanconi’s syndrome).   Most patients have low plasma caeruloplasmin (less  than 0.2 g/F) and copper concentrations (less than  12pmol/L) and high urinary copper excretion (more  than 1.2 pmol/day).   The penicillamine test is sometimes used in the  diagnosis of Wilson’s disease and is based on the  principle that penicillamine solubilizes copper and  enhances copper urinary excretion. Following a dose  of oral penicillamine, urinary copper excretion is more  than 25 pmol/day, indicative of Wilson’s disease.   Fow plasma caeruloplasmin concentrations may  also occur during the first few months of life due  to undernutrition, and in the nephrotic syndrome  due to urinary loss. Raised plasma caeruloplasmin  concentrations are found in active liver disease, in  women taking oral contraceptives, during the last  trimester of pregnancy and non-specifically when there    Figure 15.3 Patient with Wilson’s disease manifesting  the Kayser-Fleischer ring. Reproduced with kind  permission from Nyhan WL, Barshop BA and Ozand  PT. Atlas of Metabolic Diseases, 2nd edition. London:  Hodder Arnold, 2005.   is tissue damage due, for example, to inflammation  or neoplasia. These may account for the rare finding  of ‘normal’ plasma caeruloplasmin concentrations in  patients with Wilson’s disease.   Histological examination, with the demonstration of  an increased copper content, of a liver biopsy specimen  is often necessary to confirm the diagnosis.   Wilson’s disease has a recessive mode of inheritance,  but reduced plasma caeruloplasmin concentrations  may be demonstrable in heterozygotes. The  distinction between presymptomatic homozygotes  and heterozygotes is important because the former  can be treated. Treatment with copper-chelating agents  such as D-penicillamine may reduce tissue copper  concentrations.   The main function of selenium is to mediate the activity  of the enzyme glutathione peroxidase, which acts as an  antioxidant.   Deficiency can be caused by poor intake and  is most commonly seen in certain areas of China.  Patients on artificial nutrition, for example parenteral  support, are also more susceptible. Selenium deficiency  may also cause cardiomyopathy (Keshan’s disease),  osteoarthropathy (Kaschin-Beck disease), myopathy,  macrocytosis and anaemia.   Selenium may slow the progression of mild Graves’  orbitopathy (see Chapter 11).   Diagnostic laboratory tests for selenium deficiency  include the measurement of urinary or blood selenium  or finding decreased erythrocyte glutathione peroxidase  activity.   This is an enzyme cofactor, for example of superoxide  dismutase. Deficiency is associated with vitamin K  deficiency and may be seen in patients who are being  fed artificially.   Manganese excess can occur, for example in miners  of manganese ores, and can result in Parkinson-like  disease and defects of the basal ganglia. Sometimes  parenteral nutrition regimens may cause raised plasma  manganese concentration. Manganese is mainly biliary  excreted, and therefore toxicity may occur with hepatic  dysfunction or cholestasis.   Chromium is an insulin cofactor. Chromium deficiency  can occur on long-term parenteral nutrition, leading to  glucose intolerance and neuropathy.   Deficiency can occur on long-term parenteral  nutrition and may lead to tachycardia, central scotomas,  ‘night blindness’ and coma.   This is important for vitamin B n metabolism. Toxicity  is rare and has been described in dialysis patients and  heavy drinkers of beer that is contaminated with cobalt,  leading to cardiomyopathy.   It would be highly unusual to have a deficiency of the  following metals, although, for completeness, metal  poisoning is included in this chapter.   Mercury poisoning can occur from organic or inorganic  salts or elemental mercury vapour. Acute toxicity  may result in a metallic taste and respiratory distress,  nausea and vomiting. More chronic features include  neuropathy and renal dysfunction.   Acute toxicity can be treated with dimercaprol-  chelating agents, which increase excretion via urine and  bile. In chronic exposure, N-acetyl penicillamine has  been used to chelate mercury.   The diagnosis of mercury poisoning can be  established from urinary or blood mercury levels.  Sometimes hair mercury levels can be used to detect  long-term exposure.   Aluminium toxicity, although rare, is well described in  patients with renal impairment. Contamination of the  water used in dialysis fluid has been implicated in renal  osteodystrophy and dialysis dementia. Dialysis water  is now usually treated to decontaminate aluminium.  Toxicity has also been seen in individuals with excess  oral intake, such as of aluminium-containing antacids  for dyspepsia. Contamination from plastic tubing  and glass vials may occur in some long-term total  parenteral nutrition patients. Aluminium exposure  is diagnosed by plasma aluminium measurements.  Desferrioxamine has been used to chelate aluminium  in toxicity.   Cadmium toxicity can occur in industrial workers  exposed to cadmium fumes. Clinical features include  nephrotoxicity, hepatotoxicity and bone disease.  Cadmium exposure can be assessed from the levels in  blood and urine. Renal tubular damage can be monitored  by raised urinary (3 2 -microglobulin concentration.   • Vitamins are essential for biochemical processes  and can be divided into those that are fat soluble  and those that are water soluble. The former include  vitamin A, D, E and K; the latter are vitamins B  and C.   • Vitamin A and E deficiencies are more common  in developing countries. Vitamin D deficiency  can result in rickets or osteomalacia (discussed in  Chapter 6) . Vitamin K deficiency can cause bleeding  problems with prolonged prothrombin time.   • Vitamin B (thiamine) deficiency is associated with  beri beri and Wernicke-Korsakoff syndrome.   Trace elements are also essential, such as zinc,  copper, manganese and selenium. Deficiencies  can occur, usually if the patient has poor dietary  intake.   Trace element excess can occur, for example in  Wilson’s disease, in which plasma caeruloplasmin  concentration is low, resulting in excess unbound  copper.   Gastrointestinal disorders present relatively commonly  to doctors, and biochemical laboratory tests have  important roles in their investigation. This chapter  looks at gastrointestinal pathophysiology and how  chemical pathology tests may be useful in diagnosis and  treatment.   The major functions of the gastrointestinal tract are  the digestion and absorption of nutrients. Additionally,  it synthesizes certain hormones along its length that  act locally (paracrine hormones), so controlling gut  motility and the release of digestive enzymes and  secretions. The gastrointestinal tract handles about  8-9 L of fluid per day, with the majority of this derived  from endogenous secretions. Fluid reabsorption by  the gastrointestinal tract (mainly the small intestine)  is highly efficient (98 per cent), with only 100-200 mL  lost daily in the stools.   Digestion and absorption depend on food and the  products of its digestion being diluted with a large  volume of fluid, mostly an ultrafiltrate of extracellular  fluid filtered through the ‘tight junctions’ between  epithelial cells, mainly in the duodenum. Some is  reabsorbed in the proximal jejunum, along the osmotic  gradient created by reabsorption of the products of  digestion; a large amount remains in the lumen to be  reclaimed more distally.   A small amount of the nutrients from desquamated  intestinal cells also enters the lumen; most is  reabsorbed. Almost all the fat in normal faeces  is of endogenous origin, which is relevant when  interpreting the results of a faecal fat estimation.  A much smaller volume of fluid enters the lumen  by active secretion. As a result, the pH and the  electrolyte and enzyme concentrations change  during the passage of fluid through the tract in   such a way that the conditions for enzyme activity  are near optimal for digestion. Adjustment of pH  and electrolyte concentrations occurs in the distal  ileum and colon; sodium reabsorption (and therefore  water reabsorption) is enhanced by aldosterone in  the colon. The colon can metabolize undigested  carbohydrates into short-chain fatty acids such  as butyrate, propionate and acetate. These are an  important energy source for the colon.   Disturbances of water, electrolyte and hydrogen ion  homeostasis may occur in small intestinal or colonic  disease. If there is loss of fluid and electrolytes from the  upper intestinal tract because of vomiting or because  the function of intestinal cells is so perturbed that the  amount of fluid and electrolytes entering the distal  parts exceeds the reabsorptive capacity, similar changes  may occur (see also Chapter 2).   Digestive enzymes usually act on complex molecules  such as protein, polysaccharides and fat. This process  starts in the mouth, where food is broken down  by mastication and is mixed with saliva containing  a-amylase. In the stomach, acid fluid is added and  the low pH initiates protein digestion by pepsin.  The stomach also secretes intrinsic factor, essential  for vitamin B n absorption from the terminal ileum.  However, most digestion takes place in the duodenum  and upper jejunum, where alkaline fluid is added to  the now liquid intestinal contents. Pancreatic enzymes  digest proteins to small peptides and amino acids,  polysaccharides to monosaccharides, disaccharides and  oligosaccharides, and fat to monoglycerides and fatty  acids.   The main components of gastric secretion are  hydrochloric acid, pepsin and intrinsic factor, all  of which are important for normal digestion and  absorption. Loss of hydrochloric acid by vomiting, as   in pyloric stenosis, may cause a metabolic alkalosis  (Chapter 4). Gastric secretion maybe stimulated by the  following:   • The vagus nerve, which in turn responds to stimuli  from the cerebral cortex, normally resulting from  the sight, smell and taste of food. Hypoglycaemia  can stimulate gastric secretion and so can be used to  assess the completeness of a vagotomy.   • Gastrin, which is carried by the bloodstream to the  parietal cells of the stomach; its action is mediated  by histamine. Gastrin secretion is inhibited (by  negative feedback) by acid in the pylorus. Calcium  also stimulates gastrin secretion and this may explain  the relatively high incidence of peptic ulceration in  patients with chronic hypercalcaemia.   Histamine stimulates gastric secretion after binding  to specific cell surface receptors, of which there are at  least two types:   • those for which antihistamines compete with  histamine (H receptors): these are found on smooth  muscle cells,   • those on which antihistamines have no effect: these  H 7 receptors are found on gastric parietal cells.   Hypersecretion of gastric juice may cause duodenal  ulceration. However, there is overlap between the  amount of acid secreted in normal subjects and in those  with duodenal ulceration. The estimation of gastric  acidity is of very limited diagnostic value. In the very  rare Zollinger-Ellison syndrome, acid secretion is very  high due to excessive gastrin produced by a gastrinoma  - a tumour more usually involving the pancreas or  duodenum (see later).   Drugs such as ranitidine compete with histamine  for the H 7 receptors, so directly reducing acid secretion.  Proton pump inhibitors such as omeprazole and  lansoprazole have to some degree surpassed these  agents.   Hyposecretion of gastric juice occurs most  commonly in association with pernicious anaemia,  due to the formation of antibodies to the parietal cells  of the gastric mucosa. Extensive carcinoma of the  stomach and chronic gastritis may also cause gastric  hyposecretion. Plasma gastrin concentrations are  raised, because reduced acid secretion causes the loss of  negative feedback inhibition.   Tests of gastric function involving the measurement  of acid secretion have largely been superseded by  endoscopic examination of the stomach and duodenum   There may be some degree of malabsorption after  gastrectomy. Rapid passage of the contents of the small  gastric remnant into the duodenum may be associated  with the following:   • The early dumping syndrome Soon after a meal, the  patient may experience abdominal discomfort and  feel faint and nauseated. These symptoms may be  caused by the rapid passage of hypertonic fluid into  the duodenum. Before this abnormally large load  can be absorbed, water passes along the osmotic  gradient from the extracellular space into the lumen.  The reduced plasma volume causes faintness and the  large volume of fluid causes abdominal discomfort.   • The late dumping syndrome (or post-gastrectomy  hypoglycaemia) If a meal containing a high glucose  concentration passes quickly into the duodenum,  glucose absorption is very rapid, stimulating a  surge in insulin secretion. The resultant ‘overswing’  of plasma glucose concentration may cause  hypoglycaemic symptoms, typically occurring  about 2h after a meal. This is a form of reactive  hypoglycaemia (see Chapter 12).   Both these dumping syndromes can be minimized if  frequent, small, low- carbohydrate meals are taken.   There are three phases of digestion and absorption:  luminal phase, mucosal phase and post-absorptive  phase.   • the presence of nutrient in an absorbable form (and  therefore on normal digestion),   • a normal ratio of the rate of absorption to the rate  of passage of contents through the intestinal tract.   The absorptive area of the small intestine is very  large. The mucosa forms macroscopically visible folds,  increasing the area considerably. Microscopically, these  folds are covered with villi lined with absorptive cells  (enterocytes), which increase the area about eight-fold.  A large number of minute projections (microvilli)  cover the surface of each enterocyte, separated by  microvillous spaces. These increase the absorptive   area about a further 20-fold. The absorptive area is  significantly reduced if the villi are flattened, as they  are, for example, in gluten-sensitive enteropathy.  Absorption also depends on whether a molecule is:   • water soluble, so that it can be absorbed either by  active transport against a physicochemical gradient  or by passive diffusion along gradients,   • lipid soluble, enabling it to diffuse across the lipid  membranes of the enterocytes.   The release of intestinal secretions and the control  of gut motility are, in part, controlled by a series of  peptide hormones produced in the mucosa of the  gastrointestinal tract. Like other hormones, their release  is under feedback control by either the product or the  physiological response of the target tissue.   Cells that synthesize gut peptides are scattered  throughout the length of the gastrointestinal tract,  although the secretory cells are often concentrated in  one segment of the tract. Many of these peptides are  present in other organs, particularly the brain, where  they probably act as neurotransmitters.   • Gastrin is released from the gastrin-secreting G  cells in the gastric antrum in response to distension  and to protein. It stimulates the contraction of the  stomach muscles and the secretion of gastric acid.  Acid inhibits gastrin release by negative feedback.   • Cholecystokinin stimulates contraction of the gall  bladder and the release of pancreatic digestive  enzymes.   • Secretin stimulates the release of pancreatic fluid rich  in bicarbonate.   Polysaccharides, such as starch, consist either of straight  chains of glucose molecules joined by 1:4 linkages,  called amylose, or of branch chains, in which the  branches are joined by 1:6 linkages, called amylopectin.  Salivary and pancreatic O.-amvIases hydrolyse starch to  1:4 disaccharides such as maltose (glucose + glucose).  Pancreatic amylase is quantitatively the more important.  A few larger branch-chain saccharides (limit dextrans)  remain undigested.   Disaccharides [maltose, sucrose (glucose + fructose)  and lactose (glucose + galactose)] are hydrolysed to  their constituent monosaccharides by the appropriate   disaccharidases, [3-galactosidases (lactase) and  a-glucosidases (maltase, sucrase), located on the  surface of enterocytes (‘brush border’), especially in the  proximal jejunum. Sucrase also hydrolyses the 1:4, and  isomaltase the 1:6, linkages of limit dextrans.   Monosaccharides are actively absorbed in the  duodenum and proximal jejunum. Unlike for fructose,  a common active process absorbs glucose and galactose.  Carbohydrate absorption depends on:   • the presence of amylase, and therefore on normal  pancreatic function (polysaccharides only),   • the presence of disaccharidases on the luminal  membrane of intestinal cells (disaccharides),   • normal intestinal mucosal cells with normal active  transport mechanisms (monosaccharides).   Polysaccharides can be absorbed only if all three  mechanisms are functioning.   This can be used to test the integrity of the intestinal  mucosa. Xylose is a pentose that, like glucose, can be  absorbed without digestion (by facilitated diffusion),  but the metabolism of which is not hormone controlled;  therefore, unlike glucose, it is not affected by insulin  secretion.   An oral dose of xylose is absorbed by normally  functioning upper small intestinal cells. Metabolism  of the absorbed pentose is very slow and, because it  is freely filtered by the glomeruli, most of it appears  in the urine. In the xylose absorption test, either the  amount excreted in the urine during a fixed period or,  in children, the plasma concentration at a defined time  after the dose is measured.   Intestinal disease (for example blind loops) or  impaired mucosal surface area (as in coeliac disease) may  impair xylose absorption and therefore reduces excretion.  Pancreatic disease does not affect either process. Therefore  this test can distinguish between pancreatic insufficiency  and conditions affecting the intestinal mucosa.   Unfortunately, there are several sources of error in  the xylose test:   • Xylose is absorbed mostly from the upper small  intestine and absorption may therefore be normal  despite significant ileal dysfunction, for example in  Crohn’s disease.   • The urine test depends on accurate collections  over a short time interval and therefore even more  significant errors may occur than in 24-h collections.   • Mildly impaired renal function decreases glomerular  filtration of xylose and may give falsely low results in  the urine tests, or a falsely high plasma concentration.  This is a common cause of misleading results in  the elderly. The xylose absorption test should not  be performed if glomerular function is even only  marginally impaired.   • Both plasma concentrations and urine excretion  depend partly on the volume of distribution of  the absorbed xylose. In oedematous or very obese  patients, results may be falsely low.   A possible scheme for carrying out the xylose test is  now discussed.   A dose of 5 g of xylose is preferable to one of 25 g because  a high xylose concentration within the intestinal lumen  may, like a large dose of glucose, have an osmotic effect  that causes symptoms and affects the results.   • 08.00 h The bladder is emptied and the specimen  discarded. Then 5 g of xylose dissolved in 200 mL of  water is given orally. All specimens passed between  08.00 h and 10.00 h are put into a bottle labelled  number 1.   • 10.00 h The bladder is emptied and the specimen is  put into bottle number 1, which is now complete. All  specimens passed between 10.00 h and 13.00 h are  put into a bottle labelled number 2.   • 13.00h The bladder is emptied and the specimen is  put into bottle number 2, which is now complete.  Both bottles are sent to the laboratory for analysis.   In the normal subject, more than 1.5 g xylose should be  excreted during the 5h. About 50 per cent or more of  the total excretion should occur during the first 2h. In  mild intestinal malabsorption, the total 5-h excretion  may be normal, but delayed absorption is reflected  in a 2:5-h ratio of less than 40 per cent. In pancreatic  malabsorption, the result should be normal.   Protein in the intestinal lumen is derived from the  diet, intestinal secretions and desquamated mucosal  cells. Dietary protein is broken down by gastric  pepsin and in the duodenum by trypsin and other  proteolytic enzymes secreted in pancreatic juice;  pancreatic trypsinogen is converted to active trypsin   by enterokinase (enteropeptidase) located on the brush  border. The products of digestion are small peptides  and amino acids. Many peptides are further hydrolysed  by peptidases on the brush border.   Amino acids are actively absorbed in the small  intestine. Small peptides are absorbed by an active  transport mechanism, independently of amino acids  and, with a few exceptions, are hydrolysed intr acellularly.  Protein absorption depends on the presence of  pancreatic proteolytic enzymes (and therefore on  normal pancreatic function) and an intestinal mucosa  with normal active transport mechanisms.   Triglycerides are the main form of dietary fat and are  esters of glycerol with three, usually different, fatty  acids. They are insoluble in water. Cholesterol, in the  intestinal lumen, is derived from bile salts and from the  diet; only about 30 per cent of the dietary cholesterol is  absorbed (see Chapter 13).   Primary bile acids are synthesized in the liver from  cholesterol, conjugated with the amino acid glycine or  taurine in the liver, and then enter the intestinal lumen  in the bile. In the alkaline duodenal fluid their sodium  salts act as detergents, emulsifying and so facilitating the  digestion and absorption of fats. Most of the bile salts are  actively reabsorbed in the distal ileum, although some  enter the colon where they are converted by bacteria  to secondary bile salts, some of which are absorbed.  The absorbed bile salts are recirculated to the liver and  resecreted into the bile (the enterohepatic circulation).   Some bacteria within the gut lumen contain enzymes  that catalyse the deconjugation ofbile salts; unconjugated  bile salts emulsify fat less effectively than conjugated  ones and this may contribute to fat malabsorption.  Triglycerides are emulsified by bile salts within the  duodenum. They are hydrolysed by pancreatic lipase at  the glycerol/ fatty acid bond, primarily in positions 1 and  3. The end products are mainly 2-monoglycerides, some  diglycerides and free fatty acids. Colipase, a peptide  coenzyme secreted by the pancreas, is essential for lipase  activity. It anchors lipase at the fat/water interface,  prevents the inhibition of the enzyme by bile salts and  reduces its pH optimum from about 8.5 to about 6.5;  the latter is the pH in the upper intestinal lumen.   Monoglycerides and free fatty acids aggregate with  bile salts to form water-miscible micelles. The micelles   also contain free cholesterol liberated by the hydrolysis  of cholesterol esters in the lumen and phospholipids,  as well as fat-soluble vitamins (A, D, E and K). The  diameter of the negatively charged micelles is between  100 and 1000 times smaller than that of the emulsion  particles. Both their small size and their charge allow  them to pass through the narrow microvillous spaces.   Within enterocytes, triglycerides are resynthesized  from monoglycerides and fatty acids, and cholesterol  is re-esterified. Triglycerides, cholesterol esters and  phospholipids, together with fat-soluble vitamins,  combine with apolipoproteins manufactured in  enterocytes to form chylomicrons. These are suspended  in water and pass into the lymphatic circulation.   Some short-chain and medium-chain free fatty acids  pass directly through the intestinal cells into the portal  bloodstream.   • an adequate absorptive area of the intestinal mucosa  for chylomicron formation,   • the digestion of triglycerides by lipase, and therefore  on normal pancreatic function.   Faecal fat excretion should represent the difference  between the fat absorbed and that entering the  gastrointestinal tract from the diet and from the  body. The absorption of fat and, more importantly,  the addition of fat to the intestinal contents occur  throughout the small intestine. A single 24-h collection  of faeces gives very inaccurate results for two reasons:  the transit time from the duodenum to the rectum is  variable, and rectal emptying is variable and may not  be complete.   It has been suggested by some experts that the faecal  fat test is too inaccurate for clinical use and should be  abandoned. Indeed visual inspection looking for fat  globules in stools may be as sensitive and specific.   Consecutive 24-h collections give faecal fat results  that may vary by several hundred per cent. The  longer the period of collection, the more accurate the  calculated daily mean result; ideally, stools should be  collected for a minimum of 3 days.   A mean daily fat excretion of more than 18 mmol (5g)  indicates steatorrhoea. The result is not affected by diet  within very wide limits, because in the normal subject   almost all the fat is of endogenous origin. However, if  the patient is on a very low-fat diet, mild steatorrhoea  may be undetected.   The triolein breath test has also been used to assess fat  absorption. Absorbed fat is ultimately metabolized to  CO, and water. If 14 C-labelled triglyceride (triolein) is  given by mouth, the ratio of 14 C0 2 to unlabelled C0 2 can  be measured in expired air. A low ratio usually indicates  impaired fat absorption. The assay uses expensive  isotopes and requires special expertise. Although the  results correlate with faecal fat measurements, these  limitations have prevented its widespread adoption.   Vitamin B 12 can be absorbed only when it has formed a  complex with intrinsic factor, a glycoprotein secreted by  the parietal cells of the stomach (see Chapter 15). This  complex is resistant to proteolytic digestion and binds  to specific cell receptors in the distal ileum, from where  it is absorbed. Some intestinal bacteria need vitamin  B p for growth and may prevent its absorption by  competing for it with intestinal cells. Intestinal bacterial  overgrowth is associated with intestinal strictures,  diverticula and ‘blind loops’. Vitamin B p absorption  therefore depends on normal gastric secretion of  intrinsic factor, intestinal mucosa in the distal ileum  and intestinal bacterial flora.   Absorption of other water-soluble vitamins  Most of the other water-soluble vitamins (C and the  B group, except B 12 ) are absorbed in the upper small  intestine, probably by specific transport mechanisms.  Clinical deficiencies of all but folate are relatively  uncommon in the malabsorption syndromes, probably  because their absorption does not depend on that  of fat. Folate deficiency may be caused by intestinal  malabsorption, inadequate intake or increased use by  intestinal bacteria (see Chapter 15).   Calcium is absorbed mainly from the duodenum  and upper jejunum in the free ionized form;  this is enhanced by the vitamin D metabolite  1,25-dihydroxycholecalciferol, which influences  both active transport and passive diffusion. Calcium  absorption is impaired by the formation of insoluble  salts with free fatty acids and with phosphate. Much  of the faecal calcium is endogenous in origin, being  derived from intestinal secretions (see Chapter 6).   An active process that may be shared with calcium  also absorbs magnesium. Calcium and magnesium  absorption depends on:   • a low concentration of fatty acids and phosphate in  the intestinal lumen,   • the absorption and metabolism of vitamin D, and  therefore on normal fat absorption,   Iron is absorbed in the duodenum and upper jejunum  in the ferrous form (Fe 2+ ). Absorption is increased by  anaemia of any kind and probably also by vitamin C.  Some of the iron absorbed into intestinal cells enters  the plasma, but some is lost into the lumen with  desquamated cells (see Chapter 21).   Pancreatic secretions can be divided into endocrine  and exocrine components. The endocrine function  and insulin and glucagon that control the plasma  glucose concentration are discussed in Chapter 12. The  exocrine secretions are made up of two components, the  alkaline pancreatic fluid and the digestive enzymes. The  alkaline fluid is primarily responsible for neutralizing  gastric acid secretions, thus providing an optimal  environment for duodenal digestive enzyme activity.  These enzymes include the proteases trypsin, elastase  and chymotrypsin, and amylase and lipase. Some of the  proteases are secreted as precursors and are converted  to the active form within the intestinal lumen.   Gut peptides control pancreatic secretion and are  released from the duodenum in response to a rise in the  hydrogen ion concentration or to the presence of food.  They include secretin (which stimulates the release of  a high volume of alkaline fluid) and cholecystokinin  (which stimulates the release of a fluid rich in enzymes).   Plasma enzyme measurements are of limited value in  assessing exocrine function. They include the following.   This enzyme consists of two forms, one of salivary gland  origin and one of pancreatic origin. In acute pancreatitis,  total plasma amylase activity is usually significantly  increased due to release from damaged cells.   Plasma amylase is discussed more fully in Chapter 18  in the context of enzymology. Typically, plasma amylase   activity increases about five-fold in acute pancreatitis,  but plasma enzyme activities of up to, and even above,  this value may be reached in a number of other disorders,  in particular after intestinal perforation and renal failure.   Occasionally, the plasma enzyme activity in acute  pancreatitis may not be very high and usually falls rapidly  as the enzyme is excreted in the urine. Consequently, a  high plasma amylase activity is only a rough guide to  the presence of acute pancreatitis, and normal or only  slightly raised values do not exclude the diagnosis.  Plasma amylase concentrations can be spuriously  low or normal in acute pancreatitis evoked by severe  hypertriglyceridaemia. In such circumstances a raised  urinary amylase concentration may facilitate diagnosis  or clearing the plasma lipaemia by centrifugation prior to  assay. Haemorrhagic pancreatitis or chronic pancreatitis  can also show a normal plasma amylase concentration.   This enzyme is more specific for the pancreas and can  be useful to measure if the source of a raised plasma  amylase concentration is not obvious. Lipase has a  longer half-life than amylase and thus may be useful in  the late diagnosis of acute pancreatitis, when amylase  activity can fall within the reference range.   This may be used to screen for cystic fibrosis (see  later and Chapter 27) during the first 6 weeks of life.  Blockage of pancreatic ductules by sticky mucous  secretion causes high plasma trypsin concentrations.  After about 6 weeks, plasma concentrations may fall as  pancreatic insufficiency develops; normal levels do not  exclude the diagnosis.   Measurement of pancreatic enzymes and the  bicarbonate concentration in duodenal aspirates  before and after stimulation with cholecystokinin and  secretin is not very suitable for routine use because of  the difficulty in positioning the duodenal tube and in  quantitative sampling of the secretions.   ‘Tubeless tests’ have been developed that avoid the  need for intubation and that overcome the difficulties  of invasive sample collection.   A synthetic peptide, labelled with para-aminobenzoic  acid (PABA), is taken orally. After PABA has been  split from the peptide by chymotrypsin, it is absorbed  and excreted in the urine. Urinary excretion of PABA   is significantly reduced in chronic pancreatitis.  Abnormal results may occur if there is renal glomerular  dysfunction, liver disease or malabsorption, even in the  presence of normal pancreatic function. The effect of  these conditions is assessed by repeating the test with  unconjugated PABA, which eliminates the need for  digestion before absorption.   A similar, but technically simpler, test uses oral  fluorescein dilaurate. Fluorescein is released and  absorbed and the amount excreted in a timed urine  sample is measured as an indirect measure of pancreatic  function.   A low faecal pancreatic elastase determination is a  useful screening test for pancreatic malabsorption.  This can be assayed in a small ‘pea’-sized faecal sample.  Similarly, faecal chymotrypsin can also be measured.   The absence of pancreatic secretions may, by impairing  digestion, cause malabsorption. However, there are two  pancreatic diseases that are only rarely associated with  malabsorption.   Acute pancreatitis due to the necrosis of pancreatic  cells is associated with the release of enzymes into the  retroperitoneal space and bloodstream. The presence  of pancreatic juice in the peritoneal cavity causes  severe abdominal pain and shock. A vicious circle is  set up as the released enzymes digest more pancreatic  cells. Acute pancreatitis may be idiopathic, but may  follow gallstones, obstruction of the pancreatic duct or  regurgitation of bile along this duct.   Other predisposing factors include excess alcohol  ingestion and trauma to the pancreas. Hypercalcaemia  and hypertriglyceridaemia may also evoke acute  pancreatitis, as can certain drugs such as opiates. The  severity of an acute attack of pancreatitis can be assessed  by the Ranson or Glasgow criteria (Table 16.1).   Carcinoma of the pancreas  This tends to present late. Lesions at the head of the  organ may cause obstructive jaundice; extensive gland  destruction may cause late-onset diabetes mellitus.  The concentration of the tumour marker CA19-9 may  be raised in pancreatic carcinoma (see Fig. 16.1 and  Chapter 24).   A 57-year-old man presented to the surgeons in the  casualty department with an acute abdomen, severe  epigastric pain and nausea. On examination, he was  found to be hypotensive and showing abdominal  guarding. Some of his laboratory test results are as  follows:   The very high plasma amylase concentration  supports the diagnosis of acute pancreatitis, which  was confirmed by computerized tomography (CT)  imaging. The scan also showed the presence of  gallstones, which were thought to be the cause of  the acute pancreatitis. Note the hypocalcaemia,  impaired renal function and hyperglycaemia, which  can be associated with this condition. High alcohol  intake is another cause of acute pancreatitis, as can  be hypercalcaemia, hypertriglyceridaemia, trauma  and certain drugs.   Table 16.1 The Ranson/Glasgow criteria for assessing  the severity of an acute attack of pancreatitis   The mortality rate among patients with more than seven of these  criteria is near 100%.   • In intestinal malabsorption, fat digestion is usually  normal, but absorption of the products of digestion  is impaired.   • In pancreatic malabsorption, the absorptive capacity  is normal, but fat cannot be digested because there is  deficiency of digestive enzymes.   Malabsorption is usually, but not always, associated with  impaired fat absorption (steatorrhoea). No biochemical  test is sensitive enough for the early detection, or precise  enough for the differential diagnosis, of steatorrhoea;  endoscopy and ultrasound have reduced the need  for laboratory tests. However, biochemical tests are  important in detecting and monitoring the treatment  of the metabolic effects of prolonged and severe  malabsorption.   Steatorrhoea has been discussed above and can be  defined as a daily faecal fat excretion consistently more  than 18 mmol (5g) of fat, measured as fatty acids.  This is usually associated with a faecal stool weight  greater than 60 g. Unequivocal steatorrhoea can only  be demonstrated when the disease is extensive and  has usually already been elucidated by radiological or  histopathological means. Gross steatorrhoea causes  foul-smelling, bulky, pale, greasy stools, which are  difficult to flush away in the toilet.   Malabsorption may be caused by a reduction of the  absorptive area because of flattened intestinal villi,  demonstrated by microscopic examination of a mucosal  biopsy specimen, and by the following:   • Coeliac disease (gluten-sensitive enteropathy)  is caused by sensitivity to the protein oc-gliadin,  present in gluten in wheat and rye. The condition  is characterized by villous atrophy, mainly in the  proximal small intestine, which is usually reversible  following withdrawal of gluten from the diet.  Clinical symptoms usually become evident at about  1 year of age with failure to thrive and diarrhoea,  but may present at any age, including in adulthood.  Clinical features may include abdominal pain and   A 31 -year-old woman presented to the gastro-  enterologists with weight loss of about 15 kg in 1 year,  frequent diarrhoea and abdominal distension. There  was a family history of coeliac disease. On examination,  she was found to have abdominal tenderness and  looked pale. Her blood count showed a haemoglobin  level of 8.9g/dL and she had a low plasma folate  concentration. Her anti-transglutaminase antibodies  were strongly positive. A jejunal biopsy was reported  as showing villous atrophy.   The clinical history and clinical features are typical  of coeliac disease. Note also the folate deficiency. The  patient’s symptoms improved on a gluten-free diet,  with gain in body weight and normalization of her  haemoglobin.   distension, diarrhoea, tiredness, mouth ulcers,  anaemia, malabsorption and increased risk of  intestinal adenocarcinoma and lymphoma. It can  be associated also with hyposplenism, infertility,  abnormal hepatic function, dermatitis herpetiformis  and other autoimmune disorders such as type 1  diabetes mellitus, autoimmune thyroiditis and  primary biliary cirrhosis.   Antibodies to oc-gliadin or endomysium or  tissue transglutaminase (tTG) also may be detected  in plasma and their measurement can be used  in diagnosis and to monitor compliance with a  gluten-free diet. Antibodies to tTG have about  95 per cent sensitivity for coeliac disease. Exclude  immunoglobulin A (IgA) deficiency, which is more  common in patients with coeliac disease, as some  assays for coeliac screening look at IgA antibodies,  which may be falsely negative in IgA deficiency  (see Chapter 19). Indeed, IgA anti-tTG assay is a  screening test, but, in IgA deficiency, IgG anti-tTG  assay is preferable to help ascertain the need for  intestinal biopsy. However, IgG anti-tTG is not as  sensitive or specific as IgA antibodies, and biopsy  may still be clinically indicated if this test is negative.  It is important to also note that false-positive tests  can occur in other bowel diseases such as Crohn’s  disease.   • Tropical sprue, in which villous atrophy does not  respond to a gluten-free diet. There maybe a bacterial  cause because the condition sometimes responds to  broad-spectrum antibiotics and folate supplements.   • Extensive surgical resection or fistulae of the small  intestine may so reduce the absorptive area as to cause  malabsorption. Normally the small bowel spans from  260 cm to 800 cm. Any disease, trauma, radiation or  vascular event that leaves less than 200 cm of viable  small bowel remaining puts the patient at risk of  developing short-gut syndrome. The short-gut  syndrome can present with malabsorption, diarrhoea  and undernutrition. Electrolyte disturbances may  include severe hypokalaemia and hypomagnesaemia  as well as fluid depletion. Intestinal failure can be  defined as a condition in which there is inability of the  intestine to absorb nutrients and water sufficiently.   • Infiltration or inflammation of the small intestinal  wall, for example caused by Crohn’s or Whipple’s  disease or small intestinal lymphoma, may impair  absorption. Malabsorption may be aggravated by  alteration of the bacterial flora due to reduced  intestinal motility.   Food passes through the intestine more rapidly than  usual in the following circumstances.   • After gastrectomy, when normal mixing of food with  fluid, acid and pepsin does not occur in the stomach,  resulting in impaired enzyme activity within the  small intestine. An increased rate of transit through  the duodenum may contribute to the malabsorption.  However, post-gastrectomy malabsorption is rarely  severe, although iron deficiency is a relatively  common complication.   • In carcinoid syndrome, which is a rare condition  associated with excessive production of  5-hydroxytryptamine (serotonin) by tumours of  argentaffin cells usually arising in the small intestine,  or by their metastases, for example in the liver. It  may occasionally present with malabsorption, which  is probably the result of increased intestinal motility  due to the concomitant release of the gut peptide  substance P (see Chapter 24).   Pancreatic dysfunction may cause malabsorption  by reducing the secretion of digestive enzymes. The  following are some important examples:   • Chronic pancreatitis: commonly due to alcohol  excess, this is not always associated with steatorrhoea  or malabsorption. It rarely follows a severe attack of  acute pancreatitis, but is more likely after repeated  acute or subacute attacks. Diabetes mellitus requiring  insulin may occur due to islet cell damage. There  may be steatorrhoea due to the impairment of lipase  activity because of bile salt deficiency. Pancreatic  lipase activity can be reduced by up to 75 per cent  before chemical steatorrhoea occurs.   Low plasma trypsin levels are relatively specific  for chronic pancreatitis, but malabsorption does  not tend to occur until more than 90 per cent of  the pancreas has been destroyed. Similarly, there  may also be low faecal elastase or chymotrypsin  concentrations. Direct pancreatic enzyme tests are  sometimes needed to detect the disease in its early  stages.   Direct invasive tests require specialist expertise  and involve collecting duodenal aspirates by a tube  in a cannulated pancreatic duct, sometimes in  conjunction with endoscopic retrograde cholangio-  pancreatography (ERCP) after stimulation by   A 32-year-old woman with known cystic fibrosis  presented to the general physicians with copious pale,  floating and offensive-smelling stools and diabetes  mellitus. The results of some of her laboratory tests  are as follows:   A stool sample revealed very low elastase activity  with a faecal fat content of 65 mmol/ day (< 18).   The diagnosis was considered to be steatorrhoea  secondary to cystic fibrosis because of pancreatic  insufficiency. Note the hypokalaemia (due to  prolonged diarrhoea) and hyperglycaemia  (secondary to pancreatic endocrine malfunction).   exogenous secretin or cholecystokinin. Other tests may  be used to diagnose chronic pancreatitis, including  abdominal radiograph (calcification of the pancreas is  a useful sign), computerized axial tomography (CAT)  or magnetic resonance imaging (MRI) scanning,  endoscopic ultrasonography (EUS) or magnetic  resonance cholangiopancreatography (MRCP).   • Cystic fibrosis: this is an autosomal, recessively  inherited disorder of chloride transport affecting  exocrine glandular secretions. It may present in  the newborn infant with intestinal symptoms, such  as meconium ileus (failure to pass the first faeces  containing bile, intestinal debris and mucus), or in  early childhood with recurrent respiratory infections;   however, it may not be diagnosed until the patient  is an adult. Thick, viscous pancreatic and bronchial  secretions may cause malabsorption and chronic  lung disease, respectively. Cirrhosis of the liver and  sterility may develop. The diagnosis is made clinically  and by measuring the sweat electrolyte concentrations;  the chloride and sodium concentrations are usually  more than 60mmol/T. The detection of albumin in  meconium or a raised plasma trypsin concentration  during the first 6 weeks of life may be used to screen  for the disorder.   More than 200 different mutations have been  identified; the most common, which has a prevalence  of about 70 per cent, shows a 3 base pair deletion  in codon 508 of the cystic fibrosis chloride channel  gene. Prenatal diagnosis is available in some countries  using chorionic villus sampling at 8-10 weeks, but  is usually only offered if the gene defect has already  been established within the family following the  identification of an affected child (see Chapter 30).   The increased intestinal hydrogen ion activity in the  Zollinger-Ellison syndrome may inactivate pancreatic  lipase and cause precipitation of bile salts within the  gut lumen, so resulting in malabsorption (see later and  also Chapter 24).   The diagnosis of malabsorption is usually made by  clinical, haematological, histological and radiological  means, and supplemented by biochemical tests.  Malabsorption of fat occurs both in generalized  intestinal disease and in pancreatic disease. See  guidelines for the investigation of chronic diarrhoea  ( Gut 2003;52(Suppl. V):vl-vl5).   Anaemia is rarer in pancreatic than in intestinal  malabsorption because iron, vitamin B p and folate do  not depend on pancreatic enzymes for absorption. The  blood and bone marrow films typically show a mixed  iron deficiency and megaloblastic picture.   Polysaccharide absorption is impaired in both  conditions, but hypoglycaemia is rare. In pancreatic  malabsorption, in which neither disaccharidase activity  nor active monosaccharide absorption is affected, both  monosaccharides and disaccharides can be absorbed.  Hyperglycaemia suggests that pancreatic disease is the  cause of malabsorption.   Altered intestinal bacterial flora may cause  malabsorption of vitamin B and fat. This can occur in  the following conditions:   • Contaminated bowel (‘blind loop’) syndrome, which  is caused by bacterial proliferation due to impaired  intestinal motility and stagnation of intestinal contents.  It occurs when the ‘blind loops’ are the result of surgery  or in the presence of small intestinal diverticula. Some  bacteria may cause malabsorption by catalysing the  deconjugation of bile salts. As more vitamin B p and  folate may be used by the bacteria, megaloblastic  anaemia is common. A metabolic acidosis, D-lactic  acidosis, may also occur. Treatment with broad-  spectrum antibiotics may, by altering the small  intestinal bacterial flora, cause a similar syndrome.   - Culture of organisms from a specimen collected  into a special anaerobic microbiological  medium after intubation of the upper small  intestine. However, the test is invasive and  cultures may be falsely negative.   - The xylose breath test, in which 13 C/ 14 C-labelled  xylose is given orally. In the intestine, xylose is  bacterially degraded and the released labelled  CO, can be measured in the breath. This test  maybe more sensitive than the 14 C-glycocholate  breath test, in which 14 C-glycocholate is taken  orally. The intestinal bacteria split the cholate  from the labelled glycine, which is absorbed  and metabolized; 14 CO, is measured in the  expired air. False-positive results may occur in  patients with disorders of the terminal ileum.   - Increased urinary indican concentration,  which entails the assay of bacterial-derived   metabolites (usually of tryptophan) in the  urine but may not be specific and is now very  rarely measured.   • Biliary obstruction causes malabsorption of fat and  of fat-soluble substances by preventing the secretion  of bile salts into the intestinal lumen; steatorrhoea  results. The diagnosis is usually clinically obvious  due to the presence of jaundice. Retention of bile  salts, such as may occur in primary biliary cirrhosis,  may cause pruritus.   • Local diseases or surgery of the small intestine  may cause selective malabsorption of substances  absorbed predominantly at those sites. Diseases  involving the terminal ileum, such as Crohn’s  disease or tuberculosis, may impair the absorption  of bile salts and vitamin B p . Vitamin B p absorption  may also be impaired because of intrinsic factor  deficiency caused either by damage to gastric parietal  cells in pernicious anaemia or by total gastrectomy  or extensive malignant infiltration of the stomach.  Malabsorption of vitamin B p may be demonstrated  using the Schilling test (see Chapter 15).   • Disaccharidase deficiency may occur in generalized  disorders of the intestinal wall because the enzymes  are localized on the brush border of the enterocyte.  This deficiency is relatively unimportant compared  with that due to general malabsorption. Tests for  these syndromes are therefore useful only in the  absence of steatorrhoea, when selective rather than  generalized malabsorption of carbohydrate may be  present.   The symptoms of disaccharidase deficiency  are due to the effects of unabsorbed, osmotically  active sugars in the intestinal lumen. They  include faintness, abdominal discomfort and  severe diarrhoea after ingestion of the offending  disaccharide (compare with the ‘dumping  syndrome’). Diarrhoea may be severe enough  to cause volume depletion in infants. Stools are  typically acid because bacteria metabolize sugars to  acids. Unabsorbed sugars may be detectable in the  faeces by measuring ‘reducing substances’, and some  disaccharides, which may be absorbed intact, may  be detectable in the urine.   - Acquired lactase deficiency or lactose  intolerance is more common than the  congenital form and is probably the most  common type of disaccharidase deficiency.   It may not present until childhood, or even  adult life, possibly in genetically predisposed  individuals. It may also present secondary  to other intestinal disorders, such as coeliac  disease and inflammatory bowel disease.   - Lactase deficiency associated with prematurity  may resemble the congenital form. However,  the sensitivity to breast milk usually disappears  when lactase is synthesized within a few days  of birth.   - Congenital lactase deficiency (autosomal  recessive) is very rare. Infants present with  severe diarrhoea or colicky abdominal pain  soon after the introduction of milk feeds. The  syndrome is treated by removing milk and milk  products from the diet.   Sucrase and isomaltase deficiency usually  coexist. Congenital sucrase-isomaltase deficiency is  more common than congenital lactase deficiency.  Acquired sucrase-isomaltase deficiency and maltase  deficiency of any kind is very rare.   - Estimating the relevant enzymes in intestinal  biopsy tissue: this is the most reliable test  and no others may be needed, although it is  invasive.   - Measuring the plasma glucose concentration,  as in the glucose tolerance test, after giving  an oral load of the particular disaccharide.  If the disaccharide cannot be hydrolysed,  the constituent monosaccharides cannot be  absorbed and the concentrations of plasma  glucose rise very little. The result should be  compared with that of a glucose tolerance  test. If this is also flat, the abnormal response  to disaccharides is not diagnostic. If the  patient experiences typical symptoms, or if  disaccharides are excreted in the urine when the  offending sugar is given, the comparison need  not be made.   A relatively simple and sensitive test for  carbohydrate malabsorption is the hydrogen breath  test. Patients are given an oral solution of lactose.  In lactase deficiency, unabsorbed lactose is digested  by colonic flora, which results in an elevated breath  hydrogen content in expired air. Bacterial bowel  overgrowth can do the same, but the two conditions  can be distinguished by using oral glucose instead of  lactose.   In many cases of malabsorption, protein is lost  through the gut due to failure to digest and absorb  that entering it in intestinal secretions and by cell  desquamation. Isolated protein-losing enteropathy is  a very rare syndrome in which absorption is normal  but the intestinal wall is abnormally permeable to large  molecules, a condition similar to that of the glomeruli  in the nephrotic syndrome. It occurs in a variety of  conditions in which there is ulceration of the bowel or  abnormal lymphatic drainage. The clinical picture is  largely due to hypoalbuminaemia.   If there is no evidence of generalized malabsorption  and the disorder is suspected, the diagnosis can be made  after the intravenous administration of a radiolabelled  protein such as albumin or transferrin by measuring its  recovery in a timed stool collection. Alternatively, the  concentration of cq-antitrypsin, a protein resistant to  proteolytic degradation, can be measured in a known  weight of faeces; gastrointestinal bleeding may cause  false-positive results. Typically, the serum protein  electrophoretic pattern is similar to that found in the  nephrotic syndrome; the relatively high molecular  weight components of the a, -fraction are retained and  all other fractions are reduced (see Chapter 19).   Metabolic consequences of malabsorption  The following findings may occur after very prolonged  disease, but are not necessary for the diagnosis of  malabsorption. This section is best read in conjunction  with Chapter 14 (nutrition) and Chapter 15 (vitamins).   Fat-soluble vitamin deficiency may be a consequence  of impaired fat absorption due to steatorrhoea.   • Vitamin D deficiency causes impaired calcium  absorption and sometimes leads to rickets or  osteomalacia.   Amino acid and peptide malabsorption occurs in  intestinal disease, due to impaired intestinal transport  mechanisms; in pancreatic disease, the digestion of  protein is severely impaired. In either case, prolonged  disease may cause generalized muscle and tissue wasting  and osteoporosis and reduced concentration of all  plasma proteins: the low plasma albumin concentration  may cause oedema, and reduced antibody formation  (immunoglobulins) may predispose to infection.   A 60 -year- old woman with a medical history of  numerous attacks of acute pancreatitis secondary to  high alcohol intake noticed that her stools had turned  paler, associated with abdominal discomfort and  weakness. In addition she experienced paraesthesiae  of her hands and polydipsia. Abdominal examination  confirmed the abdominal discomfort and a plain  abdominal radiograph showed pancreatic calcification.  Some of her laboratory tests were as follows:   The patient has obvious features of malabsorption  with hypocalcaemia due to vitamin D deficiency. The  latter is a fat-soluble vitamin, and hypocalcaemia can  evoke neurological symptoms. She also has hypoalbu-  minaemia and diabetes mellitus secondary to pancre-  atic insufficiency. Multiple attacks of acute pancreatitis  can lead to chronic pancreatitis. She required insulin  for her diabetes mellitus, calcium and vitamin D sup-  plementation for her hypocalcaemia, and oral pancre-  atic extract (Creon) to replace her pancreatic enzymes.   Anaemia is less likely in pancreatic than in intestinal  malabsorption; iron, vitamin B p and folate do not  depend on pancreatic enzymes for absorption. The  blood and bone marrow films typically show a mixed  iron deficiency and megaloblastic picture. Anaemia  may be aggravated by protein deficiency.   The presenting clinical features in severe and long-  standing generalized malabsorption, whether intestinal  or pancreatic, may include:   - clinical, radiological, haematological and /  or biochemical findings suggestive of  undernutrition with no obvious cause.   • Has the patient a history that may suggest alteration  in the intestinal bacterial flora?   - Has the patient been on long-term broad-  spectrum antibiotics and what other relevant  medications is the patient taking?   - Ask about foreign travel or possibly contaminated  drinking water. If so, consider the possibility of  tropical sprue or other infective origin.   - Constipation with hard, dry stools makes the  diagnosis of malabsorption less likely.   - Watery stools, especially if bloodstained,  suggest colonic disease such as ulcerative colitis.   The possibility of purgative or laxative abuse  must always be considered in this context:  consider measuring the stool osmolal gap to  see if the diarrhoea is secretory or osmotic.   Is there a relationship to the type of food  eaten, for example gluten-containing products?  Check coeliac screen antibodies.   • Are there plasma electrolyte abnormalities, especially  hypokalaemia and hypomagnesaemia? Signs of  extracellular volume depletion without obvious  evidence of undernutrition favour colonic disease  as a cause of diarrhoea rather than a small intestinal  malabsorption syndrome. Check also for an acid-  base disturbance as well as hypoalbuminaemia,  hypoproteinaemia or hypogammaglobulinaemia.   • Anaemia is more likely to be due to intestinal than to  pancreatic disease:   - A hypochromic, microcytic picture may be  the result of blood loss at any level of the  gastrointestinal tract.   - A normochromic, normocytic picture is a non-  specific finding in any chronic disease.   - A dimorphic (mixed iron deficiency and  macrocytic) picture is very suggestive of  intestinal malabsorption.   - Low red cell folate and/or plasma vitamin B p  concentrations suggest intestinal malabsorption.   • If doubt remains, faecal fat estimation may help.  However, because of the difficulty of obtaining an  accurately timed faecal collection, only very high  results are of unequivocal significance; in such cases  steatorrhoea will probably be obvious visually. For  this reason many hospital laboratories no longer  perform this test. In any case, normal faecal fat  does not exclude malabsorption, as carbohydrate  absorption may be impaired.   • Slow stool elastase levels are indicative of  malabsorption of pancreatic origin.   • The xylose absorption test is sometimes helpful  in distinguishing pancreatic malabsorption from  intestinal problems. If it is unequivocally normal, an  upper intestinal lesion is unlikely. Such a result does  not, however, exclude ileal or pancreatic disease,  contaminated bowel syndrome, or even some cases  of coeliac disease.   • A pancreolauryl test or PABA test if available  may also help confirm the presence of pancreatic  insufficiency. Some authorities suggest the gold  standard test for assessing pancreatic function is the  secretin- pancreozymin test, but this is invasive and  requires specialist skill.   • If any or all of the above investigations suggest  steatorrhoea, a cause should be sought. The following  are the most definitive tests:   - Endoscopy, with histological examination of  an intestinal biopsy specimen. Flattened villi  suggest: coeliac disease or tropical sprue.   - Giardia lamblia may be detected  microscopically, and stool culture is useful in  case of other organisms.   • The diagnosis of chronic pancreatitis and of causes  of pancreatic malabsorption may also need imaging  techniques such as ERCP or MRCP. Tests such as  abdominal ultrasound or CT scanning may help,  especially if pancreatic carcinoma is suspected.  Plain abdominal radiographs may show pancreatic  calcification.   • If steatorrhoea is obvious on clinical grounds  and after visual inspection of the stool, and if the   - pancreatic disease such as chronic pancreatitis.  • Intestinal malabsorption may need further   anatomical delineation. Ileal disease may require a  Schilling test or 14 C-radiolabelled glycocholic acid  test. Bacterial intestinal overgrowth may require one  of the breath tests.   Figure 16.2 gives a summary algorithm for the  investigation of steatorrhoea (see also Box 16.1).   Laboratory investigations of plasma can be carried out  to identify some of the complications of generalized  malabsorption:    Figure 16.2 Algorithm for the biochemical investigation  of steatorrhoea (imaging and endoscopy investigations  may also be required). PABA, para-amino benzoic acid.   • Plasma electrolytes may show hypernatraemia and  impaired renal function if there is significant water  loss.   • Plasma potassium and magnesium may reveal the  presence of hypokalaemia and hypomagnesaemia.   • There may sometimes be a metabolic acidosis,  which may be due to D-lactic acidosis (if bacterial  overgrowth), or a hyperchloraemic acidosis (see  Chapter 4).   • Anaemia: haematological investigations such as  ferritin, folate and vitamin B ]V   • Rickets/osteomalacia: plasma calcium with albumin,  phosphate and alkaline phosphatase activity.   • Abnormal bleeding (vitamin K deficiency): clotting  studies such as prolonged prothrombin time.   • Low plasma immunoglobulin (hypogammaglobul-  inaemia) or low plasma cholesterol (hypocholest-  erolaemia) concentrations.   Enzyme inactivation by gastric hypersecretion  Enzyme deficiencies, e.g. enterokinase or trypsin  Decreased bile acid secretion, e.g. hepatic  disease, biliary obstruction  Intestinal stasis, e.g. scleroderma, obstruction,  blind loops   Poor absorbing surface, e.g. coeliac disease,  sprue, radiation damage, gut infiltrations such as  lymphoma   Some individuals abuse purgatives and laxatives,  and may have psychiatric problems, including  Munchausen’s syndrome. Stool samples can be screened  for purgatives such as senna or phenolphthalein by thin  layer chromatography. Also look out for associated  hypokalaemia due to gastrointestinal potassium loss  resulting from this (see Chapter 5).   Diarrhoea fluid can be classified into two types:  osmotic and secretory. The former could be due to  lactose intolerance, or osmotic laxatives containing  magnesium or lactulose. The latter could be the result  of infections, inflammatory bowel disease, carcinoid  syndrome or VIPoma, to name but a few. The osmolality  of faecal water can be calculated from:   If the measured osmolality is greater than the  calculated osmolality, an osmotically active constituent  is likely to be present, suggestive of osmotic diarrhoea  (see Chapter 2).   Helicobacter pylori is found in up to 50 per cent of  European adults and is associated with peptic ulcer,  gastric adenocarcinoma and non-Hodgkin’s lymphoma  of the stomach. There are many laboratory tests for this  organism, including microscopy, bacterial culture and  the direct urease test using gastric mucosa biopsies. Non-  invasive tests include blood, stool or saliva IgG antibody  studies to detect Helicobacter pylori infection, or breath  tests that look at labelled carbon, which is released by  this urea-splitting organism. The latter test is useful in  determining whether the organism has been eradicated.   In inflammatory bowel disease such as Crohn’s disease  elevated faecal calprotectin may occur and may be  a marker of disease activity. Calprotectin is a 36-kDa  calcium-binding protein secreted by neutrophils and  concentrations are usually normal in irritable bowel  syndrome.   Colorectal carcinoma is one of the most prevalent  cancers. Screening has been shown to reduce mortality,  but direct visualization by colonscopy is invasive, costly  and requires skilled personnel.   At present, faecal occult blood tests are being used for  screening purposes, as the tumour often releases blood  into the gastrointestinal tract. This can be observed by  point-of-care testing or laboratory tests that analyse the  blood in small faecal samples. However, false-positives  can occur in people with a high meat intake.   Future screening tests may look at altered faecal  deoxyribonucleic acid (DNA) derived from the tumour.  Plasma carcinoembryonic antigen (CEA) can be used  to monitor the treatment of colorectal carcinoma. (See  Chapter 24 for tumour markers.)   (It is suggested that this section is read in conjunction  with Chapter 24.) These tumours are very rare. They  are usually found in the pancreatic islets and cause  well-recognized clinical and pathological syndromes.   Gastrinomas, usually in the pancreatic islets, secrete  large quantities of gastrin, despite high gastric  hydrogen ion levels, and may cause the Zollinger-  Ellison syndrome. About 60 per cent of gastrinomas  are malignant. Of the remaining 40 per cent, only  about one-third (13 per cent of the total) are single,  resectable adenomas, the rest being multiple. The very  high rate of gastric acid secretion causes ulceration in  the stomach and proximal small intestine with severe  diarrhoea; inhibition of pancreatic lipase activity by the  low pH may cause steatorrhoea. This syndrome may be  associated with benign, and usually non-functioning,  adenomas elsewhere, for example in the anterior  pituitary, parathyroid and thyroid glands and in the  adrenal cortex [multiple endocrine neoplasia (MEN1)  syndrome]. The diagnosis depends on the following:   • Fasting plasma gastrin concentrations are up to  30 times the upper reference limit in Zollinger-  Ellison syndrome. Plasma concentrations more than  1000 pg/L with acid hypersecretion strongly support  this diagnosis.   • However, high plasma gastrin concentrations may  also be caused by hypochlorhydria, for example  atrophic gastritis, pernicious anaemia or post  vagotomy. H, blockers and proton pump inhibitors  are usually stopped prior to sampling.   • The Zollinger-Ellison syndrome basal gastric acid  output may be more than 15mmol/h, with large  gastric secretory volumes and gastric pH of less  than 2.0.   • In cases in which basal plasma gastrin concentrations  are equivocal, the secretin test may be useful.  Intravenous secretin (2 units/kg body weight) raises  plasma gastrin concentration to more than 200 pg/L  within 10 min if a gastrinoma is present.   • Imaging techniques such as CT, MRI and endoscopic  ultrasound may be useful, and also somatostatin  receptor scintigraphy to localize tumours and  metastases.   Glucagonomas usually arise from the a-cells of the  pancreatic islets. The presenting clinical feature is a  bullous rash, known as necrolytic migratory erythema,  sometimes accompanied by psychiatric disturbances,  thromboembolism, glossitis, weight loss, impaired  glucose tolerance, anaemia and a raised erythrocyte  sedimentation rate. Diagnosis may be confirmed by  finding a very high plasma glucagon concentration.   VIPomas are extremely rare tumours, usually of the  pancreatic islet cells, that secrete VIP (vasoactive  intestinal polypeptide), a hormone that increases  intestinal motility. They cause a syndrome in which  there is very profuse, watery diarrhoea - the Verner-  Morrison or watery diarrhoea, hypokalaemia and  achlorhydria syndrome. Plasma concentrations of VIP  are high.   In some cases, a clinical history and examination with  imaging techniques such as abdominal radiograph,  ultrasound, CT or even laparotomy may reveal a  diagnosis. However, sometimes biochemical tests may  help, particularly in the following situations:   • Plasma amylase or lipase concentration may be  raised in acute pancreatitis (see Chapter 18).   • Gallstones may present with abnormal liver function  tests, in particular plasma bilirubin and alkaline  phosphatase. They may sometimes be associated  with jaundice (see Chapter 17).   • Diabetes ketoacidosis can present with an acute  abdomen, and therefore hyperglycaemia and urinary  ketones are present (see Chapter 12).   • Hypercalcaemia is another cause of abdominal pain  (see Chapter 6).   • Severe hypokalaemia can evoke a paralytic ileus and  intestinal obstruction (see Chapter 5).   • Sickle cell disease or crisis can be investigated by  haemoglobin studies for HbS.   • Acute porphyria can present as an acute abdomen  (see Chapter 21).   • Addison’s disease can present as an adrenal crisis (see  Chapter 8).   • Heavy metal poisoning, for example lead, can cause  abdominal pain (see Chapter 21).   • Ectopic pregnancy should be considered in  women of reproductive age and can be detected   by a raised concentration of plasma [3-human  chorionic gonadotrophin and pelvic ultrasound (see  Chapter 10).   Clinical biochemistry laboratory tests can also  be used in the management of abdominal pain, for  example post-operatively. Plasma urea and electrolytes  and creatinine as well as chloride and bicarbonate  concentrations are useful, particularly if intravenous  fluids are given or an acid-base disturbance is suspected.   The routine hospital chemical pathology laboratory  has a role in the investigation of gastrointestinal  disorders, but the tests carried out in the laboratory  do not replace more specialized invasive tests such  as endoscopy or radiology.   Normal intestinal, hepatic and pancreatic function  is essential for digestion and absorption.   The gut is not merely an absorbing surface, but  also secretes hormones and is controlled by both  humoral and neural pathways.   Malabsorption can be due to various pathologies,  which should be sought and which can present as  abdominal discomfort, weight loss and steatorrhoea.   • Acute pancreatitis is a potentially lethal condition;  its diagnosis is supported by the presence of elevated  plasma amylase or lipase.   • Intestinal failure, for example gastrointestinal  fistula or short gut, can result in severe fluid loss  and electrolyte disturbance.   • Abdominal pain is common and some of its causes  are biochemical.   • The gastrointestinal tract is the source of various  tumours, the diagnosis and treatment of which can  be assisted by biochemical tests.   This chapter looks at the chemical pathology of liver  and gall bladder disorders. These are common in  clinical practice, and liver function tests constitute one  of the most frequently requested clinical biochemistry  laboratory profiles.   The liver has essential synthetic and excretory functions  and can be thought of as a large ‘metabolic factory’. It also  detoxifies and, like the kidneys, excretes the end products  of metabolism. The main blood supply to the liver is via  the portal vein. The liver is made up of hexagonal lobules  of cells (Fig. 17.1). Rows of hepatocytes radiate from  the central hepatic vein and are separated by sinusoidal  spaces, along the walls of which are interspersed hepatic  macrophages, the Kupffer cells. These phagocytic cells  are part of the reticuloendothelial system and have an  important detoxifying function. At the corners of each    Figure 17.1 Diagrammatic representation of a cross-  section of a hepatic lobule showing the relation between  the central hepatic vein and the portal tracts. Blood flows  towards the central vein, as indicated by the arrows.   lobule are the portal tracts that contain branches of  the hepatic artery, the portal vein and bile ducts. Blood  flows from the portal tracts towards the central hepatic  vein. Therefore:   • Hypoxia and toxins that are metabolized in the liver  cause damage to the centrilobular area first.   • Toxins that do not depend on hepatic metabolism  primarily affect the periphery of the lobule.   Almost all nutrients from the gastrointestinal tract  pass through the sinusoidal spaces prior to entering the  systemic circulation. The hepatic architecture may be  disturbed in cirrhosis (fibrosis).   General metabolic functions  When the glucose concentration is high in the portal  vein, it is converted to glycogen and the carbon skeletons  of fatty acids, which are transported to adipose tissue as  very low-density lipoprotein (VLDL). During fasting,  the systemic plasma glucose concentration is maintained  by the breakdown of glycogen (glycogenolysis) or by the  synthesis of glucose from substrates such as glycerol,  lactate and amino acids (gluconeogenesis). Fatty acids  reaching the liver from fat stores may be metabolized  in the tricarboxylic acid cycle, converted to ketones or  incorporated into triglycerides (see Chapter 13).   • most coagulation factors, including fibrinogen and  factors II (prothrombin), V, VII, IX, X, XI, XII and  XIII - of these, prothrombin (II) and factors VII, IX  and X cannot be synthesized without vitamin K,   • the lipoproteins, such as VLDL and high-density  lipoprotein (HDL) (see Chapter 13).   The liver has a very large functional reserve.  Deficiencies in synthetic function can be detected only if  liver disease is extensive. Before a fall in plasma albumin  concentration is attributed to advanced liver disease,  extrahepatic causes must be excluded, such as the loss  of protein through the kidney, gut or skin, or across  capillary membranes into the interstitial space, as in  even mild inflammation or infection (see Chapter 19).   Prothrombin levels, assessed by measuring the  prothrombin time, maybe reduced because of impaired  hepatic synthesis, whether due to failure to absorb  vitamin K or to hepatocellular damage. If hepatocellular  function is adequate, parenteral administration of  vitamin K may reverse the abnormality.   Excretion and detoxification  The excretion of bilirubin is considered in more detail  below. Other substances that are inactivated and  excreted by the liver include the following:   • Cholesterol - excreted in the bile either unchanged or  after conversion to bile acids.   • Amino acids - which are deaminated in the liver.  Amino groups, and the ammonia produced by  intestinal bacterial action and absorbed into the  portal vein, are converted to urea.   • Steroid hormones - which are metabolized and  inactivated by conjugation with glucuronate and  sulphate and excreted in the urine in these water-  soluble forms.   • Many drugs - which are metabolized and inactivated  by enzymes of the endoplasmic reticulum system;  some are excreted in the bile.   • Toxins - the reticuloendothelial Kupffer cells in  the hepatic sinusoids are well placed to extract  toxic substances that have been absorbed from the  gastrointestinal tract.   Efficient excretion of the end products of metabolism  and of bilirubin depends on:   Formation and excretion of bilirubin (Fig. 17.2)  At the end of their lifespan, red blood cells are broken  down by the reticuloendothelial system, mainly in the  spleen. The released haemoglobin is split into globin,  which enters the general protein pool, and haem, which  is converted to bilirubin after the removal of iron,  which is reused (see Chapter 21).   About 80 per cent of bilirubin is derived from haem  within the reticuloendothelial system. Other sources  include the breakdown of immature red cells in the  bone marrow and of compounds chemically related to  haemoglobin, such as myoglobin and the cytochromes.  Less than 300 pmol of bilirubin is produced daily from  the breakdown of erythrocytes, while the normal  liver is able to conjugate up to about 1 mmol/ day, and   Bilirubin is normally transported to the liver bound  to albumin. In this form it is called unconjugated  bilirubin, which is lipid soluble and therefore, if not  protein bound, can cross cell membranes, including  those forming the blood-brain barrier. In this form  it is potentially toxic; however, at physiological  concentrations it is all protein bound.   In the adult, about 300 pmol per day of bilirubin  reaches the liver, where it is transferred from plasma  albumin, through the permeable vascular sinusoidal  membrane. The hepatocytes can process a much  greater load than this. Bilirubin is bound to ligandin  (Y protein). From there it is actively transported to the  smooth endoplasmic reticulum, where it is conjugated  with glucuronate by a process catalysed by uridine  diphosphate glucuronyl transferase.   Bilirubin monoglucuronide passes to the canalicular  surfaces of the hepatocytes, where, after the addition of  a second glucuronate molecule, it is secreted by active  processes into the bile canaliculi. This process is largely  dependent on the active secretion of bile acids from  hepatocytes. These energy-dependent steps are the ones  most likely to be impaired by liver damage (hypoxia and  septicaemia) and by increased pressure in the biliary tract.  Other anions, including drugs, may compete for bind-  ing to ligandin, thus impairing bilirubin conjugation and  excretion. Novobiocin inhibits glucuronyl transferase,  thus exacerbating unconjugated hyperbilirubinaemia.  Bilirubin is often assayed by the Van den Bergh reaction,  which allows conjugated (direct-reacting) and unconju-  gated (indirect-reacting) bilirubin to be distinguished.   Bilirubin metabolism and jaundice  Jaundice usually becomes clinically apparent when the  plasma bilirubin concentration reaches about 50 pmol/L  (hyperbilirubinaemia), about twice the upper reference  limit. It occurs when bilirubin production exceeds the  hepatic capacity to excrete it. This may be because:   • An increased rate of bilirubin production exceeds  normal excretory capacity of the liver (prehepatic  jaundice).   • The normal load of bilirubin cannot be conjugated  and/or excreted by damaged liver cells (hepatic  jaundice).   • The biliary flow is obstructed, so that conjugated  bilirubin cannot be excreted into the intestine and  is regurgitated into the systemic circulation (post-  hepatic jaundice).   Retention of bilirubin in plasma: jaundice  Unconjugated hyperbilirubinaemia occurs if there is:   • a marked increase in the bilirubin load as a result of  haemolysis, or of the breakdown of large amounts  of blood after haemorrhage into the gastrointestinal  tract or, for example, under the skin due to extensive  bruising; in cases of haemolysis, plasma bilirubin  rarely exceeds 75pmol/T,   • impaired binding of bilirubin to ligandin or impaired  conjugation with glucuronate in the liver.   In some pathological conditions, plasma  unconjugated bilirubin levels may increase so much  that they exceed the protein-binding capacity. The  lip id- soluble, unbound bilirubin damages brain cells  (kernicterus). This is most likely to occur in newborn,  particularly premature, infants in whom the hepatic  conjugating mechanisms are immature. In addition,  the proportion of unbound, unconjugated bilirubin,  and therefore the risk of cerebral damage, increases if:   • unconjugated bilirubin is displaced from binding  sites, for example by high levels of free fatty acids or  drugs such as salicylates or sulphonamides.   Unconjugated bilirubin is normally all protein  bound and is not water soluble and therefore cannot  be excreted in the urine. Patients with unconjugated  hyperbilirubinaemia do not have bilirubinuria  (‘acholuric jaundice’) such as Gilbert’s syndrome.  Conjugated bilirubinaemia is one of the earliest signs  of impaired hepatic excretion. In most cases of jaundice  in adults, both conjugated and unconjugated fractions  of bilirubin are increased in plasma but conjugated  bilirubin predominates. Conjugated bilirubin is water  soluble and is less strongly protein bound than the  unconjugated form, and therefore can be excreted in the  urine. Bilirubinuria is always pathological. Dark urine  may be an early sign of some forms of hepatobiliary  disease.   Conjugated bilirubin enters the gut lumen in bile; it  is broken down by bacteria in the distal ileum and the  colon into a group of products known as stercobilinogen  (faecal urobilinogen). Some is absorbed into the  portal circulation, most of which is re-excreted in bile  (enterohepatic circulation). A small amount enters  the systemic circulation and is excreted in the urine  as urobilinogen, which can be oxidized to a coloured  pigment, urobilin.   Urobilinogen, unlike bilirubin, is often detectable in  the urine of normal people by testing with commercial  strip tests, particularly if the urine, and therefore the  urobilinogen, is concentrated. Urinary urobilinogen  concentration is increased in the following situations.   • When haemolysis is very severe: large amounts  of bilirubin enter the intestinal lumen and are  converted to stercobilinogen. An increased amount  of urobilinogen is formed and absorbed. If the  hepatic capacity to re-secrete it is exceeded, it is  passed in the urine.   • When liver damage impairs re-excretion of normal  amounts of urobilinogen into the bile.   The colourless, unabsorbed stercobilinogen is  oxidized to stercobilin, a pigment that contributes to  the brown colour of faeces. Pale stools may, therefore,  suggest biliary obstruction associated with an absence  of urinary urobilinogen.   Several biochemical tests constitute what are called the  ‘liver function tests’. Different tests can give different  information about hepatic dysfunction.   Strictly speaking, changes in plasma enzyme activity  generally indicate liver cell membrane damage rather  than hepatic function capacity. Because these enzymes  are also present in other tissues, changes in plasma  activities may reflect damage to those tissues rather  than to the liver (see Chapter 18).   A rise in plasma aminotransferase activities is a  sensitive indicator of damage to cytoplasmic and /  or mitochondrial membranes. Plasma enzyme  activities rise when the membranes of only very few  cells are damaged. Liver cells contain more aspartate  aminotransferase (AST) than alanine aminotransferase  (ALT), but ALT is confined to the cytoplasm, in which  its concentration is higher than that of AST. Raised  plasma transaminase concentrations are indicative of  hepatocyte damage, but do not necessarily reveal its  mechanism.   In inflammatory or infective conditions, such as  viral hepatitis, the cytoplasmic membrane sustains the  main damage; leakage of cytoplasmic contents causes   In infiltrative disorders in which there is damage to  both mitochondrial and cytoplasmic membranes, there  is a proportionally greater increase in plasma AST than  ALT activity.   The relative plasma activities of ALT and AST may  help to indicate the type of cell damage. The former is  more specific for hepatic disease; AST may be present in  skeletal muscle and is more sensitive than ALT. A plasma  AST:ALT ratio of >2 is suggestive but not diagnostic of  alcoholic liver disease and a ratio < 1 suggests chronic  viral hepatitis or hepatic steatosis (see Chapter 18).   Hepatic synthetic function  The measurement of plasma albumin and prothrombin  time may be used to assess function. The hepatic  synthetic and secretory capacities are large; only  severe and usually prolonged liver disease, for  example cirrhosis, demonstrably impairs albumin and  prothrombin synthesis.   Hypoalbuminaemia is such a common finding in  many severe illnesses that it is a less specific indicator  of impaired synthetic capacity than a prolonged  prothrombin time. A plasma albumin concentration  below the lower reference limit may imply hepatic  disease chronicity. However, there are many other  causes of a low plasma albumin concentration that are  not due to hepatic disease (see Chapter 19).   The prothrombin time may be prolonged by cholestasis:  fat-soluble vitamin K cannot be absorbed normally  if fat absorption is impaired due to intestinal bile  salt deficiency. The abnormality is then corrected by  parenteral administration of the vitamin. A prolonged  prothrombin time may also result from severe  impairment of synthetic ability if the liver cell mass  is greatly reduced; in such cases it is not corrected by  parenteral administration of vitamin K.   Hepatic excretory function  A high plasma conjugated bilirubin concentration  indicates impaired hepatic excretory function but  as this is also raised in hepatocellular disease it is not  specific for cholestasis. This may be accompanied by a  high plasma alkaline phosphatase (ALP) activity, as we  will now see. Jaundice has been described above.   Alkaline phosphatase is derived from a number of  different tissues, including the liver, the osteoblasts  in bone and the placenta. Plasma activities rise in  cholestatic liver disease because ALP synthesis is  increased and the enzyme within the biliary tract is  regurgitated into plasma. A raised ALP concentration  in the presence of a raised y-glutamyl transferase  (GGT) concentration implies that the ALP is of hepatic  origin.   y-Glutamyl transferase is an enzyme derived from the  endoplasmic reticulum of the cells of the hepatobiliary  tract. As this reticulum proliferates, for example in  response to the prolonged intake of alcohol and of drugs  such as phenobarbital and phenytoin, synthesis of the  enzyme is induced and plasma GGT activity increases.  Therefore, raised plasma activities do not necessarily  indicate hepatocellular damage, but may reflect enzyme  induction or cholestasis.   Biochemical tests can be used to investigate hepatic  disorders, the mechanisms underlying which can be  divided into three main groups; these often coexist, but  one usually predominates in any particular condition  (Table 17.1).   • Liver-cell damage is characterized by the release of  enzymes (AST and ALT) from damaged hepatocytes.  Plasma ALT and AST activities are increased.   • Cholestasis is characterized by retention of  conjugated bilirubin and of ALP, and by increased  ALP synthesis at the sinusoidal surface. Plasma  conjugated bilirubin levels and ALP activities are  increased.   • Reduced mass of hepatocytes, if considerable,  is characterized by a reduction in albumin and  prothrombin synthesis. The plasma albumin  concentration is reduced and the prothrombin time  is prolonged.   Fresh urine analysis may confirm the presence of  bilirubin (conjugated hyperbilirubinaemia) and  urobilinogen   Urine Multistix include stabilized, diazotized  2,4-dichloraniline, which reacts with bilirubin to form  azobilirubin. The test will detect about 3 pmol/L of  bilirubin. Drugs, such as large doses of chlorpromazine,  may give false-positive results.   Urine Multistix also include paradimethylamino-  benzaldehyde, which reacts with urobilinogen. Urine  Multistix do not react with porphobilinogen. This test  will detect urobilinogen in urine from some normal  individuals. False-positive results may occur after  taking drugs such as para-aminosalicylic acid and some  sulphonamides.   Raised total plasma bile acid concentrations in the  third trimester of pregnancy associated with pruritus  are suggestive of obstetric cholestasis, which can lead  to both maternal and fetal morbidity and mortality.  Elevation of plasma ALT concentration may follow the  increase in the concentration of plasma bile salts (see  Chapter 10).   secretory) function are relatively insensitive indicators  of liver disease. There are a number of new tests that are  being devised to improve the accuracy of the diagnosis  of hepatic disorders. Tests of hepatocellular activity have  been proposed, such as galactose elimination capacity,  the aminopyrine breath test, indocyanine green  clearance, and monoethylglycinexylidide (MEGX)  production. All these tests are indirect measures of  hepatic activity that rely on measuring compounds or  their metabolites after they have been acted on by the  liver. As yet, they do not have a place in routine clinical  diagnosis.   • intrahepatic, in which bile secretion from the  hepatocytes into the canaliculi is impaired, due to:   • extrahepatic, due to obstruction to the flow of bile  through the biliary tract by:   - pressure on the tract from outside by malignant  tissue, usually of the head of the pancreas,   It is essential to distinguish between intrahepatic  and extrahepatic causes of cholestasis, as surgery may  be indicated for the latter but is usually contraindicated  for intrahepatic lesions. The biochemical findings may  be similar:   • Bilirubin concentrations in plasma may be normal  if only part of the biliary system is involved by  intrahepatic lesions such as cholangitis, early  primary biliary cirrhosis or primary or secondary  tumours. The unaffected areas can secrete  bilirubin.   • Alkaline phosphatase activity is a sensitive test  for cholestasis. Increased synthesis of ATP in the  affected ducts increases the activity of this enzyme in  plasma. If this is the only abnormal finding, it must  be shown to be of hepatic origin before it is assumed  to indicate liver disease.   Patients with prolonged and more widespread  cholestasis may present with severe jaundice and  pruritus due to the deposition of retained bile salts in  the skin; the plasma bilirubin concentration may be  more than 800pmol/T. More rarely, there is bleeding  due to malabsorption of vitamin K, with consequent  prothrombin deficiency. Cholesterol retention may  cause hypercholesterolaemia. Dark urine and pale  stools suggest biliary retention of conjugated bilirubin.   The jaundice caused by extrahepatic obstruction due  to malignant tissue is typically painless and progressive,  but there may be a history of vague persistent back pain  and weight loss. By contrast, intraluminal obstruction  by a gallstone may cause severe pain, which, like the  jaundice, is often intermittent. Gallstones may not  always cause such symptoms. If a large stone lodges in  the lower end of the common bile duct, the picture may  be indistinguishable from that of malignant obstruction.   Although most of the findings are directly  attributable to cholestasis, biliary back pressure may  damage hepatocytes, and plasma aminotransferase  activities may increase. Unless the cause is clinically  obvious, evidence of dilated ducts due to extrahepatic  obstruction should be sought using tests such as  ultrasound, computerized tomography (CT) scanning  or cholangiography.   This is a rare autoimmune disorder that occurs most  commonly in middle-aged women. Destruction and  proliferation of the bile ducts produce a predominantly  cholestatic picture, with pruritus and a plasma ATP  activity that may be very high, jaundice develops late in  most patients. Mitochondrial antibodies are detectable  in the plasma of more than 90 per cent of cases; the  plasma immmunoglobulin M (IgM) concentration  is usually raised. Patients may also manifest  hypercholesterolaemia, xanthelasma (see Chapter 13),  other autoimmune disorders and osteoporosis.   Prolonged parenteral nutrition may be associated  with a progressive increase in plasma ATP activity  and a subsequent rise in the plasma aminotransferase  concentrations. The cause is not known, although  biliary sludging may occur and the biochemical changes  may return to normal when the parenteral feeding is  discontinued. Cyclical parenteral nutrition may help  the situation when patients are fed intermittently (see  Chapter 14).   A 52-year-old woman was referred to the hepatology  clinic because of jaundice, pruritus, hepatomegaly,  xanthelasma and the following abnormal liver test  results:   Subsequent studies, including liver biopsy, showed  the patient to have primary biliary cirrhosis. Note  the predominant cholestatic biochemical picture  with raised plasma ALP and GGT activities. This  condition is associated with hyperlipidaemia, hence  the xanthelasma, and is more common in middle-  aged women with other autoimmune disorders.  There may also be raised plasma IgM concentration  and osteoporosis and osteomalacia.   The biochemical findings in acute hepatitis are  predominantly those of cell membrane damage with an  increase in plasma ALT activity greater than that of AST.  There may be a superimposed cholestatic picture and,  in very severe cases, impaired prothrombin synthesis.   Viral hepatitis may be associated with many viral  infections, such as infectious mononucleosis (Epstein-  Barr virus), rubella and cytomegalovirus. However, the  term is most commonly used to describe three principal  types of viral infection in which the clinical features of  the acute illness are very similar, although they have a  different incubation period:   • Hepatitis A (‘infectious hepatitis’), transmitted by  the faecal-oral route as a food-borne infection, is  relatively common in schools and other institutions  and has an incubation period of between 15 and 45  days. Relapses may occur, but it rarely progresses to  chronic hepatitis.   • Hepatitis B (‘serum hepatitis’) is transmitted by  blood products and other body fluids; it occurs   A 22-year-old woman who was an intravenous drug  addict was referred to the hepatology clinic because  of the following abnormal liver test results:   Further investigations, including hepatitis screen,  showed her to be hepatitis B positive.   Note the grossly elevated plasma aminotransferase  activities, indicative of extensive hepatocyte damage.  Intravenous drug addicts are at increased risk  of hepatitis B infection. The urinary bilirubin is  positive, as the hyperbilirubinaemia is predominantly  conjugated, that is, water soluble.   more sporadically than hepatitis A. It has a longer  incubation period, of between 40 and 180 days.  Some patients may be anicteric; some may develop  fulminant hepatitis or chronic active hepatitis and  later cirrhosis and hepatocarcinoma. They may  become asymptomatic carriers of the disease.   • Hepatitis C (non-A, non-B hepatitis), which may be  the result of sexual transmission or the transfusion  of blood products, has an incubation period of  between 15 and 50 days. It may progress to cirrhosis.   In all types there may be a 3- to 4-day history of  anorexia, nausea and tenderness or discomfort over the  liver before the onset of jaundice. Some patients remain  anicteric. Plasma aminotransferase activities are very  high from the onset of symptoms; they peak about  4 days later, when jaundice becomes detectable, but  may remain elevated for several months. Once jaundice  appears, some of the initial symptoms improve.   In the early stages there is often a cholestatic  element, with pale stools due to reduced intestinal  bilirubin, and dark urine due to a rise in plasma  conjugated bilirubin concentration; un conjugated  bilirubin concentrations also increase due to impaired  hepatocellular conjugation.   Plasma bilirubin concentrations rarely exceed  350 pmol/L, and the plasma ALP activity is only   moderately raised, or even normal. If hepatocellular  damage is severe and extensive, the prothrombin time  may be increased and, in patients with cholestasis,  malabsorption of vitamin K may be a contributory  factor.   Testing for viral antigens, or for antibodies synthesized  in response to the virus, can be used to diagnose viral  hepatitis.   Hepatitis A viral (HAV) antibodies of the IgM class  are detectable in the plasma of patients at the onset of  symptoms. The presence of an IgG anti-HAV antibody  is suggestive of previous infection. Most cases of  hepatitis A recover completely.   Hepatitis B viral (HBV) infection, during the  prodromal illness, can be diagnosed by the presence in  the plasma of a viral surface antigen (HB Ag) and a core  antigen (HB), an internal component of the virus. These  antigens are short lived. During the next few weeks,  an antibody response occurs, with the appearance of  plasma antibodies to the viral core (anti-HB c ), to HB e  and finally to the surface antibody (anti-HB s ), which  maybe used to document previous infection (Fig. 17.3).   The presence of the HB e antigen correlates with  infectivity, and its disappearance is a good prognostic  sign; HB Ag may persist, especially in patients with an  impaired immune response, and indicates chronicity  associated with raised plasma aminotransferase activities.   Hepatitis C viral (HCV; non-A, non-B hepatitis)  infection is often diagnosed by exclusion. Anti-HCV  anti-bodies may be detected in plasma about 12 weeks   Figure 17.3 Serological and biochemical changes  following infection with hepatitis B virus. anti-H B c ,  antibody to the viral core; anti-H B s , antibody to viral  surface; ALT, alanine aminotransferase; HB s Ag, viral  surface antigen.   after exposure to the virus in about 50 per cent of  patients.   It should be noted that there are other possible  viruses that can cause liver dysfunction such as hepatitis  D and E. Hepatitis D is a ribonucleic acid (RNA)  subviral satellite and needs hepatitis B to propagate,  while hepatitis E is a RNA virus spread more commonly  by the oral-faecal route.   Alcoholic hepatitis occurs in heavy drinkers, often  after a period of increased alcohol intake. Although  the clinical features may mimic acute viral hepatitis,  the plasma aminotransferase activities and bilirubin  concentration are not usually as markedly elevated,  although GGT may be.   There is no perfect laboratory marker of alcohol  abuse. A raised mean corpuscular red cell volume  (MCV), hypertriglyceridaemia, hyperuricaemia and  elevated plasma GGT are clues, but are not specific.  Increased plasma desialylated or carbohydrate-  deficient transferrin of greater than 2 per cent or  raised plasma sialic acid levels have been proposed as  markers of alcohol abuse, as sialic acid metabolism may  be perturbed in the presence of high concentrations  of alcohol. Note that the consumption of more than  80 g a day of alcohol may raise GGT concentrations,  not necessarily by hepatic damage, but by enzyme  induction.   Various drugs and other toxins are hepatotoxic,  sometimes directly and sometimes due to a  hypersensitivity reaction; in the latter case, the damage  is not dose related. The clinical picture may resemble  that of acute viral hepatitis or cholestasis. A drug  history is an essential part of the assessment of a patient  presenting with liver disease. Table 17.2 lists some of  these agents.   The finding of persistent, (usually only slightly) raised  plasma aminotransferase activities, sometimes with  chronic or recurrent symptoms suggesting liver disease,  may be due to several disorders. It may be the only  abnormal biochemical finding.   This is a term used to describe the finding of raised  plasma aminotransferase activities without clinical   signs or symptoms and without a significant change  in activity over many years. The activities rarely exceed  three times the upper reference limits. Jaundice is  unusual.   This is caused by active hepatocellular destruction with  episodes of relapses and remissions. It may progress to  cirrhosis. It occurs at any age, but is most common in  women. It may:   • be associated with, or a consequence of, viral  infections such as HBV or HCV, or may be drug  induced,   • be part of an autoimmune process that sometimes  involves more than one organ,   The earliest findings that differentiate it from  chronic persistent hepatitis are an increasing plasma  IgG concentration, perhaps detected by a rising plasma  y-globulin concentration, and the presence of smooth   muscle and antinuclear antibodies. As the disease  progresses, more cells are destroyed and the plasma AST  activity may rise to or exceed that of ATT; slight jaundice  may develop. If there is significant hepatocellular  destruction, the plasma albumin concentration falls.   Cirrhosis is the end result of many inflammatory  and metabolic diseases involving the liver, including  prolonged toxic damage, usually due to alcohol. In  ‘cryptogenic cirrhosis’, the cause is unknown. The  fibrous scar tissue distorts the hepatic architecture,  and regenerating nodules of hepatocytes disrupt the  blood supply, sometimes increasing the pressure in the  portal vein, causing portal hypertension. Blood may be  shunted from the portal into the hepatic vein, bypassing  the liver.   In the early stages there may be no abnormal  biochemical findings. During phases of active cellular  destruction, the plasma AST, and sometimes ALT,  activities rise. In advanced cases, the biochemical    A 50-year-old known alcoholic man attended the  general medical clinic because of ascites and the  following abnormal liver test results:   The abnormal liver test results and  hypoalbuminaemia together with ascites supported  the diagnosis of cirrhosis, secondary to his alcohol  problem. Hypoalbuminaemia may be due to many  disorders, such as gross proteinuria, but in the  presence of hepatic disease suggests a reduction in  hepatic synthetic capacity typical of cirrhosis.   findings are mostly associated with a reduced  functioning cell mass. The vascular shunting allows  antigenic substances, which have been absorbed from  the intestine, to bypass the normal hepatic sinusoidal  filtering process, and to stimulate increased synthesis  of IgG and IgA, producing the typical serum protein  electrophoretic pattern of (3— y fusion (see Chapter 19).   Portal hypertension and impaired lymphatic  drainage lead to the accumulation of fluid in the  peritoneal cavity (ascites). This may be aggravated by  hypoalbuminaemia, which may also cause peripheral  oedema. In advanced cirrhosis, the findings of  hepatocellular failure develop.   There are a number of causes of ascites including  cirrhosis, malignancy or infection, nephrotic syndrome,  hypothyroidism, pancreatitis and cardiac failure.  Primary hepatocellular carcinoma may develop in a  cirrhotic liver.   The Child-Pugh classification system is a way of  grading the severity of cirrhosis in the face of portal  hypertension (Table 17.3). Survival for patients with  grade C cirrhosis is usually less than 1 year.   Liver damage severe enough to cause obvious clinical  signs of impaired hepatocellular function may be  caused by severe hepatitis or advanced cirrhosis, or may   follow an overdose of a liver toxin such as paracetamol  (acetaminophen). The biochemical findings may  include any or all of those of acute hepatitis. Jaundice is  progressive. In the final stage, the number of hepatocytes,  and so the total amount of aminotransferases released,  may be so reduced that plasma activities fall despite  continuing damage to the remaining cells. This finding  should not be interpreted as a sign of recovery. Other  features may include the following:   • Hypovolaemia and hypotension, which are due to  loss of circulating fluid in ascites and in the oedema  fluid formed because of hypoalbuminaemia, and  which may be aggravated by vomiting. The resultant  low renal blood flow may have two consequences:   - increased antidiuretic hormone (ADH) and  secondary hyperaldosteronism, causing elec-  trolyte disturbances, especially hypokalaemia,  and sometimes dilutional hyponatraemia (see  Chapter 2),   - renal circulatory insufficiency, causing oliguria,  a high plasma creatinine concentration and  uraemia despite reduced urea synthesis.   • Impaired hepatic deamination of amino acids, causing  accumulation of amino acids in plasma with overflow  amino aciduria and sometimes hyperammonaemia.  If the reduced formation of urea from amino acids is  not balanced by renal retention due to the decrease  in glomerular filtration rate (GFR), the plasma urea  concentration may be low.   Treatment of end-stage liver disease  Liver transplantation maybe the only possible treatment  for end-stage liver disease. Complications include graft  failure, hepatic artery thrombosis, infection and acute  and chronic rejection. Both acute rejection (which  occurs in up to 80 per cent of recipients) and chronic  rejection (occurring in about 10 per cent of cases) are   associated with a rise in plasma bilirubin concentration  and ALP activity; chronic rejection may be irreversible.  The indications for hepatic transplantation may include  prolonged prothrombin time and plasma bilirubin  more than 300 pmol/L.   Hepatic infiltration and malignant disease  Invasion of the liver by secondary carcinoma, or  infiltration by lymphoma or granulomas such  as sarcoidosis, may be associated with abnormal  biochemical tests. Sometimes the only abnormal  finding is raised plasma AST activity; the ALT activity  may also be raised to a lesser extent or there may be  GGT elevation. The picture may reflect cholestasis,  with or without jaundice.   Metabolic function is rarely demonstrably impaired.  If a primary hepatocellular carcinoma develops,  either in a cirrhotic liver or de novo, the plasma  aminotransferase and ALP activities usually rise rapidly,  and plasma a-fetoprotein concentrations are often very  high; this latter finding is not diagnostic of primary  hepatic malignancy (see Chapter 24).   A group of rare metabolic disorders, most of which  are inherited, is associated with liver disease, especially  cirrhosis.   Idiopathic haemochromatosis is a genetically  determined disorder in which slightly increased  intestinal absorption of iron over many years produces  large iron deposits of parenchymal distribution,  including the liver (see Chapter 21).   OCj -Antitrypsin deficiency (see Chapter 19) is  associated with neonatal hepatitis in individuals with  the PiZZ phenotype, which progresses to cirrhosis  in childhood. The condition can also present in  adulthood, and often there is basal emphysema  particularly in smokers (Fig. 17.4).   This autosomal recessive disorder, due most  commonly to a deficiency of galactose- 1 -phosphate  uridyltransferase, may cause cirrhosis of the liver if  untreated. Liver transplantation may be indicated if  hepatocellular carcinoma, a complication of cirrhosis,  develops (see Chapter 27).   A 70 -year- old man with known colonic carcinoma,  operated on 2 years previously, was found by his  general practitioner to have the following liver test  results:   An abdominal ultrasound scan showed multiple  space-occupying lesions within the liver.   The patient was eventually found to have widespread  hepatic secondaries from his colonic tumour and  died a few months later. The combination of raised  plasma ALP and GGT activities is suggestive of  hepatic space-occupying lesions, particularly in the  absence of major cholestasis.   A healthy 43-year-old man, on no medication, had  the following results from tests performed during a  private healthcare screening programme:   Normal full blood count, reticulocytes, plasma  haptoglobin concentration and blood film.   The raised concentration of plasma bilirubin is  predominantly unconjugated. There is no evidence  of haemolysis and the other liver function tests are  normal. A likely diagnosis is of Gilbert’s syndrome.  This is a common condition and its diagnosis is based  on the exclusion of liver disease and haemolysis in the  presence of a modest concentration of unconjugated  hyperbilirubinaemia.   This is a rare, recessively inherited disorder caused by  reduced biliary excretion of copper and by impaired  hepatic incorporation of copper into caeruloplasmin.  The symptoms are due to the excessive accumulation  of copper in the liver, brain and kidneys and present  with evidence of acute liver failure, chronic hepatitis  or cirrhosis in children or in young adults (see also  Chapter 14).   This rare disorder presents as acute hepatitis, associated  with marked encephalopathy, severe metabolic acidosis  and hypoglycaemia in children typically between  the ages of 3 and about 12 years. There is acute fatty  infiltration of the liver. The plasma aminotransferase  activities are high, but plasma bilirubin levels are only  slightly raised.   The aetiology is uncertain, but the condition may be  precipitated by viral infections, such as influenza A or  B, drugs such as salicylates and sodium valproate, and  certain toxins; it has been recommended that children  should not be given aspirin. One possible mechanism  is that there is uncoupling of mitochondrial oxidative  phosphorylation. A number of inherited metabolic  disorders, particularly those involving fatty acid  oxidation, may present with a Reye-like syndrome in  children under the age of about 3 years.    Figure 17.4 Patient with severe aj-antitrypsin  deficiency, showing hepatosplenomegaly, who  developed cirrhosis and oesophageal varices.  Reproduced with kind permission from Nyhan WL and  Barshop BA. Atlas of Inherited Metabolic Diseases,  3rd edition. London: Hodder Arnold, 2012.   Non-alcoholic steatotic hepatitis or fatty liver  Non-alcoholic fatty liver disease (NAFLD) refers to  a spectrum of liver disease ranging from fatty liver  (steatosis) to non-alcoholic steatotic hepatitis (NASH)  through to cirrhosis (irreversible, fibrosis and scarring).  This is associated with insulin resistance (see Chapter  12). It is one of the most common causes of abnormal  liver function tests. Plasma aminotransferases are  usually raised and may relate to body mass index. It is  important to exclude other liver disorders, and hepatic  ultrasound may show increased echogenicity of the liver.  The biochemical features of NAFLD may improve with  dietary measures and treatment of hyperlipidaemia  (see Chapter 13).   This syndrome occurs when cirrhosis and often portal  hypertension presents in conjunction with renal  dysfunction. It is thought to be due to impaired renal  perfusion due to vasoconstriction of renal arteries.  Usually the creatinine clearance is less than 40 mL/min  and plasma creatinine is greater than about 130 pmol/L,  with a urine volume of less than 500 mL/ day and  urinary sodium less than lOmmol/L.   Some drugs such as methotrexate can cause hepatic  fibrosis. Conventional liver function tests are insensitive  to liver fibrosis. Increased serum procollagen-3  N-terminal peptide (P3NP) is a marker of fibrosis and  may reduce the need for liver biopsy.   There are many causes of haemolysis, including sickle-  cell anaemia, thalassaemia and spherocytosis, and it  can also be drug or autoimmune induced. In adults,  unconjugated hyperbilirubinaemia is usually mild  because of the large reserve of hepatic secretory capacity.  The plasma bilirubin concentration is usually less than  70 pmol/L. Erythrocytes contain high amounts of AST  and lactate dehydrogenase (LDH t and LDHJ (see  Chapter 18). Blood reticulocytes may be raised, with  abnormal blood film and a low plasma haptoglobin  concentration. A haemolytic component may be seen  in alcoholic hepatitis known as Zieve’s syndrome.  Urinary bilirubin concentration is usually not raised in  haemolysis (acholuric jaundice).   Red cell destruction, together with immature hepatic  processing of bilirubin, may cause a high plasma level  of unconjugated bilirubin in the newborn infant; so-  called physiological jaundice is common. Normal full-  term babies may show jaundice between days 2 and 8  of life.   Physiological jaundice rarely exceeds lOOpmol/L.  Jaundice on the first day of life is invariably pathological,  as are levels of bilirubin exceeding 100 pmol/L or if  the hyperbilirubinaemia is conjugated. As a result  of haemolytic disease, the plasma concentration of  unconjugated bilirubin may be as high as 500pmol/L  and may exceed the plasma protein-binding capacity;  free unconjugated bilirubin may be deposited in the  brain, causing kernicterus. Neonatal jaundice and its  treatment are discussed more fully in Chapter 26.   The inherited hyperbilirubinaemias  There is a group of inherited disorders in which either  unconjugated or conjugated hyperbilirubinaemia is the  only detectable abnormality.   This is a relatively common (3-7 per cent of the  population) familial condition, which may be present  at any age but usually develops after the second decade.  Plasma un conjugated bilirubin concentrations are  usually between 20 pmol/L and 40 pmol/L and rarely  exceed 80 pmol/L. They fluctuate, and may rise during  intercurrent illness, dehydration, menstruation and  fasting. The condition is probably harmless but must  be differentiated from haemolysis and liver disease.  It often becomes evident when plasma bilirubin  concentrations fail to return to normal after an attack  of hepatitis, or during any mild illness, which, because  of the jaundice, may be misdiagnosed as hepatitis.  Conjugated bilirubin is less than 20 per cent of the  total bilirubin.   Mutations in the hepatic uridine diphosphate  glucuronyl transferase ( UGT ) gene are present, but  not all patients with the polymorphism develop the  phenotype. Hepatic UGT activity is decreased to  approximately 30 per cent of normal in individuals  with Gilbert’s syndrome. Decreased activity has been  attributed to an expansion of thymine-adenine repeats  in the promoter region of the UGT1A1 gene, the  principal gene encoding this enzyme.   Diagnosis of Gilbert’s syndrome is by exclusion  of haemolysis and other hepatic disorders. It should  be remembered that sometimes thyrotoxicosis  can cause raised unconjugated bilirubin due to  reduced UGT activity and so can reabsorption of a  large haematoma due to haemoglobin breakdown.  Prolonged fasting (48 h with calorie intake restricted  to 300kcal/day) may result in a rise, predominantly  in the unconjugated bilirubin fraction, of around  100 per cent if Gilbert’s syndrome is present.  Increases are also seen after the administration of  50 mg intravenous nicotinic acid. However, these  dynamic tests are not without side effects and are  rarely used.   This is due to a rare deficiency of hepatic UGT, and is  a more serious condition. It usually presents at birth.  The plasma unconjugated bilirubin may increase to  concentrations that exceed the binding capacity of  albumin and so cause kernicterus. The defect may  be complete (type I), and inherited as an autosomal  recessive condition, or partial (type II), and inherited  as an autosomal dominant condition.   In type II drugs that induce enzyme synthesis,  such as phenobarbital, may reduce plasma bilirubin  concentration.   This is probably harmless and is due to defective  excretion of conjugated bilirubin, but not of bile acids.  It is characterized by slightly raised plasma conjugated  bilirubin levels that tend to fluctuate. Because the  bilirubin is conjugated, it may be detectable in the  urine. Plasma ALP activities are normal. There may  be hepatomegaly, and the liver is dark brown in  appearance due to the presence of a pigment with the  staining properties of lipofuscin. The diagnosis may be  confirmed by the characteristic staining of a specimen  obtained by liver biopsy.   Rotor’s syndrome is similar in most respects to Dubin-  Johnson syndrome, but the liver cells are not pigmented  (Box 17.1).   Four bile acids are produced in humans. Two of these,  cholic acid and chenodeoxycholic acid, are synthesized   Alcohol, haemochromatosis, Wilson’s disease  Gallstones, cholangiocarcinoma, bile duct strictures,  pancreatic tumours   Meta bolic disorders, e.g. oq-antitrypsin, Reye’ssyndrome,  fatty liver, intrahepatic cholestasis of pregnancy  Diffusion infiltration, e.g. sarcoid, lymphoma, amyloid  Inborn errors, e.g. Dubin-Johnson syndrome, Rotor’s  syndrome   in the liver from cholesterol and are called primary  bile acids. They are secreted in bile as sodium salts,  conjugated with the amino acid glycine or taurine  (primary bile salts). These are converted by bacteria  within the intestinal lumen to the secondary bile salts,  deoxycholate and lithocholate, respectively (Fig. 17.5).   Secondary bile salts are partly absorbed from the  terminal ileum and colon and are re-excreted by the liver  (enterohepatic circulation of bile salts). Therefore, bile  contains a mixture of primary and secondary bile salts.   Deficiency of bile salts in the intestinal lumen leads  to impaired micelle formation and malabsorption of  fat (see Chapter 13). Such deficiency may be caused by  cholestatic liver disease (failure of bile salts to reach the  intestinal lumen) or by ileal resection or disease (failure  of reabsorption causing a reduced bile salt pool). Bile  salts are also important biochemical modulators and  activate various nuclear receptors.   Between 1 L and 2 L of bile is produced daily by the  liver. This hepatic bile contains bilirubin, bile salts,  phospholipids and cholesterol, as well as electrolytes  in concentrations similar to those in plasma. Small  amounts of protein are also present.   In the gall bladder there is active reabsorption of  sodium, chloride and bicarbonate, together with an   Figure 17.5 Synthesis of bile acids in the liver and  their conversion to secondary bile salts in the intestine.   isosmotic amount of water. Consequently, gall bladder  bile is 10 times more concentrated than hepatic bile;  sodium is the major cation and bile salts the major  anions. The concentrations of other non-absorbable  molecules, such as conjugated bilirubin, cholesterol  and phospholipids, also increase.   Although most gallstones contain all biliary  constituents, they consist predominantly of one.  Only about 10 per cent contain enough calcium to be  radio-opaque and in this way they differ from renal  calculi. They can be shown on gall bladder ultrasound  (Fig. 17.6).   Pigment stones are found in such chronic haemolytic  states as hereditary spherocytosis. Increased breakdown  of haemoglobin increases bilirubin formation and  therefore biliary secretion. The stones consist mostly of  bile pigments, with variable amounts of calcium. They  are small, hard and dark green or black, and are usually  multiple. Rarely, they contain enough calcium to be  radio-opaque.   Cholesterol is most likely to precipitate if bile is  supersaturated with it; further precipitation on a  nucleus of crystals causes progressive enlargement.  Not all patients with a high biliary cholesterol  concentration suffer from bile stones. Changes in  the relative concentrations of different bile salts may  favour precipitation. The stones may be single or  multiple. They are described as mulberry-like and  are either white or yellowish; the cut surface appears  crystalline.   There is no clear association between  hypercholesterolaemia and the formation of cholesterol  gallstones, although both may be more common in  obese individuals. However, there may be an increased  incidence in patients taking some lipid-lowering drugs,  such as the fibric acid derivatives.   Most gallstones contain a mixture of bile constituents,  usually with a cholesterol nucleus as a starting point.  They are multiple-faceted, dark-brown stones with a  hard shell and a softer centre and may contain enough  calcium to be radio-opaque.   Gallstones may remain silent for an indefinite length  of time and be discovered only at laparotomy for an  unrelated condition. They may, however, lead to various  clinical consequences.   • Acute cholecystitis', obstruction of the cystic duct  by a gallstone causes chemical irritation of the gall  bladder mucosa by trapped bile and secondary  bacterial infection.   • Obstruction of the common bile duct occurs if a stone  lodges in it. The patient may present with biliary   colic, obstructive jaundice (Table 17.4), which is  usually intermittent, or acute pancreatitis if the  pancreatic duct is also occluded.   • Rarely, gallstones may be associated with gallstone  ileus or carcinoma of the gall bladder.   The most common treatment for symptomatic gallstones  is cholecystectomy, either open or laparoscopic. The  following are some alternative approaches:   • Dissolving the gallstone: the bile acid ursodeoxycholic  acid reduces the relative saturation of bile with  cholesterol and so facilitates dissolving cholesterol  stones, particularly small, uncalcified ones.   • Shock-wave lithotripsy: the stone is shattered into tiny  fragments and can then pass through the cystic duct.   The commonly available biochemical laboratory  tests for the diagnosis of liver disease involve the  measurement of plasma levels of:   The initial selection of investigations depends on the  age of the patient, the history and clinical features.   Jaundice as a presenting feature (Fig. 17.7)  Whereas a patient with chronic liver disease may present  with jaundice, the differential diagnosis of jaundice with  bilirubinuria (due to conjugated bilirubin) in a previously    well patient is usually between acute hepatocellular  damage and cholestasis. A suggested scheme is as follows  (for neonatal jaundice, see Chapter 26):   - recent exposure to hepatitis or infectious  mononucleosis, including a sexual and  occupational history,   - associated symptoms such as abdominal pain,  pruritus, weight loss or anorexia and nausea,   - recent changes in the colour of the urine or  stools,   - Predominantly unconjugated hyperbilirubi-  naemia with plasma conjugated bilirubin  levels less than about 10 per cent of the total,  and with little or no bilirubinuria, may suggest  haemolysis as a cause. Haemolysis is supported  by a raised reticulocyte count and diagnostic  blood film, reduced plasma haptoglobin  and raised plasma lactate dehydrogenase  concentrations.   - If haemolysis is excluded, consider Gilbert’s  syndrome provided other liver tests are normal  and other hepatic disorders have been excluded.   • A fresh urine sample should be examined. This test  may show the presence of bilirubin if conjugated  hyperbilirubinaemia is present. Dark-yellow or  brown urine suggests biliary obstruction. An absence   of urinary urobilinogen is seen in biliary obstruction.  Reagent strips are available for testing for bilirubin  and urobilinogen in urine.   • Pale stools suggest biliary obstruction as a cause of  jaundice.   • Whatever the results of the urine and stool inspection  and testing, request plasma aminotransferase, ALP  and GGT assays:   - In hepatitis, there is a predominant increase in  the concentrations ofplasma aminotransferases;  usually the plasma ALT activity is higher than  the AST activity. If hepatitis or infectious  mononucleosis is suggested by the history,  request serological tests.   - In cholestasis, there is predominant elevation  of the plasma ALP and GGT activities. Bile duct  dilatation should be sought using ultrasound  or other radiological tests:   • if the bile ducts are dilated, there is  obstruction that may require surgery,   • if the plasma ALP activity is high but  dilated ducts are not demonstrated, there is  probably intrahepatic cholestasis.   • Other tests, such as ferritin and iron saturation  (haemochromatosis), and autoantibody (such as  smooth muscle, p -anti-neutrophil cytoplasmic  antigen, mitochondrial, nuclear antibodies) and  immunoglobulin levels, may also be indicated.   • If acute alcoholic hepatitis is suspected, contributory  evidence may be the finding of a disproportionately  high plasma GGT activity compared with those of the  aminotransferases. There may also be macrocytosis,  hypertriglyceridaemia and hyperuricaemia.   • In obstructive j aundice (biliary obstruction) , the plasma  ALP is usually more than four to five times and GGT  more than 10 times normal. Liver/biliary ultrasound is  useful to distinguish between obstructive jaundice with  dilated biliary ducts or undilated ducts. Endoscopic  retrograde cholangiopancreatography (ERCP) or  percutaneous cholangiography may be indicated.   • If the diagnosis is in doubt, a liver biopsy may be  indicated, although there may be a risk of bleeding  and therefore the prothrombin time should be  measured beforehand.   - a previous history of hepatitis, intravenous  drug use, occupational and sexual history,   • Request tests for plasma bilirubin, aminotransferases  (ALT and AST), albumin, GGT and ALP:   - A raised plasma ALT activity higher than that  of the AST may be due to reversible alcoholic  hepatitis, to chronic persistent hepatitis, or to  early chronic active hepatitis.   - A raised plasma AST activity higher than  that of ALT may be due to cirrhosis or severe  chronic active hepatitis.   - A high plasma ALP activity with raised GGT  concentration suggests cholestasis.   - Aminotransferase levels of more than 10 times  normal suggest primary hepatocyte damage, as  in viral hepatitis or caused by drugs/toxins.   • An alcohol-related aetiology is supported by a  macrocytosis and plasma GGT concentration, but  neither test is fully diagnostic. See above.   • Detectable plasma mitochondrial or smooth-muscle  antibodies are suggestive of primary biliary cirrhosis  or chronic active hepatitis, respectively.   • A raised plasma ferritin concentration with high  iron saturation (see Chapter 21) may reveal  haemochromatosis.   • Plasma protein electrophoresis and immunoglobulin  assay may help in the diagnosis of:   - chronic active hepatitis - a high plasma IgG  concentration and normal IgA,   A low plasma albumin concentration (hypo-  albuminaemia) in the face of abnormal liver function  tests can be seen in cirrhosis implying chronicity.   • A prolonged prothrombin time implies poor hepatic  synthetic capacity, for example clotting factors, and  is prognostic in paracetamol overdose. It is also  important if a liver biopsy is considered.   • Significant infiltration of the liver by tumour  cells, or by granulomas such as sarcoidosis   (possibly with raised plasma angiotensin-  converting enzyme activity), may occur. In this  situation raised plasma AST activity may be the  most sensitive test, despite a normal plasma ALT  activity. Hepatic space-occupying lesions may  additionally present with raised GGT and ALP  concentrations, and a liver ultrasound is useful to  detect these.   • A fatty liver may be revealed by liver ultrasound  as this may show increased echogenicity. This may  be associated with hypertriglyceridaemia, obesity   • If primary hepatocellular carcinoma is suspected,  the plasma a-fetoprotein level may also be high.   • Low plasma copper and caeruloplasmin  concentrations may suggest Wilson’s disease (see  Chapter 15), and plasma a antitrypsin deficiency  can result in cirrhosis (see Chapter 19).   • Radionuclide scans or other imaging procedures  (CT or MRI) may be useful. A liver biopsy may be  indicated to clarify a histological diagnosis.   • The liver has an enormous synthetic capacity and is  involved in numerous metabolic pathways, including  vitamin storage, amino acid deamination, bile salt and  cholesterol synthesis, and the production of various  proteins such as clotting factors and hormones.   • Compounds ‘released’ from the liver into the plasma  can be used as markers of liver damage, including  bilirubin, ALT, AST, GGT and ALP.   • Hyperbilirubinaemia can be due to raised  unconjugatedorconjugatedbilirubin concentration.  The former maybe due to haemolysis and the latter  to hepatic or extrahepatic causes.   • Raised plasma aminotransferase activities suggest  hepatocyte damage and raised hepatic ALP  concentration is associated with cholestasis.   • Plasma GGT is a sensitive marker of hepatic  damage but its activity can also be raised as a result  of drug enzyme induction, hypertriglyceridaemia  and increased alcohol intake.   • A prolonged prothrombin time and low plasma  albumin concentration may both reflect reduced  hepatic synthetic capacity.   This chapter discusses the principles of clinical  enzymology, which have already been encountered in  some of the preceding chapters. Enzymology can be  defined as the assay of an enzyme(s) in body fluids,  usually blood, that can be used diagnostically or to  monitor a clinical condition.   An enzyme is a protein that catalyses one or more  specific biochemical reactions. It is usually easier to  measure enzyme activity in body fluids, by monitoring  changes in either substrate or product concentrations,  than to measure enzyme protein concentration  directly, although this is sometimes done. However,  measurement of the enzyme protein concentration is  more specific and less prone to analytical variation.   Generally, enzymes are present in cells at much  higher concentrations than in plasma. Some occur  predominantly in cells of certain tissues, where they  may be located in different cellular compartments such  as the cytoplasm or the mitochondria. ‘Normal’ plasma  enzyme concentrations reflect the balance between the  rate of synthesis and release into plasma during cell  turnover, and the rate of clearance from the circulation.  The enzyme activity in plasma may be:   • higher than normal, due to the proliferation of cells,  an increase in the rate of cell turnover or damage  or in enzyme synthesis (induction), or to reduced  clearance from plasma,   • lower than normal, due to reduced synthesis,  congenital deficiency or the presence of inherited  variants of relatively low biological activity -  examples of the latter are the cholinesterase variants.   Sometimes macroenzymes are found, that is to say,  a high-molecular-weight form of a native enzyme.  Often these are enzymes [such as lactate dehydrogenase  (LDH), creatine kinase (CK) and alkaline phosphatase  (ALP)] complexed with immunoglobulins and are  more common in individuals with autoimmune  disease. It is important to recognize macroenzymes as  they can sometimes cause diagnostic confusion. As we   will now see, changes in plasma enzyme activities may  be useful to detect and localize tissue cell damage or  proliferation, or to monitor the treatment and progress  of disease.   Plasma enzyme levels depend on the extent of cell  damage and the rate of release from damaged cells,  which, in turn, depends on the rate at which damage  is occurring.   In the absence of cell damage, the rate of release  depends on the degree of induction of enzyme synthesis  and the rate of cell proliferation.   These factors are balanced by the rate of enzyme  clearance from the circulation.   Acute cell damage, for example in viral hepatitis,  may cause very high plasma aminotransferase activities  that reduce as the condition resolves. By contrast,  the liver may be much more extensively involved in  advanced cirrhosis but the rate of cell damage is often  low, and consequently plasma enzyme activities may  be only slightly raised or within the reference range. In  very severe liver disease, plasma enzyme activities may  even fall terminally when the number of hepatocytes is  grossly reduced (see Chapter 17).   Relatively small enzymes, such as amylase, can be  cleared by the kidneys. Thus, plasma amylase activity  may be high as a result of renal glomerular impairment  rather than pancreatic damage. However, most  enzymes are large proteins and may be catabolized  by plasma proteases before being taken up by the  reticuloendothelial system.   In healthy individuals, each enzyme has a fairly  constant and characteristic biological half-life, a fact  that maybe used to assess the time since the onset of an  acute illness. After a myocardial infarction, for example,  plasma levels of CK and aspartate aminotransferase  (AST) fall to normal before those of LDH, which has a  longer half-life (see Chapter 22).   Most of the enzymes commonly measured to assess tissue  damage are present in nearly all body cells, although  their relative concentrations in certain tissues may differ.  Measurement of the plasma activity of an enzyme known  to be in high concentration within cells of a particular  tissue may indicate an abnormality of those cells, but the  results will rarely enable a specific diagnosis to be made.  For example, if there is circulatory failure after a cardiac  arrest, very high plasma concentrations of enzymes  originating from many tissues may occur because of  hypoxic damage to cells and reduced rates of clearance.   The distribution of enzymes within cells may  differ. Alanine aminotransferase (ALT) and LDH are  predominantly located in cytoplasm, and glutamate  dehydrogenase (although this is not usually measured  clinically) in mitochondria, whereas AST occurs in both  these cellular compartments. Different disease processes  in the same tissue may affect the cell in different ways,  causing alteration in the relative plasma enzyme activities.   The diagnostic precision of plasma enzyme analysis  may be improved by the following:   • Serial enzyme estimations The rate of change of  plasma enzyme activity is related to a balance  between the rate of entry and the rate of removal  from the circulation. A persistently raised plasma  enzyme activity is suggestive of a chronic disorder  or, occasionally, impaired clearance.   • Isoenzyme determination Some enzymes exist in  more than one form; these isoenzymes may be  separated by their different physical or chemical  properties. If they originate in different tissues,  such identification will give more information than  the measurement of plasma total enzyme activity;  for example, CK may be derived from skeletal or  cardiac muscle, but one of its isoenzymes is found  predominantly in the myocardium.   • Estimation of more than one enzyme Many  enzymes are widely distributed, but their relative  concentrations may vary in different tissues. For  example, although both ALT and AST are abundant  in the liver, the concentration of AST is much greater  than that of ALT in heart muscle.   Before attributing a change in plasma enzyme activity  to a specific disease process, it is important to exclude  the presence of factitious or non-specific causes.   Slight rises in plasma ALT and AST activities are  common, non-specific findings in many illnesses.  Moderate exercise, or a large intramuscular injection,  may lead to a rise in plasma CK activity; isoenzyme  determination may identify skeletal muscle as the tissue  of origin.   Some drugs, such as the anticonvulsants phenytoin  and phenobarbital, may induce the synthesis of the  microsomal enzyme y-glutamyl transferase (GGT),  and so increase its plasma activity in the absence of  disease.   Plasma enzyme activities may be raised if the rate  of clearance from the circulation is reduced. In the  absence of hepatic or renal disease, this may occur if,  for example, the plasma enzyme forms complexes with  immunoglobulins, known as a macroenzyme.   Various enzymes can form clinically significant  macroenzymes including amylase, LDH, ALP and  CK.   The total concentration of all plasma enzyme proteins  is less than 1 g/L. The results of enzyme assays are not  usually expressed as concentrations, but as activities.  Changes in concentration may give rise to proportional  changes in catalytic activity, but the results of such  measurements depend on many analytical factors,  including:   The definition of ‘international units’ does not take  these factors into account, and the results from different  laboratories, which are apparently expressed in the  same units, may not be directly comparable. Therefore,  plasma enzyme activities must be interpreted in relation  to the reference ranges from the issuing laboratory.  In some countries such as the UK there are plans to  harmonize or converge laboratory reference ranges for  certain analytes.   Plasma AST activity is moderately higher during the  neonatal period than in adults. Plasma ALP activity  of bony origin is higher in children than in adults and  peaks during the pubertal bone growth spurt before  falling to adult levels. A second peak occurs in the  elderly.   Plasma GGT activity is higher in men than in women.  Plasma CK activity is also higher in males, probably in  part due to their increased muscle bulk.   Plasma CK activity is higher in black people and Afro-  Caribbeans than in white people.   Plasma ALP activity rises during the last trimester of  pregnancy because of the presence of the placental  isoenzyme. Several enzymes, such as AST and CK, rise  moderately in plasma during and immediately after  labour or strenuous exercise.   Plasma enzyme activities should therefore be  interpreted in relation to the sex-, race-/ethnicity- and  age-matched reference ranges of the issuing laboratory.   Individual enzymes of clinical importance are  considered in the following section. Applications of  their assays in defined clinical situations are discussed  later in the chapter and in other chapters in this book.   The aminotransferases (ALT and AST) are enzymes  involved in the transfer of an amino group from a  2-amino acid to a 2-oxoacid; they need the cofactor  pyridoxal phosphate for optimal activity. They are  widely distributed in the body. (See Chapter 17, which  deals with hepatic disease, for further details.) The  aminotransferases are used as part of the biochemical  liver profile.   Aspartate aminotransferase (glutamate oxaloacetate  aminotransferase, GOT) is present in high  concentrations in cells of cardiac and skeletal muscle,  liver, kidney and erythrocytes. Damage to any of these  tissues may increase plasma AST levels.   • Artefactual : due to in vitro release from erythrocytes  if there is haemolysis or if separation of plasma from  cells is delayed.   • Physiological : during the neonatal period (about 1.5  times the upper adult reference limit).   • Marked increase (may be greater than 5-10 times the  upper reference limit or URL):   • Moderate to slight increase (usually less than five  times URL):   - Hepatic steatosis [fatty liver or non-alcoholic  fatty liver disease (NAFLD)],   Note that AST is not specific for hepatic disease.  Alanine aminotransferase   Alanine aminotransferase (glutamate pyruvate  aminotransferase, GPT) is present in high  concentrations in liver and, to a lesser extent, in skeletal  muscle, kidney and heart.   • Moderate to slight increase (usually less than five  times URL):   - surgery or extensive trauma and skeletal muscle  disease (much less affected than AST),   Note that ALT is more specific for hepatic disease  than AST.   Lactate dehydrogenase catalyses the reversible  interconversion of lactate and pyruvate. The enzyme is  widely distributed in the body, with high concentrations  in cells of cardiac and skeletal muscle, liver, kidney, brain  and erythrocytes; measurement of plasma total LDH  activity is therefore a non-specific marker of cell damage.   • Artefactual : due to in vitro haemolysis or delayed  separation of plasma from whole blood.   - some haematological disorders: in blood  diseases such as megaloblastic anaemia, acute  leukaemias and lymphomas, very high levels  (up to 20 times the URL in adults) may be  found. In cases of lymphoma LDH can be used  as a tumour marker. Smaller increases occur  in other disorders of erythropoiesis, such as  thalassaemia, myelofibrosis and haemolytic  anaemias, renal infarction or, occasionally,  during rejection of a renal transplant.   • Moderate to slight increase (usually less than five  times URL):   Five main isoenzymes can be detected by electrophoresis  and are referred to as LDH 1 to LDH 5 . LDHj, the fraction  that migrates fastest towards the anode, predominates  in cells of cardiac muscle, erythrocytes and kidney. The  slowest moving isoenzyme, LDH 5 , is the most abundant  form in the liver and in skeletal muscle.   Whereas in many conditions there is an increase  in all fractions, the finding of certain patterns is of  diagnostic value:   • Predominant elevation of LDH and LDH 2 (LDH t  more than LDH 2 ) occurs after myocardial infarction,  in megaloblastic anaemia and after renal infarction.   • Predominant elevation of LDH, and LDH 3 occurs  in acute leukaemia; LDH 3 is the main isoenzyme  elevated as a result of malignancy of many tissues.   • Elevation of LDH 5 occurs after damage to the liver or  skeletal muscle.   It is rarely necessary to quantify LDH isoenzyme  activity. A rise in LDH t is most significant in the  diagnosis of myocardial infarction. However, as  LDHj and, to a lesser extent LDH 2 and LDH 3 , can use  2-hydroxybutyrate as well as lactate as substrate, some  laboratories assay hydroxybutyrate dehydrogenase  (HBD) as an index of LDH activity. Lactate  dehydrogenase was used in the delayed diagnosis  of a myocardial infarct (troponin has largely taken  over this role from LDH) and also as a marker for  certain tumours, for example lymphomas, and to help  determine haemolysis (see Chapters 17 and 22).   Creatine kinase is most abundant in cells of cardiac and  skeletal muscle and in brain, but also occurs in other  tissues such as smooth muscle.   Creatine kinase consists of two protein subunits, M  and B, which combine to form three isoenzymes, BB  (CK-1), MB (CK-2) and MM (CK-3).   • CK-MM is the predominant isoenzyme in skeletal  and cardiac muscle and is detectable in the plasma  of normal subjects.   • CK-MB accounts for about 35 per cent of the total  CK activity in cardiac muscle and less than 5 per cent  in skeletal muscle; its plasma activity is always high  after myocardial infarction. The use and limitations  of CK-MB estimation are considered in Chapter  22. It may be detectable in the plasma of patients  with a variety of other disorders in whom the total  CK activity is raised, but this accounts for less than  6 per cent of the total CK activity.   • CK-BB is present in high concentrations in the brain  and in the smooth muscle of the gastrointestinal and  genital tracts. Increased plasma activities may occur    during parturition. Although they have also been  reported after brain damage, for example trauma  or cerebrovascular accident, and in association  with malignant tumours of the bronchus, prostate  and breast, measurement is not of proven value for  diagnosing these conditions. In malignant disease,  plasma total CK activity is usually normal.   There are also other forms of CK. One is a  mitochondrial form seen in hepatic disease, certain  tumours and critically ill patients. There are also  type 1 and type 2 macroenzyme forms of CK. Type 1  macroenzyme is associated with autoimmune disease  such as rheumatoid arthritis and is thought to be  CK complexed with IgG; type 2 macroenzyme is an  oligomer of mitochondrial CK.   - plasma CK is generally higher in Africans than  in Caucasians.   • Moderate to slight increase (usually less than five  times URL):   - physical exertion - there may be a significant  rise in plasma activity after only moderate  exercise, muscle cramp or following an  epileptic fit,   A 45-year-old man was prescribed a statin  by his general practitioner because of  hypercholesterolaemia. A week after commencement  on the drug, he complained of severe muscle aches.  The following are the results of the tests requested by  his general practitioner:   Alanine aminotransferase (ALT) 82U/L (< 42)  Asparate aminotransferase (AST) 98U/L (< 45)  Alkaline phosphatase 113U/L (<250)   The grossly elevated CK activity supports the  diagnosis of rhabdomyolysis. This is a rare  complication of statin drug usage. It is important also  to monitor renal function, as the myoglobin released  from muscle is nephrotoxic. Intracellular ions such  as potassium are also released from the muscle into  the circulation, resulting in hyperkalaemia. Note that  the plasma AST and ALT activities are raised, as these  can be found in muscle as well as in liver tissue.   Plasma CK activity is raised in all types of muscular  dystrophy, but not usually in neurogenic muscle  diseases such as poliomyelitis, myasthenia gravis,  multiple sclerosis or Parkinson’s disease.   Rhabdomyolysis can be defined as an acute increase  in plasma CK concentration greater than 10 times  the upper limit of normal. Severe muscle breakdown  results in grossly elevated plasma CK concentrations,  sometimes uptolOOOOOU/L. This can be due to trauma,  severe exertion, alcohol, heat, electrolyte disturbances  and drugs such as statins. Plasma myoglobin (see  Chapter 21) is elevated and, being of low molecular  weight, is filtered through the renal glomeruli and can  precipitate out in the renal tubules, resulting in acute  kidney injury.   Urinary myoglobin can be inferred by the red-  brown colour and positive urine dipstick test for  haem in the absence of erythrocytes as judged  by microscopy. Intravascular volume expansion  with saline and sometimes mannitol-alkaline  diuresis my help reduce the risk of rhabdomyolysis.  Rhabdomyolysis is associated with hyperkalaemia and  hyperphosphataemia due to the release of intracellular  ions from myocytes, and calcium can be sequestered,  resulting in hypocalcaemia.   This is unusual but may include cachetic states associated  with reduced muscle mass, for example alcoholism,  undernutrition and patients in intensive care.   This glycolytic enzyme has been measured in plasma as  a marker of muscle disease. However, generally it offers  no major advantage over CK and is now rarely used.   Amylase (molecular weight 45kDa) breaks down  starch and glycogen to maltose. It is present at a high  concentration in pancreatic juice and in saliva and may  be extracted from other tissues, such as the gonads,  Fallopian tubes, skeletal muscle and adipose tissue.  Being of relatively low molecular weight, it is excreted  in the urine.   Estimation of plasma amylase activity is mainly  requested to help in the diagnosis of acute pancreatitis,  in which the plasma activity maybe very high. However,  it may also be raised in association with other intra-  abdominal and extra-abdominal conditions that cause  similar acute abdominal pain; thus a high result is not  a specific diagnostic marker for acute pancreatitis (Fig.  18.1; see also Chapter 16). If the plasma amylase activity  fails to fall after an attack of acute pancreatitis, there  may be leakage of pancreatic fluid into the lesser sac  (a pancreatic pseudocyst); although C-reactive protein  (CRP) may be a more useful marker of resolving  uncomplicated acute pancreatitis. Plasma activity may  be near normal in chronic or haemorrhagic pancreatitis.   - perforated peptic ulcer, especially if there is  perforation into the lesser sac.   • Moderate to slight increase (usually less than five  times URL).   • after injection of contrast medium into  salivary ducts for sialography.   In some patients, high plasma amylase activity is  due to low renal excretion of a macroenzyme form,  despite normal glomerular function. The condition is  symptomless and it is thought that the enzyme is bound  to IgA, giving a complex of molecular weight about  270 kDa. This harmless condition may be confused  with other causes of hyperamylasaemia. If amylase and  creatinine are assayed in simultaneous plasma and urine  samples, an amylase clearance to creatinine clearance  ratio can be calculated. If the result is multiplied by 100, a  ratio of less than 0.02 is suggestive of macroamylasaemia.  Electrophoretic techniques can also be used to determine  the presence of macroamylasaemia.   Plasma amylase is derived from the pancreas and  salivary glands. It is rarely necessary to identify the  isoenzyme components in plasma, but they can be  distinguished by electrophoretic techniques, or by  using an inhibitor derived from wheat germ. Possible  indications for isoenzyme determination include:   • the coexistence of mumps or renal failure, which  complicate the interpretation of high activities due  to acute pancreatitis;   • the possibility of chronic pancreatic disease, in which  low activities may be found.   Sometimes, when it is difficult to interpret plasma  amylase results, it may be more useful to measure  plasma lipase This enzyme is also derived from the  pancreas but is more specific for pancreatic pathology.  In addition, lipase has a longer half-life than amylase  and therefore may be more useful in the diagnosis of  late-presenting acute pancreatitis.   The ALPs are a group of enzymes that hydrolyse  organic phosphates at high pH. They are present in  most tissues but are in particularly high concentration  in the osteoblasts of bone and the cells of the  hepatobiliary tract, intestinal wall, renal tubules and  placenta.   In adults, plasma ALP is derived mainly from bone  and liver in approximately equal proportions; the  proportion due to the bone fraction is increased when  there is increased osteoblastic activity that may be  physiological (Fig. 18.2).   - During the last trimester of pregnancy, the  plasma total ALP activity rises due to the  contribution of the placental isoenzyme.  Plasma ALP concentration may increase by  up to five times and usually returns to normal  levels by 1 month post partum.   - In preterm infants, plasma total ALP activity is  up to five times the URL in adults, and consists  predominantly of the bone isoenzyme.   - In children, the total activity increases by about  two to five times during the pubertal bone  growth spurt. There is a gradual increase in the  proportion of liver ALP with age.   - In the elderly, the plasma bone isoenzyme  activity may increase slightly.    Figure 18.2 Algorithm for the investigation of a raised plasma alkaline phosphatase. GGT, y-glutamyl transferase.   - primary hyperparathyroidism with extensive  bone disease (usually normal but may be  slightly elevated),   - space-occupying lesions, tumours, granulomas  and other causes of hepatic infiltration.   • Inflammatory bowel disease: the gut ALP isoenzyme  can be increased in ulcerative colitis.   A 23 -year-old man was investigated in the  gastroenterology clinic because his plasma amylase  was 445 U/L (< 200) and some of his blood results  were as follows:   No other investigations revealed any abnormality,  including upper gastrointestinal endoscopy and  abdominal ultrasound. A urine analysis did not find  any amylase activity.   The question here is what is the explanation for the  raised plasma amylase activity? Amylase is normally  present in the urine because of its relatively small  molecular size. Its absence in urine and serum amylase  electrophoretic studies confirmed macroamylasaemia.  This is a large form of amylase, usually immunoglobulin  A bound, that is not renally cleared.   A 64-year-old man with lung carcinoma attended  the oncology clinic. Some of his blood results were  as follows:   Alkaline phosphatase (ALP) 426 U/L (<250)  y-Glutamyl transferase (GGT) 50 U/L (<55)  Albumin 36g/L (35-45)   The question here is what is the source of the raised  ALP activity? The normal GGT activity makes  a hepatic source unlikely. Similarly, the normal  plasma calcium concentration and bone scan do  not make a bone source likely either. However,  ALP isoenzymes showed the presence of a Regan  isoenzyme, thought to be ectopically released from  his lung carcinoma.   with malignant disease, especially carcinoma of the  bronchus. There is also a Nago isoenzyme released by  certain tumours.   Transient very high levels of ALP (up to 30 times  URL) have been recorded in children, but the clinical  significance of this finding is unknown. This has  been called transient hyperphosphatasaemia and may  be either the bone or the liver isoenzyme. It may be  associated with abdominal symptoms and usually  resolves within a few months.   Plasma total ALP activity is not usually increased in  myelomatosis, despite the radiographic appearance of  multiple ‘punched-out’ osteolytic lesions. The lesions  are in the marrow cavity, not the bone substance, and  osteoblastic activity is not stimulated. However, ALP  activity may be raised if there is liver involvement  or, more rarely, if there is healing of very extensive  pathological fractures.   A low plasma ALP concentration is less usual, but may  be caused by the following:   • Hypophosphatasia, an autosomal recessive disorder,  associated with rickets or osteomalacia.   • Treatment of hyperlipidaemia with afibrate drug, for  example bezafrbrate. This observation can be useful  to determine drug compliance.   Bone disease with increased osteoblastic activity and  liver disease with involvement of the biliary tracts are  the most common causes of an increased total ALP  activity.   Rarely, the cause is not apparent and further tests  may be helpful. The isoenzymes originating from  the cells of bone, liver, intestine and placenta may be  separated by electrophoresis, but interpretation may be  difficult if the total activity is only marginally raised.  The placental and ‘Regan’ isoenzymes are more stable  at 65°C than the bone, liver and intestinal isoenzymes,  and heat inactivation may help to differentiate the heat-  stable from the heat-labile fraction.   The placental isoenzyme does not cross the placenta  and is therefore not detectable in the plasma of the  newborn infant (Fig. 18.3).   Acid phosphatase (ACP) is found in cells of the prostate,  liver, erythrocytes, platelets and bone. The main  indication for estimation was to help diagnose prostatic  carcinoma and to monitor its treatment. Estimation has  been largely replaced by the measurement of plasma  prostate-specific antigen (PSA), a protein derived from  the prostate (see Chapter 24). This test is more specific  and sensitive for diagnosis and monitoring treatment  and has essentially rendered the plasma ACP assay  obsolete in the diagnosis and management of prostatic  carcinoma.   Haemolysed blood samples should also be avoided,  as ACP is found in erythrocytes. Normally, ACP drains  from the prostate, through the prostatic ducts and into  the urethra, and very little can be detected in plasma.  In extensive prostatic carcinoma, particularly if it has  spread extensively or has metastasized, plasma ACP  activity rises, probably because of the increased number  of prostatic ACP- containing cells. Another problem  with plasma ACP is that its concentration can increase  after rectal examination.   Figure 18.3 Electrophoresis separation of alkaline phosphatase (ALP) isoenzymes. Liver ALP runs more anodal  than bone and heat and inhibition studies can be used to determine the various isoenzyme forms. Reproduced  with kind permission of Sebia.   The release of ACP from blood cells in vitro may occur  even in unhaemolysed samples and many methods have  been devised in an attempt to measure only the prostatic  fraction, without complete success. One method makes  use of the fact that L-tartrate inhibits prostatic ACP; the  assay is performed with and without the addition of  L-tartrate; the difference in activity between the results  (the tartrate-labile fraction) is mainly prostatic ACP.  Other methods measure the prostatic enzyme protein  concentration directly by immunoassay.   - artefactually raised following rectal examination,  acute retention of urine or passage of a urinary  catheter, due to pressure on prostatic cells,   - artefactually in a haemolysed specimen, or  following rectal examination, acute retention  of urine or passage of a catheter,   - some cases of metastatic bone disease, especially  with osteosclerotic lesions,   The assay is not of major diagnostic value in these  conditions, although there is current research looking  at the potential use of bone-derived ACP as a marker of  osteoclastic activity in osteoporosis.   y-Glutamyl transferase occurs mainly in the cells of  liver, kidneys, pancreas and prostate. Plasma GGT  activity is usually higher in males than in females.   • Induction of enzyme synthesis, without cell damage,  by drugs or alcohol. Many drugs (most commonly  the anticonvulsants, phenobarbital and phenytoin)  and alcohol induce proliferation of the endoplasmic  reticulum.   • Cholestatic liver disease, when changes in GGT  activity usually parallel those of ALP. In the  cholestatic jaundice of pregnancy, plasma GGT  activities may not increase.   • Hepatocellular damage, such as that due to infectious  hepatitis; measurement of plasma aminotransferase  activities is a more sensitive indicator of such  conditions.   Slightly raised activities (up to about two to three  times URL) are particularly difficult to interpret. Very  high plasma GGT activities, out of proportion to those  of the aminotransferases, may be due to:   A patient should never be labelled an alcoholic  because of a high plasma GGT activity alone. (For a  fuller description of liver disease see Chapter 17.)   A 23 -year-old woman had her wisdom teeth removed  under general anaesthesia, with suxamethonium  as an inducing agent. Post-operatively she required  prolonged mechanical ventilation for 4h. Some of  her laboratory test results were as follows:   The patient has had an anaesthetic reaction.  Although the plasma cholinesterase activity is within  the reference range, further cholinesterase studies  showed low dibucaine and fluoride numbers. The  data are suggestive of a variant cholinesterase (UA).  This metabolizes suxamethonium more slowly than  the ‘normal’ cholinesterase enzyme, resulting in  prolonged post-operative apnoea.   • Ingestion, or absorption through the skin, of such  anticholinesterases as organophosphates.   The muscle relaxants suxamethonium (succinyl  choline, ‘scoline’) and mivacurium are usually broken  down by plasma cholinesterase, and this limits the  duration of their action. Giving these agents to patients  with a low cholinesterase activity (usually due to an  enzyme variant) may result in a prolonged period of  apnoea (‘scoline apnoea’); such patients may need  prolonged ventilatory support after an operation.   The abnormal cholinesterase variants may be  classified by measuring the percentage inhibition of the  enzyme activity by dibucaine (dibucaine number) or by  fluoride (fluoride number). A low dibucaine number  and/or fluoride number with or without low plasma  activity is suggestive of a cholinesterase variant, for  example A, K, J, F types. There is also a silent gene type.   The identification of patients susceptible to  suxamethonium, and of their affected relatives, is  important. Close blood relatives should be traced and  investigated to identify their genotype, and so to predict  the chance of future anaesthetic risk. All affected  individuals should carry a warning card or a ‘Medic  Alert’ bracelet and should notify the anaesthetist if they  require an anaesthetic.   Reduced red cell acetylcholinesterase activity can be  useful as a measure of exposure to organophosphates,  for example certain pesticides, and this activity can be  used to monitor people at risk of organophosphate  exposure.   Angiotensin-converting enzyme  Angiotensin-converting enzyme (ACE) is a dipeptidyl  carboxypeptidase and cleaves dipeptides from the  free carboxy end of various polypeptides, including   angiotensin I and bradykinin. One of its actions is  releasing angiotensin II by cleaving the dipeptide  histidyl-leucine from angiotensin I. The major site of  ACE production is the pulmonary endothelium.   This enzyme can be measured in plasma as a marker  of disease activity in sarcoidosis. It can be elevated in  other lung conditions and therefore is not specific for  sarcoidosis and has limited diagnostic value. However,  high plasma ACE activity may decline on treatment of  sarcoidosis with steroids.   Classic allergy such as bronchospasm and urticaria  generally produces allergen-specific IgE to certain  agents, with IgE-producing mast-cell degranulation.   Tryptase is a serine protease found in mast cells. In  cases of mast-cell degranulation, as occurs in systemic  anaphylaxis, tryptase levels rise within 1 h and remain  elevated for 4-6 h. In patients with life-threatening  systemic reactions to diverse allergens (such as  venoms, latex and drugs), the finding of elevated  plasma tryptase levels (more than 1 pg/L) between 1  and 6h after the event is highly sensitive and specific  for confirming mast-cell degranulation as the cause of  the event.   Plasma enzyme changes in liver disease are discussed  in Chapter 17 and are important in the diagnosis of  various liver disorders.   In the muscular dystrophies, for example Duchenne’s,  plasma levels of the muscle enzyme CK (isoenzyme  CK-MM) and also of LDH and the aminotransferases  (mainly AST) are increased as a result of leakage from  the diseased cells. Results of plasma CK estimation  are more specific than LDH and AST for muscle  disease.   Points to consider in the interpretation of CK levels  in Duchenne’s muscular dystrophy are:   • Activities are highest (may be greater than 5-10  times the URL) in the early stages of the disease;  later, when much of the muscle has wasted, they are  lower and may even be normal.   • Activities are higher following muscular activity  immediately after rest (because of the release of CK   The clinical diagnosis of the Duchenne type of  muscular dystrophy can be confirmed by measuring  the plasma CK activity, examining a muscle biopsy  specimen and by deoxyribonucleic acid (DNA) analysis  in the majority of cases (see Chapter 30).   Carriers of the disorder can often be detected by  DNA analysis of the dystrophin gene and by finding  raised plasma CK activities. The rise is only moderate,  and non-specific causes of raised enzyme activities  must be excluded.   Less marked changes in plasma CK, LDH and AST  are found in some patients with the other muscle  disorders described above.   Plasma total enzyme activities may be raised, or an  abnormal isoenzyme detected, in several neoplastic  disorders:   • Malignancies may be associated with a non-  specific increase in plasma LDH and, occasionally,  aminotransferase activity.   • Plasma aminotransferase and ALP estimations may  be of value to monitor the treatment of malignant  disease. Raised ALP levels may indicate secondary  deposits in liver or in bone. Liver deposits may  also cause an increase in plasma LDH or GGT  concentration.   • Tumours occasionally produce a number of enzymes,  some of which are placental or placental-like ALP   (Regan or Nago isoenzymes), LDH t (HBD) or  CK-BB, assays of which may be used as an aid to  diagnosis or for monitoring treatment.   • Although plasma prostatic (tartrate-labile) ACP  activity rises in some cases of malignancy of the  prostate gland, plasma PSA determination has now  essentially replaced its clinical use.   Very high activities of LDH may be found in  megaloblastic anaemias and leukaemias and in other  conditions in which bone marrow activity is abnormal.  Typically there is much less change in the plasma AST  than in the LDH activities. Severe in vivo haemolysis  produces changes in both AST and LDH activities,  which mimic those of myocardial infarction.   The diagnosis of a myocardial infarction is usually made  on the clinical presentation and electrocardiographic  findings and confirmed by the characteristic changes  in plasma enzyme activities or troponins. The latter  now have superseded enzyme analysis in this context  and enzymes are rarely measured (see Chapter 22).   It should be evident that enzymology is important  in diagnostic processes. (The reader is also referred to  Chapter 16 on gastrointestinal and pancreatic disorders,  Chapter 17 concerning liver function tests, Chapter 22  on cardiovascular chemical pathology and Chapter 24  on tumour markers.) Enzymes can also be measured  in other body fluids, such as faeces, that is, elastase (see  Chapter 16), and pleural fluid, for example adenosine  deaminase (see Chapter 23).   • Enzymes can be measured in body fluids,  mainly plasma, to aid the clinical diagnosis and  management of certain conditions. This is called  clinical enzymology.   • Enzymes may exist as isoenzymes (molecular variants  of enzymes), which may be present in different  tissues. Examples are ALP (hepatic, bone, intestinal  and placental isoenzymes) and CK (striated muscle,  MM; brain, BB; and cardiac muscle, MB).   Certain aminotransferases (AST and ALT) and also  GGT can be used in the investigation of hepatic  disease.   Clinical enzymology has limitations, as generally  plasma enzyme activity lacks specificity. Isoenzyme  determination or measuring a number of enzymes  may increase diagnostic accuracy.   Some enzymes may complex with larger molecules,  such as immunoglobulins, forming macroenzymes.   Plasma albumin and total protein are often included  as part of a biochemistry department’s assay profile,  and other more specialized plasma proteins such as  C-reactive protein (CRP) may also be measured in  certain circumstances. There are also a number of  clinical conditions that involve abnormalities of plasma  and urinary proteins, some of which are discussed in  this chapter.   The amount of protein in the vascular compartment  depends on the balance between the rates of synthesis and  catabolism or loss. However, also important is the relative  distribution between the intravascular and extravascular  compartments, as the concentration depends on the  relative amounts of protein and water in the vascular  compartment. Therefore, abnormal concentrations do  not necessarily reflect defects in protein metabolism.   Hepatocytes synthesize many plasma proteins; those of  the complement system are also made by macrophages.  Immunoglobulins are mainly derived from the B  lymphocytes of the immune system.   Most plasma proteins are taken up by pinocytosis into  capillary endothelial cells or mononuclear phagocytes,  where they are catabolized. Small-molecular-weight  proteins are lost passively through the renal glomeruli  and intestinal wall. Some are reabsorbed, either directly  by renal tubular cells or after digestion in the intestinal  lumen; others are catabolized by renal tubular cells.   Functions of plasma proteins  Peptide hormones and blood clotting factors contribute  quantitatively relatively small, but physiologically  important, amounts of plasma protein. Some plasma  proteins originate from cell breakdown.   Control of extracellular fluid distribution  The distribution of water between the intravascular and  extravascular compartments is influenced by the colloid  osmotic effect of plasma proteins, predominantly  albumin (see Chapter 2).   Albumin and specific binding proteins transport  hormones, vitamins, lipids, bilirubin, calcium, trace  metals and drugs. Combination with protein allows  poorly water-soluble substances to be transported  in plasma. The protein-bound fraction of many of  these substances is physiologically inactive, unlike the  unbound fraction.   Inflammatory response and control of infection  The immunoglobulins and the complement proteins  form part of the immune system and the latter,  together with a group of proteins known as acute-  phase reactants, for example CRP, are involved in the  inflammatory response.   Methods of assessing plasma proteins  Plasma proteins may be expressed either as  concentrations (for example g/L) or as activities of those  proteins that have defined functions, such as clotting  times for prothrombin or enzyme activity (see Chapter  18). The distinction is important when abnormal  forms of protein are present at normal concentration  but with impaired function, such as Cl inhibitor (see  Classic pathway). Laboratories are able to measure total  serum protein concentration, assay specific proteins  and also identity and quantify serum proteins by serum  electrophoresis.   Acute changes in proteins are often due to loss from, or  gain by, the vascular compartment of protein-free fluid,  rather than of protein. Only marked changes of major   constituents, such as albumin and immunoglobulins, are  likely to alter total protein concentrations significantly.  The acute-phase response can result in rapid albumin  extravasation and thus hypoalbuminaemia.   Plasma total protein concentrations may be  misleading for other reasons. They may be normal in  the presence of quite marked changes in the constituent  proteins. For example, a fall in plasma albumin  concentration may be approximately balanced by a  rise in immunoglobulin concentrations. Also, most  individual proteins apart from albumin contribute  little to the total protein concentration; therefore, quite  a large percentage change in the concentration of one  may not cause a detectable change in the total protein  concentration.   Raised plasma total protein concentrations may  be due to loss of protein-free fluid, or excessive stasis  during venepuncture, or a major increase in the  concentration of one or more of the immunoglobulins,  including paraproteins. The difference between serum  total protein and albumin concentration is termed  the serum globulin concentration. Low plasma total   protein concentrations may be due to dilution, for  example if blood is taken near the site of an intravenous  saline infusion, hypoalbuminaemia or severe  immunoglobulin deficiency.   Electrophoresis is a technique that separates  compounds such as proteins according to their  different electrical charges. It is usually performed by  applying a small amount of serum to a strip of cellulose  acetate or agarose and passing a current across it for a  standard time. In this way, five main groups of proteins  - namely albumin and the a , CX 2 , P and y globulins -  may be distinguished after protein staining and may  be visually compared with those in a normal control  serum. Each of the globulin fractions contains several  proteins (Fig. 19.1).   The following description applies to the normal  appearance, in adults, of the principal bands seen after  electrophoresis on cellulose acetate:    Figure 19.1 The normal serum electrophoretic pattern. In this example the globulin has separated into p 2 and p 2  fractions. This finding is not uncommon, especially in stored specimens.   • Albumin, usually a single protein, makes up the most  obvious band.   • cx 2 -globulins consist mainly of a, -macroglobulin and  haptoglobin.   • |3 - globulins often separate into two; |3 consists  mainly of transferrin with a contribution from low-  density lipoprotein (LDL), and |i, consists of the C3  component of complement.   • Y -globulins are immunoglobulins; some immuno-  globulins are also found in the a, and |3 regions.   If plasma rather than serum is used, fibrinogen  appears as a distinct band in the (3— y region. This  may make interpretation difficult. Blood should be  allowed to clot and serum used, rather than plasma, if  electrophoresis is to be performed.   Some abnormal electrophoretic patterns are  characteristic of a particular disorder or group of  related disorders, while others indicate non-specific  pathological processes. For example, the oq band, which  contains haptoglobin, may be reduced if there is in vivo  haemolysis and may split into two if in vitro haemolysis  has occurred.   Reduction may occur in severe undernutrition,  sometimes due to malabsorption, unless accompanied  by infection and haemodilution. An increase may occur  in haemoconcentration.   Tissue damage usually triggers the sequence of  biochemical and cellular events associated with  inflammation. The biochemical changes include the  stimulation of synthesis of the so-called acute-phase  proteins, with a rise in the oq-globulin and oq-globulin  fractions. The plasma concentrations of these proteins  reflect the activity of the inflammatory response,  and their presence is responsible for the rise in the  erythrocyte sedimentation rate (ESR) and increased  plasma viscosity characteristic of such a response.   In chronic inflammation, the usual increase in  immunoglobulin synthesis may be visible as a diffuse  rise in y-globulin. If there is an active inflammatory  reaction, the increased density in the y-globulin region  is associated with an increase in the oq and oq fractions  of the acute-phase response.   The changes in the concentrations of plasma proteins  in liver disease are discussed in Chapter 17. They are  usually ‘non-specific’, but in cirrhosis a characteristic  pattern is sometimes seen. Albumin and often oq-  globulin concentrations are reduced and the y-globulin  concentration is markedly raised, with apparent fusion  or ‘bridging’ of the |3 and y bands because of an increase  in plasma IgA concentrations.   Plasma protein changes depend on the severity of  the renal lesion. In early cases, a low plasma albumin  concentration may be the only abnormality, but the  typical pattern in established cases is reduced albumin,  cq -globulin and sometimes y-globulin bands and an  increase in CX, -globulin concentration due to a relative  or absolute increase in the high-molecular-weight cq-  macroglobulin. If the syndrome is due to conditions  such as systemic lupus erythematosus (SLE), the  y-globulin concentration may be normal or raised.   The cx band consists almost entirely of cq -antitrypsin  and its absence or an obvious reduction in its density   suggests oq-antitrypsin deficiency. Occasionally, cq-  antitrypsin variants may present with a split cq band.   Hypogammaglobulinaemia shows reduced plasma  y-globulins and is discussed in the sections on  immunoglobulins.   Multiple myeloma and paraproteinaemia may show  a band or bands on the electrophoretic strip and are  also discussed later.   Albumin, with a molecular weight of about 65kDa, is  synthesized by the liver. It has a normal plasma biological  half-life of about 20 days. About 60 per cent in the  extracellular fluid is in the interstitial compartment.  However, the concentration of albumin in the smaller  intravascular compartment is much higher because of  the relative impermeability of the blood vessel wall. This  concentration gradient across the capillary membrane is  important in maintaining plasma volume (see Chapter 2) .   • the bisalbuminaemias, in which two forms of  albumin are present; there are usually no clinical  consequences,   • analbuminaemia, in which there is deficient synthesis  of the protein; clinical consequences are slight, and  oedema, although present, is surprisingly mild.   An abnormally high plasma albumin concentration  is found only artefactually in a sample taken with  prolonged venous stasis or after loss of protein-free fluid.   A low plasma albumin concentration may be due to  dilution or to redistribution. True albumin deficiency  may be caused by a decreased rate of synthesis, or by an  increased rate of catabolism or loss from the body.   Dilutional hypoalbuminaemia may, as in the case of  total protein, result from artefactual changes due to  taking blood from the arm into which an infusion is  flowing, administration of an excess of protein-free  fluid or fluid retention, usually in oedematous states or  during late pregnancy.   Redistribution of albumin from plasma into the  interstitial fluid space results from:   • Recumbency: plasma albumin concentrations maybe  5-10 g/L lower in the recumbent than in the upright  position because of the redistribution of fluid.   • Increased capillary membrane permeability: this is the  usual cause of the rapid fall in plasma concentration  found in many circumstances, for example post-  operatively and in most illnesses.   The slight fall in plasma albumin concentration  found in mild illness may be due to a combination of  the above two factors.   Normally, about 4 per cent of the body albumin is  replaced each day. Hepatic impairment, such as cirrhosis,  causes hypoalbuminaemia if the rate of synthesis of new  amino acids is inadequate to replace those deaminated  during metabolism; most of the amino nitrogen is then  lost as urinary urea. Hypoalbuminaemia may therefore  be due to:   • malabsorption, resulting in impaired absorption of  dietary peptides and amino acids,   • impairment of synthesis, due to chronic liver  dysfunction, for example cirrhosis.   Because of its relatively lowmolecular weight, significant  amounts of albumin and other low-molecular-weight  proteins are lost in conditions associated with increased  membrane permeability. The plasma concentration of  albumin is higher than that of the other proteins and its  loss is therefore more obvious. Albumin loss may occur  through:   • the skin, because of extensive burns or skin diseases  such as psoriasis; a large part of the interstitial fluid  is subcutaneous,   Catabolism (and therefore nitrogen loss) is increased in  many illnesses, including sepsis and hyperthyroidism. This  may worsen the hypoalbuminaemia due to other causes.   • Fluid distribution Albumin is quantitatively the  most important protein contributing to the plasma   colloid osmotic pressure. Oedema may occur in  severe hypoalbuminaemia (see Chapter 2).   • Binding functions Albumin binds calcium, bilirubin  and free fatty acids. (See Chapter 6 for a discussion  of albumin-adjusted calcium.)   A number of drugs, such as salicylates, penicillin  and sulphonamides, may become albumin bound.  The albumin-bound fractions are physiologically and  pharmacologically inactive. A marked reduction in  plasma albumin, by reducing the binding capacity,  may increase the plasma free concentration of those  substances leading to toxic effects.   Albumin-bound drugs may, if administered  together, compete for binding sites, thus increasing free  concentrations; an example of this is the simultaneous  administration of salicylates and the anticoagulant  warfarin, thus potentiating the effect of the latter (see  Chapter 25).   The acute-phase response  The body responds to tissue damage and to the presence  of infecting organisms or other foreign substances by  a complex, inter-related series of cellular and humoral  responses. These act together to initiate and control  the inflammatory reaction and so to remove damaged  tissues and foreign substances. Defence mechanisms  can be separated into two main groups.   • The inflammatory response: this is non-specific,  requires changes in the permeability of membranes  and depends on humoral factors such as acute-  phase reactants, complement or cytokines released  in response to inflammation and on a phagocytic  cellular response dependent on the function of  polymorphonuclear leucocytes and monocytes.   • The immune response: in this type of response, cellular  or humoral factors act on specific foreign organisms.  It depends on the function of lymphocytes, which  can be differentiated into:   - B-lymphocytes, activated by antigens and by  lymphokines, which develop into plasma cells  in the bone marrow and synthesize and secrete  immunoglobulins.   - T-lymphocytes, which, in the thymus, acquire  a range of surface antigens that play a vital  role in T-cell function after migration to other  lymphoid tissue. These include cytotoxicity  and delayed hypersensitivity. They also have  a regulatory function as either helper or  suppressor cells. Each group can be further   subdivided using antibodies against the cell  surface markers; this is known as cluster  differentiation (Fig. 19.3).   The inflammatory response and the ability to kill  foreign organisms may be impaired if there is deficiency  of either cellular or humoral components.   The innate or natural immune system, which is  phylogenetically older than so-called acquired or adaptive  immunity, is a rapid first-line defence mechanism based  on non-lymphoid tissue components. The acute-phase  response is part of this system and results in pronounced  changes in the concentration of plasma proteins in  response to a variety of stresses, including infection,  tissue injury and inflammation. The concentration of  some of these proteins increases (positive acute-phase  proteins), such as CRP, fibrinogen and plasma amyloid  A, whereas that of others decreases (negative acute-phase  proteins), such as albumin and transferrin (Fig. 19.4.)   IFN, interferon; IL, interleukin; TNF, tumour necrosis  factor. Reproduced with kind permission from Candlish  JK and Crook M. Notes on Clinical Biochemistry.  Singapore: World Scientific Publishing, 1993.    Figure 19.4 Some proteins of the acute-phase response.  A, albumin; CRP, C-reactive protein; HG, haptoglobin;   T, transthyretin (previously called pre-albumin). The  latter pair can be termed negative acute-phase proteins.  Reproduced with kind permission from Candlish JK and  Crook M. Notes on Clinical Biochemistry. Singapore:  World Scientific Publishing, 1993.   The non-specific changes in plasma protein  concentrations occurring in response to acute or  chronic tissue damage are caused by increased protein  synthesis in the liver in response to peptide mediators  or cytokines.   Cytokines regulate the immune and inflammatory  responses. They are classified, depending on their  principal biological functions, into groups such as inter-  leukins (ILs), interferons (IFNs) and tumour necrosis  factors (TNFs). They activate receptors on adjacent cells  (paracrine) or on the same cell (autocrine), but those  involved in the inflammatory response, such as IT-1,  IL-6 and TNF-cz, affect distant (endocrine) organs.   • Activators of other inflammatory pathways  such as CRP, so called because it reacts with the  C-polysaccharide of pneumococci. This protein  combines with bacterial polysaccharides or phos-  pholipids released from damaged tissue to become  an activator of the complement pathway. Plasma  concentrations of CRP rise rapidly in response to  acute inflammation and its assay is particularly  useful in the early detection of acute infection.   • Inhibitors, such as Otj- antitrypsin, which control the  inflammatory response and so minimize damage to  host tissue.   • Scavengers, such as haptoglobin, which binds  haemoglobin released by local in vivo haemolysis  during the inflammatory response.   The concentrations of plasma fibrinogen and of  several complement components also increase. An  acute-phase response increases blood viscosity and  therefore the ESR. The plasma albumin concentration  falls and secondary decreases of complement and  haptoglobin occur if they are used up excessively.  Haptoglobin maybe undetectable if much haemoglobin  is released from red cells due to intravascular haemolysis  or haemorrhage into tissues.   The non-specific nature of the response means that  the measurement of individual acute-phase proteins is  rarely helpful as an aid to diagnosis. The demonstration  of a rise in plasma CRP concentration is more sensitive  and specific than measurement of the ESR and is most  often raised in bacterial infections and in other acute  inflammatory conditions (Fig. 19.4).   • To detect an infection Plasma CRP concentrations  are not usually increased in viral infections, but the  highest levels are seen in bacterial infections. It has  a particular place in diagnosing neonatal infections.  In immunosuppressed patients, the usual systemic  response to infection such as neutrophil leucocytosis  and fever may not be present, and therefore a raised  plasma CRP concentration of greater than lOmg/E  suggests infection.   • As a guide to the severity of connective tissue disease  activity Such conditions include rheumatoid  arthritis, juvenile chronic polyarthritis (Still’s  disease) and polymyalgia rheumatica. Systemic lupus  erythematosus shows less of a CRP rise, but this may  be useful to distinguish infection in SLE from an  acute disease flare-up (plasma concentrations more  than 60mg/L indicate the former).   • In the diagnosis of inflammatory bowel disease Plasma  CRP levels more than 50 mg/L are more indicative of  Crohn’s disease than ulcerative colitis.   • As an indicator of cardiovascular risk Raised plasma  CRP concentrations using a high-sensitivity assay  are a reputed cardiovascular risk factor (see Chapter  22), as subclinical inflammatory processes have  been implicated in atherosclerosis. Plasma levels  less than 1 mg/L constitute low cardiovascular risk;  patients with levels more than 3 mg/L are at high  risk of cardiovascular disease. Owing to problems   of biological variation at these low concentrations  repeat determinations are useful.   Procalcitonin is a 14-kDa protein encoded by the  Calcl gene. Blood levels of procalcitonin are increased  in systemic inflammation and procalcitonin has been  proposed as a possibly better marker of bacterial sepsis  than CRP.   Macrophages and hepatocytes synthesize a group of  proteins called the complement system, which is part  of the innate immune system. Sequential activation  reduces complement plasma concentrations, but in  many instances this is compensated for by increased  synthesis of acute-phase reactants. The products  of activation attract phagocytes to the area of   inflammation (chemotaxis), which, by increasing the  permeability of the capillary wall to both cellular and  chemical components, allows them to reach affected  cells. Activation of the complete complement pathway  on foreign cell surfaces results in their lysis and, together  with immunoglobulins, some of the complement  components act as opsonins, which enhance  phagocytosis. Because of inhibitors, complement  usually circulates in an inactive form.   Clinically, the most important of the complement  proteins is C3. Two main pathways are concerned with  its activation (Fig. 19.5). Activation of either results in  low plasma C3 concentrations.   In the classic pathway, C3 is activated by the products  of a pathway initiated by the activation of Cl, usually    Figure 19.5 The complement pathway, much simplified. Ag, antigen; Ab, antibody; IgA, immunoglobulin A.   by antigen-bound IgG or IgM (immune complexes),  by protein within the cell walls of many strains of  Staphylococcus aureus, or by CRP. C4 (and C2) are used  during the resultant sequence of events, which also  activates the alternative pathway via C3; plasma C3 and  C4 concentrations fall. Once the formation of immune  complexes stops, plasma C3 and C4 concentrations are  restored. The enzyme Clq esterase inhibits the serine  protease Cl (which is composed of Clq, Clr and Cls).   In the alternative pathway, which is the more important  of the two, C3 is activated by IgA immune complexes,  and substances such as lipopolysaccharides and  peptidoglycans in the walls of certain bacteria and  viruses, factor B and properdin are involved. C3b  is formed, which in turn activates more C3. Other  products of C3b cause vasodilatation and cell lysis.  The cyclical process is self-perpetuating and plasma C3  concentrations fall. If the initial stimulus is removed,  inhibitors such as those formed in the acute-phase  reaction control the reaction and C3 concentrations  return to normal; factors H and I inhibit the enzyme C3  convertase. The alternative pathway is most important  in the absence of preformed antibodies.   Recently, a third pathway of C3 activation has been  described involving mannose-binding lectin, one of  the colectin proteins. After C3 activation, the terminal  complement system consists of other activated proteins  such as C5-9, collectively known as the membrane  attack complex. This is involved in puncturing cell  membranes.   In immune-complex disease, such as SLE, circulating  immune complexes persist. The products of C3, released  by continued activation of the classic pathway, may  damage blood vessels, joints and the kidneys. Plasma  concentrations of both C3 and C4 are low. Persistently  low plasma C3 concentrations may help to distinguish  chronic mesangiocapillary glomerulonephritis, with a  poor prognosis, from the less serious and self-limiting  acute post-streptococcal glomerulonephritis, in which  plasma C3 concentrations return to normal within a  few months.   Two types of phagocytic cells help to combat infection.  Polymorphonuclear neutrophil leucocytes migrate into  sites of acute inflammation and represent a non-  specific first line of defence against pyogenic bacteria.  Mononuclear phagocytes circulate in blood as monocytes.   They migrate to sites of chronic inflammation, where  they develop into macrophages. Under T-cell control,  macrophages are activated and develop a greatly  enhanced ability to phagocytose and digest pathogenic  organisms.   The most important functions of T cells in the  inflammatory response are the initiation and mainten-  ance of chronic cellular inflammatory responses  to invading organisms and the control of immune  inflammation by providing helper and suppressor  influences on other cells. B cells need the co-operation  of T cells (helper cells) to make antibodies to complex  antigens. T-cell modulation of inflammatory and  immune responses is mediated by chemical messengers  called lymphokines. T cells also have a cytotoxic  function directed towards destroying the body’s own  cells infected by intracellular pathogens such as viruses.   Immunoglobulins, synthesized by B lymphocytes, are  incorporated into cell membranes, where they act as  antigen receptors. On exposure to a specific antigen  in the presence of T-helper cells, these B lymphocytes  proliferate and differentiate into plasma cells  synthesizing and secreting immunoglobulins.   The basic immunoglobulin is a Y-shaped molecule  (depicted schematically in Fig. 19.6).   Usually four polypeptide chains are linked by  disulphide bonds. There are two heavy (H) and two light  (L) chains in each unit. The H chains in a single unit are  identical and determine the immunoglobulin class of  the protein. Heavy chains y, a, p, 5 and £ occur in IgG,  IgA, IgM, IgD and IgE respectively. The L chains are of  two types, K and X. In a single molecule, the L chains are    Figure 19.6 Diagram of an immunoglobulin monomer.  L, light; H, heavy.   of the same type, although the immunoglobulin class as  a whole contains both types.   There are two antigen-combining sites per  monomer, together known as the F(ab) 2 piece. These  lie at the ends of the arms of the Y; both H and L chains  are necessary for full antibody activity. The amino acid  composition of this part of the chain varies in different  units (variable region). When the F(ab) 2 site combines  with antigen, conformational changes are transmitted  through the hinge region of the heavy chain, and the Fc  segment of the molecule becomes activated and reacts  with Fc receptors on a number of immune cell types  resulting in immune activation.   The rest of the H and T chains is less variable  (constant region, which includes the Fc segment). The  activated Fc segments of the H chains are responsible  for such properties as the ability to bind complement or  actively to cross the placental membrane. The H chains  are associated with a variable amount of carbohydrate;  IgM has the highest content.   Some immunoglobulin molecules contain more than  one basic unit bound together by ‘J’ chains; for example,  the IgM molecule consists of five units. Because of the  variation in size and therefore in density, the classes can  be separated by ultracentrifugation. They are classified  by their sedimentation coefficient (Svedberg units, S),  the S value of a protein increasing with increasing size.  The S values of immunoglobulins, together with their  most important functions and properties, are shown  in Table 19.1. However, now with the development  of reliable immunoassays, ultracentrifugation is  not used in routine clinical practice to quantify  immunoglobulins.   In response to infection, each plasma cell produces an  immunoglobulin of a single class.   Immunoglobulin G concentrations rise slightly later  in response to soluble antigens such as bacterial toxins.  Owing to their relatively low molecular weight, they can  diffuse into the interstitial fluid to fight against tissue  infection. Within a few weeks of the initial infection,  raised plasma concentrations of all immunoglobulins  may be demonstrated and may be detectable as a  diffusely increased y zone on the electrophoretic strip.  There are four subclasses (IgG 1-4) in humans.   Immunoglobulin M is synthesized first in response to  particulate antigens such as blood-borne organisms.  Because of the relatively large size of IgM, it mostly  remains in the vascular compartment. This, together  with the speed of synthetic response, makes it the  first line of defence among immunoglobulins against  invading organisms. The fetus can synthesize IgM, and  high plasma concentrations at birth usually indicate  that there has been an intrauterine infection.   Immunoglobulin A is synthesized predominantly  submucosally and is present in intestinal and  respiratory secretions, sweat, tears and colostrum. It is  affected more than other immunoglobulins in diseases  of the gastrointestinal and respiratory tracts. Secretory  IgA is a dimer, in which two subunits are joined by a   Table 19.1 The properties and functions of the plasma immunoglobulins (Ig)   Infections, particularly if chronic, activate B  lymphocytes and cause a polyclonal immunoglobulin  response. However, immunoglobulin estimation adds  little to the observation of increased y-globulin visible  in the serum electrophoretic pattern.   In certain conditions (some of which are listed  in Table 19.2), one or more immunoglobulin class  predominates. Although there is considerable  overlap, individual immunoglobulin estimation may  occasionally help in the diagnosis of some of these  conditions.   Immunoglobulin E is synthesized by plasma cells  beneath the mucosae of the gastrointestinal and  respiratory tracts and in the lymphoid tissue of the  nasopharynx. Circulating IgE is rapidly bound to cell  surfaces, particularly those of mast cells and basophils;  plasma concentrations are therefore very low.   The combination of antigen with this cell-bound  antibody stimulates cells to release biologically  active mediators and accounts for such immediate  hypersensitivity reactions as occur in hay fever, the most  severe form of hypersensitivity being anaphylactic shock.   Raised plasma IgE concentrations are found in  several diseases with an allergic (atopic) component,  such as some cases of eczema, asthma due to allergens  (‘extrinsic’) and parasitic infestation (Table 19.3).   The diagnosis of an allergic disorder is usually made  from the history, but skin testing and the measurement  of plasma specific IgE concentrations may be needed  as total serum IgE concentrations are relatively  insensitive for allergy diagnosis. In skin testing, a small  drop of dilute allergen is injected subcutaneously;  a positive ‘wheal and flare’ develops within a few  minutes (immediate hypersensitivity). This test is  contraindicated in patients with a history of eczema or  anaphylaxis, and plasma IgE may be assessed to detect  specific allergens.   Deficiencies of components of the  inflammatory response and of proteins  of the immune system   Deficiencies of the inflammatory or immune response  of the host defence system may result in an increased  risk of infection and lack of immune regulation. Plasma  immunoglobulins reflect only the humoral phase of  the immune system; their measurement does not assess  cellular immunity. Normal plasma immunoglobulin  concentrations do not exclude an immune deficiency  state; they may, in fact, be raised, as in the acquired  immunodeficiency syndrome (AIDS). The human  immunodeficiency virus (HIV) binds to CD4 cells and  becomes internalized.   As a general rule, any patient with a severe, protracted,  unusual or recurrent infection should be investigated  for an underlying deficiency in ‘defence’ mechanisms.   Dysgammaglobulinaemia is defined as an abnormality  in the structure or distribution in the class(es) of  immunoglobulins.   Immunoglobulin subclass determination is rarely  needed clinically. However, IgG subclass deficiency may  be associated with certain recurrent respiratory tract  infections.   Hypogammaglobulinaemia can be defined as a decrease  in plasma immunoglobulins. Total plasma protein  concentration is usually low. A marked reduction  in plasma immunoglobulin concentrations may be  detectable as obvious hypogammaglobulinaemia in the  serum electrophoretic pattern, but usually measurement  of individual proteins is needed to make a diagnosis. The  effects of deficiencies of individual immunoglobulins  are related to their functions and distribution:   • IgA deficiency may be symptomless, or may be  associated with recurrent, mild respiratory tract  infections or intestinal diseases.   • IgG deficiency may result in recurrent pyogenic  infections of tissue spaces, especially in the lungs  and skin, by toxin-producing organisms such as  staphylococci and streptococci.   Primary immunoglobulin deficiency is less common  than that which is secondary to other disorders. Various  classifications have been proposed for these deficiencies,  some of which are discussed below.   In the newborn infant, circulating IgG is derived  from the mother by active placental transfer. Plasma  concentrations decrease over the first 3-6 months of  life and then gradually rise as endogenous IgG synthesis  increases. In some infants, onset of synthesis is delayed  and ‘physiological hypogammaglobulinaemia’ may  persist for several months.   During pregnancy, most of the IgG transfer across  the placenta takes place in the last 3 months. Severe  deficiency may therefore develop in very premature  babies because the concentration of IgG derived from  the mother falls before the endogenous concentration  rises.   Primary IgA deficiency in plasma, saliva and other  secretions is relatively common, with an incidence  of about 1 in 500 of the population. Most cases are  symptom free.   Several rare syndromes, usually familial,  have been described. In infantile sex-linked  agammaglobulinaemia (Bruton’s disease), which  occurs only in males, there is almost complete  absence of B- cells and circulating immunoglobulins,  while cellular (T-cell) immunity is normal. Clinical  consequences are increased susceptibility to various  infections, commonly of the respiratory tract. Other  syndromes have varying degrees of immunoglobulin  deficiency and impaired cellular immunity and can  occur in either sex.   Low plasma immunoglobulin concentrations are  most common in patients with malignant disease,  particularly of the haemopoietic and immune  systems, and often precipitated by chemotherapy or  radiotherapy; they are an almost invariable finding  in patients with myelomatosis (immune paresis). In  severe protein-losing states, such as the nephrotic  syndrome or protein-losing enteropathy, low plasma  immunoglobulin concentrations (especially of IgG) are  due partly to the loss of relatively low-molecular-weight  immunoglobulins and partly to increased catabolism.  Other causes are shown in Box 19.1.   Patients with T-cell deficiency present with severe,  protracted infections caused by viruses, fungi or  rarely detected organisms such as Pneumocystis carinii  and Cryptococcus. It is important to identify T-cell  deficiencies, because the administration of live vaccines   Malignancy, including chronic lymphocytic leukaemia,  lymphomas, paraproteinaemia  Chronic kidney disease  Chronic infections   and blood products is then contraindicated. T-cell  deficiency may be classified as:   • primary (congenital) deficiencies, which are rare,  often severe and usually present soon after birth;  they range from total or partial T-cell depletion to  loss of function,   • secondary (acquired) deficiencies, associated with  metabolic disorders such as diabetes mellitus,  undernutrition, drug abuse, auto-immunity,  malignancy and immunosuppressive treatment or  radiation; secondary T-cell deficiency is relatively  common, often mild and presents at any age.   In severe combined immunodeficiency (SCID)  syndrome, T-cell function is affected, often also with B-cell  abnormalities. This syndrome can be caused by a number  of genetic conditions that interfere with lymphocyte  development and function, including SCID-X1, a defect  of the y-chain of interleukin receptors, and adenosine  deaminase deficiency, which allows a lymphocyte  toxic build-up of adenosine diphosphate, guanosine  triphosphate and deoxyadenosine triphosphate.   T cells may be decreased in number or may function  abnormally. The total lymphocyte count maybe normal  despite severe T lymphocytopenia, such as occurs in  acquired deficiencies, or malignancy, as in chronic  lymphocytic leukaemia. T-cell function maybe assessed  by determining cell-surface markers and therefore  changes in the different T-cell subsets. Changes in  the ratio of helper to suppressor cells occur in many  conditions, but are profound in AIDS, in which the  virus specifically affects the CD4 cell population and  numbers can go below 200 cells/mm 3 .   Other proteins involved in complement activation or  as acute-phase reactants may be deficient in addition  to immunoglobulins. The most clinically significant are  discussed below.   Complement deficiencies, for example of C5-9, are  very rare, although they may predispose individuals  to infections, for example meningococcal. Cl, C2  and C4 deficiency are associated with SLE. Mannose-  binding lectin deficiency increases the risk of Neisseria  meningitidis and Streptococcus pneumoniae infection.  C5 deficiency may lead to Leiner’s disease, associated  with diarrhoea, seborrhoeic dermatitis and wasting.   In hereditary angioneurotic oedema, Cl esterase  inhibitor is deficient or of low activity. The condition  is characterized by episodes of increased capillary  permeability and consequent oedema of the  subcutaneous tissues and mucous membranes of the  upper respiratory or gastrointestinal tract. Laryngeal  oedema may be fatal. Plasma Cl esterase inhibitor  concentration or activity can be measured.   Complement deficiences can be assessed by  measuring individual complement components and  also CH50 (a measure of the complement activity).   oq-Antitrypsin normally controls the proteolytic action  of lysosomal enzymes from phagocytes (protease  inhibitors, Pis), and is an acute-phase reactant. Plasma  concentrations rise 2-3 days after trauma or acute  infection.   There are more than 50 genetic variants of oq-  antitrypsin, inherited as autosomal codominant  alleles. The most common allele is PIM with a genotype  MM. At least seven of the phenotypes associated with  low functional plasma a - antitrypsin concentrations,  the most important of which is PI null, are associated  with complete deficiency of the protein. Others  include PIS and PIZ, in which protein accumulates in  the liver because it cannot be secreted after synthesis,  and PIM Duarte, in which plasma concentrations  of the protein may be normal but there is deficient  functional activity.   cq-Antitrypsin deficiency may be suspected after  serum electrophoresis if the oq band is much reduced,  absent or split; the condition may present clinically  with the following:   • Hepatic cirrhosis, occurring in 10-20 per cent of  subjects, such as PIZ homozygotes, in whom the  protein cannot be secreted by hepatocytes. The  condition may present as hepatitis in the neonatal  period or as cirrhosis in children or young adults  (see Chapter 17).   • Pulmonary disease: the unopposed action of  proteases from phagocytes in the lung may, by  destroying elastic tissue, cause basal panlobular  emphysema in adults, aged between 30 and 40 years,  who are homozygous for the abnormal alleles. The  condition may be exacerbated by cigarette smoking,  air pollution or infection.   The diagnosis of oq- antitrypsin deficiency can  usually be confirmed by demonstrating that the   plasma (/^antitrypsin concentration is low; the  phenotype or genotype should then be identified.  Blood relations should be investigated and all those  with abnormal plasma concentrations should receive  genetic counselling and be advised not to smoke.   Each B-cell clone is highly specialized and synthesizes  a single class and type of immunoglobulin. The  normal B-cell response to an antigenic stimulus,  such as infection, is synthesis of a range of different  immunoglobulins by groups (clones) of cells. This is  known as a polyclonal response and causes a diffuse  hypergammaglobulinaemia in the electrophoretic  pattern. If one of these cells proliferates to form a clone  of like cells, a single protein will be produced in excess.  The monoclonal proliferation of B cells is often, but not  always, malignant.   The term paraprotein (or also called M protein) refers  to the appearance of an abnormal, narrow, dense band  on the electrophoretic strip. It is found most commonly  in the y region, but may be anywhere from the a, to  the y region. Paraproteins can often be shown to be  monoclonal.   Although the presence of a paraprotein is strongly  suggestive of a malignant process, it is not diagnostic.  Paraproteins may be found in the following malignant  conditions:   • myelomatosis, which accounts for most of the cases  of malignant paraproteinaemia,   The consequences of malignant B-cell proliferation  Some of the clinical and laboratory findings are similar  in all malignant B-cell tumours. Whether they occur  depends on the concentration of paraprotein, the  presence or absence of immune paresis and the presence  of Bence tones protein (BfP). However, malignant  tumours of B- cells can exist without all or, rarely, any  of these findings.   Unless the concentration of paraprotein is very high,  these findings may not be present. Very high plasma  concentrations, which are suggestive of malignancy,  may be associated with a very elevated ESR or CRP and  also with the following:   • In vivo effects of increased plasma viscosity with  sluggish blood flow in small vessels. This may result  in retinal vein thrombosis with impairment of vision,  cerebral thrombosis, or even peripheral gangrene  (hyperviscosity syndrome).   • An increased blood viscosity that may be noticeable  during venepuncture; it may also be difficult  to prepare blood films. Hyperviscosity is most  common in macroglobulinaemia but may occur in  myelomatosis.   • A high plasma total protein concentration despite  a normal or low plasma albumin concentration;  the difference between the total plasma protein  concentration and albumin concentration is termed  the globulin concentration.   • Spurious hyponatraemia (pseudohyponatraemia)  may occur due to the space-occupying effect of the  protein and the effect on indirect ion electrodes (see  Chapter 2).   • Bence Jones protein is usually, but not invariably,  found in the urine of patients with malignancy  of B- cells (Fig. 19.7); it can also be found in the  absence of malignant disease. It consists of free  monoclonal L chains, or fragments of them, which  have been synthesized much in excess of H chains,  implying a degree of dedifferentiation. Because of  its relatively low molecular weight (20-40 kDa), the  protein is filtered at glomeruli and only accumulates  in plasma if there is glomerular impairment or if it  polymerizes. It may damage renal tubular cells and  form large casts, producing the ‘myeloma kidney’. It  may also be a precursor for amyloid deposition in  tissues.   • Abnormal ratio of serum free K to free A, light chains.   The synthesis of immunoglobulins from other clones  of cells may be suppressed by the proliferation of  a single clone. In such cases the band, other than  the narrow paraprotein, is reduced or absent and  concentrations of the other immunoglobulins are low  (Fig. 19.7).   Figure 19.7 Serum and urinary protein electrophoretic  patterns in myelomatosis. Patient A with paraprotein  and immune paresis in serum and Bence Jones protein  (BJP) band in urine. Patient B with paraprotein and  immune paresis in serum and with heavy BJP on the  right and leakage of albumin and other low-molecular-  weight proteins (glomerular permeability) in urine.   • normochromic normocytic anaemia, a common  presenting feature of any malignant disease,   • haemorrhages, perhaps due to complexing of  coagulation factors by the paraprotein,   • Raynaud’s phenomenon, which may occur if the  paraprotein is a cryoglobulin,   (3,-Microglobulin is a low-molecular-weight protein  (11.8kDa) that forms part of the human lymphocyte  antigen on the surface of all nucleated cells. The  protein is readily filtered by glomeruli, and plasma  concentrations are normally low. In myelomatosis the  plasma -microglobulin concentration is an index  of the extent of the disease and of the prognosis.   Occasionally, raised plasma concentrations may occur  in a number of haematological malignancies. (3 n -  Microglobulin is a surrogate marker for the overall  body tumour burden.   Myelomatosis is caused by the malignant proliferation  of plasma cells throughout the bone marrow. The  condition becomes increasingly frequent after the age  of 50; it can occur before the age of 30, but is rare. The  sex incidence is about equal. The clinical features are  due to malignant proliferation of plasma cells and  disordered immunoglobulin synthesis and/or secretion  from the cell.   Bone pain may be severe and is due to pressure from  the proliferating cells. Radiographs may show discrete  punched-out areas of radiotranslucency, most frequently   A 72-year-old man presented to his general  practitioner with back pain and weakness. The  following are the results of some of his laboratory  tests:   Note the impaired renal function, hypercalcaemia,  hypoalbuminaemia and raised plasma globulins  and raised total plasma protein concentration.  The patient was also found to be anaemic, with a  haemoglobin concentration of 9.3g/dL. Dipstick  urine testing showed proteinuria. Serum and urinary  protein electrophoresis showed an IgG K-paraprotein  with immune paresis and Bence Jones proteinuria.  Skeletal bone survey showed lytic bone lesions, and  bone marrow biopsy supported the diagnosis of  multiple myeloma.   in the skull, vertebrae, ribs and pelvis. There may be  generalized osteoporosis. Pathological fractures may  occur. Histologically there is little osteoblastic activity  around the lesion, which arises in the marrow rather  than in the bone itself; consequently, the plasma alkaline  phosphatase activity is normal unless there is liver  involvement, in which case the raised level is of hepatic,  not bony, origin. A normal plasma alkaline phosphatase  activity, in cases with bone lesions, suggests myelomatosis  rather than bony metastases. Hypercalcaemia may occur  and also renal failure. The latter may occur as a result of  Bence Jones proteinuria as well as the hypercalcaemia.  Amyloidosis is also associated.   The immunoglobulin most frequently increased is IgG,  and, less commonly, IgA (about 2.5:1). Occasionally,  IgM, IgD or IgE is found, the last two being very rare.  Bence Jones protein may sometimes be present in  plasma if renal failure is present. In about 20 per cent  of cases, no paraprotein is detected in the plasma but  BJP is detected in urine. Rarely, neither a paraprotein  nor BJP can be found. In either case there is usually  immune paresis. In IgD myelomatosis an increase in  y-globulin concentration may not be detectable by  routine electrophoresis. The paraprotein should be  typed and the plasma concentration monitored to  follow progress. The serum free light chain ratio (k to  X) may also be abnormal. This ratio is normally about  0.26-1 .65 and can be useful in monitoring patients with  low levels of paraprotein (monoclonal or ‘M’ protein)  and of early treatment response and disease relapse.   The proportion of plasma cells in the bone marrow is  increased and many of these cells are atypical (‘myeloma  cells’). Examination of a bone marrow aspirate must be  carried out before myelomatosis is diagnosed or excluded.   Rarely, myeloma involves soft tissues, without marrow  changes (extramedullary plasmacytoma). Although the  protein abnormalities of myelomatosis are often found,  the behaviour and prognosis of the two conditions are  different. Spread of soft-tissue plasmacytoma is slow  and tends to be local. Local excision of the solitary  tumour may be effective.   Waldenstrom’s macroglobulinaemia is caused by a  malignancy of B cells, with increased synthesis of IgM;   the cells resemble lymphocytes rather than plasma  cells. There is generalized lymphadenopathy. Like  myelomatosis, macroglobulinaemia usually occurs in  older patients, over the age of 60, but, unlike myelomatosis,  it is more common in men than in women. Symptoms  of the ‘hyperviscosity syndrome’ are commoner than in  myelomatosis, probably because of the large size of the  IgM molecule; skeletal manifestations are rarer.   • IgM monoclonal paraprotein in the y-region on  protein electrophoresis. The plasma IgA concentration  is usually reduced, but that of IgG may be raised.   • The bone marrow aspirate or lymph node biopsy  contains atypical lymphocytoid cells.   The heavy-chain diseases are a rare group of disorders  characterized by the presence of an abnormal protein  in plasma or urine, identifiable as part of the H chain  (a, y or p). The clinical picture is that of:   • intestinal lymphomatous lesions, with severe  malabsorption ( (/-chain disease); the condition  usually affects young adults of Mediterranean  origin;   • generalized lymphadenopathy with recurrent  infections in the elderly (y-chain disease);   Cryoglobulins are proteins that precipitate when cooled  below body temperature. They may be associated with  diseases known to produce paraproteins. About half  of them can be shown to consist of a monoclonal  immunoglobulin (usually IgM or IgG). The patient  usually presents with other symptoms of the underlying  disease and the cryoglobulin is found during  investigation. Occasionally, intravascular precipitation  may occur at temperatures above 22°C and, if the  concentration of protein is high, presenting symptoms  and signs may be skin lesions such as purpura and  Raynaud’s phenomenon. In some cases the protein  is polyclonal and includes complement; these cases  may be associated with immune-complex disease.  Occasionally no underlying abnormality can be found  (essential cryoglobulinaemia).   To determine cryoglobulinaemia, the blood should be  collected in prewarmed tubes at 37°C and transported  quickly to the laboratory. Aliquots can be taken, one at  room temperature (22°C) and the other at 37°C. The  cryoglobulin protein can be quantified by subtracting  the difference between the 37°C sample and the  supernatant of the 22°C sample, as at 22°C the protein/  gel would be expected to precipitate, as lower than body  temperature.   If a paraprotein is found on electrophoresis, investigation  for one of the diseases discussed above should be initiated.  In up to 30 per cent of hospital cases, and probably  more in the ‘well population’, no cause can be found.  The condition, which may be transient, has been called  ‘benign’ or ‘essential’ paraproteinaemia or monoclonal  gammopathy of undetermined significance (MGUS). The  diagnosis should be made provisionally and the patients  followed up; they may later develop myelomatosis or  macroglobulinaemia. This condition is more common  in the elderly; BJP is not usually present and serum  paraprotein (or M protein) is often less than 30 g/L with  no immune paresis or amyloidosis and no evidence of  myeloma organ or tissue impairment and with the bone  marrow less than 10 per cent of clonal plasma cells.   Amyloid diseases are secondary protein structure  diseases in which insoluble protein fibrils accumulate  extracellularly. At least 20 different types of fibrils  have been described in human amyloidosis, each with  a different clinical picture. All types of tissue amyloid  consist of a major fibrillar protein that defines their  type. All amyloid types show certain features:   • soluble in water and in buffers of low ionic strength,   • amorphous eosinophilic appearance on light  microscopy after haematoxylin and eosin staining,   • green fluorescence seen under polarized light after  Congo red staining,   Amyloidosis is usually classified biochemically.  The amyloidoses are referred to with a capital A (for  amyloid) followed by an abbreviation for the fibril  protein. For example, in cases formerly termed primary  amyloidosis and in myeloma-associated amyloidosis,  the fibril protein is an immunoglobulin light chain or   light-chain fragment (abbreviated L); thus, patients  with these amyloidoses are now said to have light-  chain amyloidosis (AL). In instances previously termed  senile cardiac amyloidosis and in cases formerly called  familial amyloid polyneuropathy, the fibrils consist  of the transport protein transthyretin (TTR); these  diseases are now termed ATTR. (See Chapter 11.)   The loss of most plasma proteins through the  glomeruli is restricted by the size of the pores in, and  by a negative charge on, the basement membrane that  repel negatively charged protein molecules. Alteration  of either of these factors by glomerular disease may  allow albumin and larger proteins to enter the filtrate.  Low-molecular-weight proteins are filtered even under  normal conditions; most are absorbed and metabolized  by tubular cells. Normal subjects excrete up to 0.08 g  of protein a day in the urine, amounts undetectable by  usual screening tests. Proteinuria of more than 0.15 g a  day almost always indicates disease.   Significant proteinuria may be due to renal disease  or, more rarely, may occur because large amounts of  low-molecular-weight proteins are circulating and  therefore being filtered. Blood and pus in the urine and  also urinary infection give positive tests for protein (see  also Chapter 3).   Glomerular proteinuria is due to increased glomerular  permeability, as in nephrotic syndrome. Albumin is  usually the predominant protein in the urine.   Transient proteinuria may be associated with physical  exertion, trauma, cardiac failure, fever and other acute  illness.   Proteinuria is usually more severe in the upright  than in the prone position. The term ‘orthostatic’ or  ‘postural’ has been applied to proteinuria, often severe,  which disappears at night. Overnight urine collection  shows normal albumin excretion (i.e. less than 50 mg  during the 8-h period). It appears to be glomerular  in origin and is most common in adolescents and  young adults, typically those who are tall and thin. It  may be associated with severe lordosis. Renal function  is normal and proteinuria is usually less than 1 g/day.   Although it is often harmless, evidence of renal disease  may occur after some years.   Sensitive immunological assays have shown the normal  daily excretion of albumin to be less than 0.05 g.  Patients with diabetes mellitus who excrete more  than this, but whose total urinary protein excretion is  ‘normal’, are said to have microalbuminuria and to be  at greater risk of developing progressive renal disease  than those whose albumin excretion is normal. This  can be assessed from the urinary albumin to creatinine  ratio (ACR) The incidence of this complication may be  reduced by optimization of glycaemic control and also  blood pressure using angiotensin-converting enzyme  (ACE) inhibitors (see Chapter 12). Microalbuminuria  can also occur in other diseases, such as inflammatory  bowel disease or rheumatoid arthritis.   Tubular proteinuria may be due to renal tubular damage  from any cause, especially pyelonephritis. If glomerular  permeability is normal, proteinuria is usually less than  1 g/day and consists mainly of low-molecular-weight  globulins and not albumin. Low-molecular-weight   A 65-year-old man with type 2 diabetes mellitus  had glycated haemoglobin (HbA lc ) of 10.0 per cent  (86 mmol/mol) and random blood glucose  concentration of 23.8mmol/L in the diabetes clinic.  He was taking metformin 500 mg twice a day. His  blood pressure was 180/100 mmHg. The urinary  albumin to creatinine ratio (ACR) was 9.9 g/mol. A  year prior to this, his urinary ACR was normal, at less  than 2.5 g/mol.   The patient has microalbuminuria, which was  confirmed on repeat urine sampling. This can  progress to proteinuria and eventually chronic kidney  disease. The aim of the clinician should be to try to  optimize glycaemic control. In this case, the patient’s  HbA lc was high, reflecting poor control, and was  improved by increasing his metformin and adding  the sulphonylurea gliclazide, which brought his HbA k  to 8 per cent (64 mmol/mol). He is hypertensive,  which also needs treating, and he was started on an  angiotensin- converting enzyme inhibitor.   (/-globulins and |3-globulins are sensitive markers  of renal tubular damage. Tubular proteinuria can be  diagnosed by measuring certain low-molecular-weight  proteins in urine, such as retinol -bin ding protein (RBP),  A- a c e t \ T |1 - 1 ) - g I u c o s a m i n i d a s e or C^-microglobulin.   However, because the P 2 -microglobulins in urine  are unstable, other proteins, such as RBP, may be better  indicators of tubular damage. Tubular proteinuria  is associated with Fanconi’s syndrome and with  glycosuria, amino aciduria, hyperphosphaturia  (resulting in hypophosphataemia) and renal tubular  acidosis. This can be primary or secondary, for example  to heavy metals and drugs such as cisplatin.   This occurs when proteins of low molecular weight are  filtered normally by the glomerulus and reabsorbed at  the proximal tubule but are produced in amounts greater  than the reabsorptive capacity of the proximal tubule.  Overflow proteinuria can be due to the production  of BJP, to severe haemolysis with haemoglobinuria,  or to severe muscle damage (rhabdomyolysis) with  myoglobinuria. In the last two cases, the urine may be  red or brown in colour.   Bence Jones proteinuria can be inferred by comparison  of urinary and serum electrophoretic patterns, by  immunochemical assays and confirmation of free light  chains by immunofixation. Bence Jones protein is the  only protein of a molecular weight lower than albumin  that is likely to be found in significant amounts in  unconcentrated urine in the absence of haemoglobinuria  or myoglobinuria. The presence of a band in urine that is  denser than that of albumin, especially if not present in  the serum, is suggestive of BJR   Apparent proteinuria has occasionally been found  because the patient, or someone else, has added egg  white or animal blood to their urine for example.   An established case of the nephrotic syndrome is  characterized by proteinuria, hypoalbuminaemia,  oedema and hyperlipidaemia. The clinical condition is  caused by increased glomerular permeability, resulting  in a daily urinary protein loss of, by definition, more  than 3g (although some definitions state different  values). There may be hypertension and evidence of  other impaired renal function. Some of the causes are  shown in Box 19.2.   A 47-year-old man attended the medical out-patient  department because of bilateral ankle oedema and  hyperlipidaemia. A number of investigations were  requested, with the following results:   Estimated glomerular filtration rate (eGFR)  65mL/min per 1.73 m 2  Total protein 55 g/L (60-75)   A 24-h urinary total protein determination was  7.8 g and a urine spot protein to creatinine ratio was  756 mg/mmol.   The patient had nephrotic syndrome presenting  with peripheral oedema, hypoproteinaemia,  hypoalbuminaemia and severe hyperlipidaemia. Note  that the plasma creatinine and urea concentrations  may not be too abnormal unless chronic kidney  disease ensues. A renal biopsy showed that the patient  had focal segmental glomerulonephritis.   Proteinuria in the nephrotic syndrome usually ranges  from 3 g or more a day (or a urine protein to creatinine  ratio of >300 mg/mmol; see Chapter 3). The relative  proportion of different proteins lost is an index of the  severity of the glomerular lesion. In mild cases, albumin  (molecular weight 65 kDa) and transferrin (molecular  weight 80 kDa) are the predominant urinary proteins,  but op-antitrypsin (molecular weight 50 kDa) is also  present. With increasing glomerular permeability,  larger proteins such as IgG (molecular weight 160 kDa)  appear.   The serum electrophoretic picture depends on the  severity of the renal lesion. In early cases a low serum  albumin concentration may be the only abnormality,  but the typical pattern includes reduced albumin,  op- and y-globulin bands, with an increase in cp-   Most types of glomerulonephritis, usually due to  deposition of circulating immune complexes in the  glomeruli in about 80 per cent of cases  In children, ‘minimal change’ glomerulonephritis is  the most common cause   Inferior vena caval or renal vein thrombosis  Drugs and toxins, e.g. gold, penicillamine, cisplatin   Figure 19.8 Serum and urinary protein electrophoretic  patterns from patients with the nephrotic syndrome.  Patient A has selective glomerular proteinuria, and  patient B has non-selective proteinuria.   globulin because of an increase in the high-molecular-  weight op-macroglobulin (Fig. 19.8). The y-globulin  concentration may be normal or raised. Urinary   electrophoresis gives some idea of the severity of the  glomerular lesion.   The differential protein clearance is a more precise  measure of the selectivity of the lesion. The clearance  of a low-molecular-weight protein, such as transferrin  or albumin, is compared with that of a larger one,  such as IgG. The result is usually expressed as a ratio,  obviating the need for timed urine collections. A ratio  of IgG to transferrin or albumin clearance of less than  0.2 indicates high selectivity (predominant loss of small  molecules) and has a more favourable prognosis than a  higher ratio; such cases usually respond well to steroid  or cyclophosphamide therapy.   The consequences of the protein abnormalities are  oedema caused by a fall in the intravascular colloid  osmotic pressure, due to hypoalbuminaemia (see  Chapter 2), and reduction in the concentration of  protein-bound substances due to loss of carrier pro-  teins; it is important not to misinterpret low plasma  total concentrations of calcium, thyroxine, cortisol and  iron.   In mild cases, plasma LDL concentrations increase,  with consequent hypercholesterolaemia probably due  to the increased rate of protein synthesis, including  that of apolipoprotein B (apoB) lipoprotein, in the liver  (see Chapter 17). In more severe cases, a rise in plasma  very low-density lipoprotein (VLDL) (triglycerides)  concentration may cause plasma turbidity. Fatty casts  may be detectable in the urine.   In the early stages, glomerular permeability is high  and the plasma urea and creatinine concentrations are  normal, although uraemia may develop. At this stage,  protein loss is reduced and plasma concentrations of  protein and lipid may revert to normal, but this does  not indicate recovery. Secondary hyperaldosteronism  may also occur (see Chapters 2 and 3).   It is essential that the sample is fresh. There are  limitations of screening tests, such as the following.   • The tests were mainly developed to detect albumin  and may be negative in the presence of other  proteins, such as B)R   • Because the tests depend on protein concentrations,  very dilute urine may give negative results despite  significant proteinuria.   One example of a urinary protein screening test is  Albustix. The test area of the reagent strip is impregnated  with an indicator (tetrabromophenol blue) buffered to   pH 3. At this pH it is yellow in the absence of protein.  Protein forms a complex with the dye, stabilizing it in  the blue form; it is green or bluish-green if protein is  present. The colour after testing is compared with  the colours on the chart provided, which indicate the  approximate protein concentration. The strips should  be kept in the screwtop bottles, in a cool place and the  instructions should be followed carefully. However, due  to some of the problems associated with such methods  in some countries there has been a move away from  such testing, preferring instead the spot urine ACR.  False-positive results occur:   • if the specimen is contaminated with vaginal or  urethral secretions, including haematuria, semen or  menstrual fluid,   • in strongly alkaline (infected or stale) urine, when  buffering capacity is exceeded; a green colour in this  case is a reflection of the alkaline pH,   • if the urine container is contaminated with  disinfectants such as chlorhexidine.   False-negative results occur if acid has been added  to the urine as a preservative (for example for the  estimation of urinary calcium).   • Normally urinary total protein is about 50-1 50 mg/  day. Rather than a 24-h urinary protein collection,  it may be more convenient to use a spot urine ACR  or in pregnancy a spot urinary protein to creatinine  (P:C) ratio. Normally the latter is less than 15 mg /  mmol. Remember that urine dipstick tests can give  false-positive results (see above).   • Exclude urinary tract infection; a midstream urine  test may be useful.   • Is the proteinuria transient? Confirm on at least two  or three occasions.   • Renal function tests, i.e. plasma urea and creatinine,  should be performed and it is also important to check  plasma albumin and protein for hypoalbuminaemia  and hypoproteinaemia.   • Take a drug history for renal toxic agents, for example  gold or penicillamine.   • Clinical history and examination may reveal systemic  illnesses that can cause proteinuria (see above).  Check plasma prostate-specific antigen in men to  help assess for prostatic disease.   • Consider orthostatic or postural proteinuria when  an early morning urine sample is negative for protein  but pre-bed samples show proteinuria.    Figure 19.9 Algorithm for the investigation of  proteinuria. * = A spot urine protein/creatinine ratio  (>300) can also be used instead of a 24-h urine  protein collection (see text).   • A 24-h protein measurement (now rarely used)  and/or random spot urinary P:C ratio are then  necessary to quantify the proteinuria. A high degree  of proteinuria (for example more than 3 g/day or P:C  more than 300 mg/mmol) is indicative of nephrotic  syndrome. A selectivity index can then be calculated  for the proteinuria to see whether or not it is non-  selective. It has been recommended that a spot urine  ACR is the preferred screening test due to better assay  robustness, although there is some debate whether  this is true if exclusively a tubular proteinuria where  other proteins aside of albumin may be lost.   • If proteinuria is confirmed, urinary protein  electrophoresis may be useful to determine the   pattern of proteinuria, that is, whether there  is glomerular or tubular pathology. Overflow  proteinuria may also be revealed, such as Bence Jones  proteinuria, myoglobinuria or haemoglobinuria.   • Does the patient have diabetes mellitus and what  is the degree of proteinuria? Diabetic nephropathy  is also associated with diabetic retinopathy, that is,  representing increased microvascular permeability.   • Other tests may be indicated, such as autoantibodies,  including antiglomerular membrane antibodies,  streptococcal antibodies and also complement C3  and C4. In addition, renal imaging, for example  ultrasound, and renal biopsy may be required.   This chapter ends with the clinical indications for  measuring serum or plasma protein and albumin.   Blood for protein estimations, including for immuno-  globulins, should be taken with a minimum of venous  stasis, otherwise falsely high results may be obtained.   Protein electrophoresis should be performed on  serum from clotted blood, rather than on plasma,  because the presence of fibrinogen may mask, or be  interpreted as, an abnormal band or protein.   Blood for cryoglobulin measurement should be  collected in a syringe warmed to 37°C, and should  be maintained at this temperature until it has been  tested. Failure to observe this precaution may result  in false-negative findings, because the cryoprecipitate  is incorporated into the blood clot on cooling. It is  important to contact your laboratory for advice before  taking the blood.   The following are the more common indications for  plasma protein and albumin estimations:   • To investigate oedema Very low plasma albumin  and total protein concentrations in the presence of  oedema indicate that hypoalbuminaemia may be the  cause. Conditions causing severe hypoalbuminaemia  include the nephrotic syndrome and hepatic  cirrhosis, both of which have typical serum  electrophoretic patterns. The diagnosis of nephrotic  syndrome depends on finding gross proteinuria.   • To assess changes in hydration Changes in plasma  total protein or albumin concentrations over short  periods of time are almost certainly due to changes   in hydration, or to changes in capillary permeability  (see Chapter 2). Dehydration may evoke raised  plasma albumin and protein concentrations, whereas  the converse is true in overhydration.   • To help assess nutritional status Plasma albumin  concentrations are sometimes used to help  assess nutritional status. However, low albumin  concentrations, although associated with  undernutrition, are not specific as they can be low  for other reasons (see Chapter 14).   • To investigate suspected myelomatosis or  macroglobulinaemia The difference between the  total plasma protein and albumin concentration is  called the ‘globulin’ concentration. This is raised in  clinical situations when the globulin concentration  is raised, such as in myelomatosis.   • Raised plasma globulin also occurs in liver disease,  for example autoimmune hepatitis.   Protein electrophoresis should be carried out on  serum to detect a paraprotein or M protein and,  importantly, also on urine to detect BJP. The diagnosis  of myelomatosis should be confirmed by microscopic   inspection of a bone marrow aspirate or biopsy and  usually also a skeletal survey. Plasma immunoglobulin  concentrations should be measured to assess the degree  of immune paresis.   • To evaluate apparently abnormal concentrations  or changes in concentrations of a protein-bound  substance Estimation of plasma albumin  concentration should always accompany that of total  plasma calcium. If changes in this or other protein-  bound substances parallel those of albumin, they are  probably due to changes in protein concentrations.   • As part of the investigation of hypogamma-  globulinaemia A low total plasma protein  concentration, if not due to hypoalbuminaemia,  may be due to hypogammaglobulinaemia. Plasma  immunoglobulin concentrations can be measured  as part of the assessment of the adequacy of the  immune system.   Note that these tests assess only humoral immunity:  T-cell abnormalities and their investigations are outside  the scope of this book.   • Abnormalities of plasma and urinary proteins are  relatively common in clinical practice, and therefore  knowledge of protein biochemistry is necessary for  a full understanding of these conditions.   • Diagnostically, certain proteins can be specifically  assayed or protein electrophoresis of serum and/or  urine can be performed to show pattern changes.   • The acute-phase response and inflammation are  essential mechanisms to protect the body. B cells  are concerned primarily with immunoglobulin   synthesis and humoral immunity, whereas T cells  are involved in cellular immunity.   Multiple myeloma is a plasma cell malignancy  associated with paraproteinaemia, Bence  Jones proteinuria, osteolytic bone lesions and  hypercalcaemia.   Proteinuria canbedueto either glomerular or tubular  disorders. The nephrotic syndrome is associated  with peripheral oedema, hypoalbuminaemia and  hypoproteinaemia.   Abnormalities of purine metabolism are often found  in clinical practice, notably hyperuricaemia and  gout. After exploring purine metabolic pathways,  this chapter discusses the various disorders of purine  metabolism, including their clinical features, diagnosis  and treatment.   Normal purine metabolism  Urate is the endproductofpurinemetabolisminhumans.  In most other mammals, it is further metabolized to the  more water-soluble allantoin. Humans lack uricase and  thus their uric acid levels are higher. It is because of  the poor solubility of urate that humans are prone to  the clinical effects of hyperuricaemia, such as gout and  renal damage. The purines adenine and guanine are  constituents of nucleic acid from deoxyribonucleic acid  (DNA) and ribonucleic acid (RNA). The purines used  by the body for nucleic acid synthesis may be derived  from the breakdown of ingested nucleic acid, mostly  from cell-rich meat, or they may be synthesized de novo  from small molecules. About two-thirds of the body’s  urate (3-4 mmol/ day) is produced endogenously, with  one-third coming from exogenous dietary purines  (1-2 mmol/day).   The upper part of Figure 20.1 summarizes some of the  more important synthetic steps.   The first step in purine synthesis is condensation  of pyrophosphate with phosphoribose to form  phosphoribose diphosphate (phosphoribosyl  pyrophosphate, PRPP).   The amino group of glutamine is incorporated into  the ribose phosphate molecule and pyrophosphate is  released. Amidophosphoribosyl transferase catalyses this  rate-limiting or controlling step. The enzyme is subject  to feedback inhibition by increasing concentrations of  purine nucleotides; thus the rate of synthesis is slowed   when its products increase. The control of this rate-  limiting step may be impaired in primary gout.   Glycine is added to phosphoribosylamine. The rate  of purine synthesis is increased in primary gout. In  Figure 20.1, the atoms in the glycine molecule have  been numbered to correspond with those in the purine  and urate molecules, and the solid lines further indicate  the final position of the amino acid in these molecules.   Via a series of metabolic steps, purine ribonucleotides  (purine ribose phosphates) are formed, which control  the second step in the synthetic pathway - the formation  of phosphoribosylamine. Ribose phosphate is cleaved  off, thereby releasing the purines. Some cytotoxic drugs  inhibit various stages of the pathway, thus preventing  DNA formation and cell growth.   Purines synthesized in the body, those derived from  the diet and those liberated by endogenous catabolism  of nucleic acids may be oxidized to urate or reused for  nucleic acid synthesis.   Purines oxidized to urate Some adenine is oxidized  to hypoxanthine, which is further oxidized to xanthine.  Guanine can also form xanthine. Xanthine, in turn,  is oxidized to form urate. The oxidation of both  hypoxanthine and xanthine is catalysed by xanthine  oxidase in the liver. Thus the formation of urate from  purines depends on xanthine oxidase activity: gout may  be treated using an inhibitor of this enzyme (allop urinol).   Purines reused for nucleic acid synthesis Some xanthine,  hypoxanthine and guanine can be resynthesized to purine  nucleotides by pathways involving, among other enzymes,  hypoxanthine-guanine phosphoribosyl transferase  (HGPRT) and adenine phosphoribosyl transferase (APRT).   Urate is filtered through the glomeruli and most  is reabsorbed in the proximal tubules. More than  80 per cent of that formed in urine is derived from more    Figure 20.1 Summary of purine synthesis and breakdown, showing the steps of clinical importance. APRT,  adenine phosphoribosyl transferase; GluNH 3 + , glutamine; G I u , glutamate; HGPRT, hypoxanthine-guanine  phosphoribosyl transferase.   distal tubular secretion. Urinary excretion is slightly  lower in males than in females, which may contribute  to the higher incidence of hyperuricaemia in men.   Renal secretion may be enhanced by uricosuric  drugs (e.g. probenecid or sulfinpyrazone), which  block tubular urate reabsorption. Tubular secretion  of urates is inhibited by organic acids, such as lactic  and oxoacids, and by ketones and thiazide diuretics.  Seventy-five per cent of urate leaving the body is   in urine. The remaining 25 per cent passes into the  intestinal lumen, where it is broken down by intestinal  bacteria, the process being known as uricolysis.   Generally speaking, hyperuricaemia can occur by two  main mechanisms: increasedproduction (overproducers)  and/or decreased excretion (underexcretors).   The factors that may contribute to hyperuricaemia  (summarized in Fig. 20.2) are:   Increased synthesis, due to impaired feedback control,  is probably the most important mechanism causing  primary hyperuricaemia, whereas abnormalities of the  other steps are causes of secondary hyperuricaemia.   Consequences of hyperuricaemia  Urate is poorly soluble in plasma. At plasma pH, most  urate is ionized at position 8 of the purine ring (see  Fig. 20.1). This anionic group is associated with the  predominant extracellular cation, sodium. Ionization  of uric acid decreases as the pH falls, and it therefore  becomes less soluble; at a urinary pH below about 6,  uric acid may form renal calculi.   Crystallization in joints, especially those of the feet,  produces the classic picture of gout. Urate precipitation  at these sites causes an inflammatory response with  leucocytic infiltration, and it is thought that lactic acid  production by these cells causes a local fall in pH: this   5. Decreased in renal failure,  thiazide diuretic therapy, some  cases of primary hyperuricaemia  and acidosis   converts urate to uric acid, which is less soluble than  urate, and a vicious circle is set up in which further  precipitation, and therefore further inflammation,  occurs. In acute attacks of gouty arthritis, local factors are  more important than the plasma urate concentration,  which is usually normal during the attack.   Precipitation may occur in subcutaneous tissues,  especially of the ears, and in the olecranon and  patellar bursae and tendons. Such deposits are called  gouty tophi (Fig. 20.3). A potentially serious effect of  hyperuricaemia is precipitation of urate in the kidneys  and renal calculi, causing progressive renal damage.  For this reason it has been recommended that even  asymptomatic cases should probably be treated if the  plasma urate concentration is consistently higher  than 600pmol/L. Hyperuricaemia can be primary or  secondary.   In some cases a hereditary component is present,  probably involving abnormalities of urate metabolic  pathways.   Primary hyperuricaemia and gout are very rare in  children and unusual in women of child-bearing age.  Plasma urate concentrations are low in children and  rise in both sexes at puberty, more so in males than in  females; concentrations are higher in males. Women  become more prone to hyperuricaemia and gout in the  post-menopausal period.    Figure 20.3 Acute gout. Note gouty tophi on both  big toes, affecting the metatarsophalangeal joints.  Reproduced with kind permission from Kinirons M and  Ellis H. French’s Index of Differential Diagnosis, 15th  edition. London: Hodder Arnold, 2011.   • A high-meat diet contains a relatively high  proportion of purines.   • Alcohol has been shown to decrease renal excretion  of urate. This may be because it increases lactic acid  production, which inhibits urate secretion.   None of these factors is likely to precipitate gout in a  normal person, but, like thiazide diuretics, may do so in  a subject with a hyperuricaemic tendency.   Purine synthesis is increased in about 25 per cent of  cases of primary hyperuricaemia due to overactivity of  amidophosphoribosyl transferase, which controls the  formation of phosphoribosylamine.   Reduced renal tubular secretion of urate has also been  demonstrated in other cases of primary hyperuricaemia.  Many subjects may have both increased synthesis and  decreased excretion.   Familial juvenile gouty nephropathy is a rare  autosomal dominant condition leading to progressive  renal failure.   Lesch-Nyhan syndrome is an exceedingly rare  X-linked, recessively inherited disorder of urate  metabolism caused by a reduced activity of HGPRT.  Severe hyperuricaemia occurs in young male children.  Hypoxanthine and other purines cannot be recycled  to form purine nucleotides, and probably produce  more urate. The syndrome is associated with mental  deficiency, a tendency to self-mutilation, aggressive  behaviour, athetosis and spastic paraplegia. Partial  deficiency of HGPRT is known as Kelley-Seegmiller  syndrome. Hyperuricaemia can also occur with  increased 5-PRPP synthase activity.   • In rapidly growing malignant tissue, especially in  leukaemias, lymphomas and polycythaemia rubra  vera.   - After the treatment of large malignant tumours  by radiotherapy or cytotoxic drugs (tumour   lysis syndrome). This can cause massive sudden  release of urate, which may crystallize in and  block renal tubules causing acute oliguric  renal dysfunction. During such treatment,  allopurinol should be given and, if glomerular  function is not impaired, fluid intake kept high.   - As a result of trauma, including that of surgery;  endogenous urate release is increased.   - During starvation or prolonged fasting: the  patient’s own tissues may be used as an energy  source, with increased urate release. Starvation  may be associated with mild ketoacidosis,  and protein catabolism releases acidic amino  acid residues. The acidosis, by inhibiting  the secretion of urate, may aggravate the  hyperuricaemia. During prolonged fasting,  plasma urate concentrations may rise.   • Thiazide diuretics : although hyperuricaemia is  relatively common during diuretic treatment, clinical   A 62-year-old man was treated with chemotherapy  by the oncologists for non-Hodgkin’s lymphoma.  His biochemistry results post-chemotherapy were as  follows:   Estimated glomerular filtration rate (eGFR) 48 mL/  min per 1.73 m 2   Where there is rapid growth of dividing tumour  cells and consequent elevated nucleic acid turnover,  increased purine metabolism and hyperuricaemia can  occur. This is a case of tumour lysis syndrome, which  can occur after chemotherapy where there is rapid  cell destruction and release of purine metabolites and  intracellular ions such as potassium and phosphate,  resulting in hyperkalaemia and hyperphosphataemia.   gout is a rare complication. It may, however, be  precipitated in those patients with a gouty tendency.   • Other drugs, including low doses of salicylates,  ciclosporin, nicotinic acid, methoxyflurane,  levodopa, ethambutol and pyrazinamide.   • Renal glomerular dysfunction: before the diagnosis  of primary hyperuricaemia is made, the plasma  concentration of creatinine should be assayed  on the same specimen as urate, to exclude renal  dysfunction as a cause. It may be difficult to decide  which abnormality is the primary one because  hyperuricaemia can cause renal dysfunction. The  urate to creatinine concentration ratio in a spot urine  sample may help distinguish hyperuricaemia due  to reduced renal excretion from renal impairment  secondary to hyperuricaemia. A ratio of less than  0.7 implies that renal impairment is responsible  for the hyperuricaemia. A ratio of greater than 0.7  suggests urate nephropathy. Clinical gout is rare in  the secondary hyperuricaemia of renal disease.   • Dyslipidaemia and impaired glucose tolerance or type  2 diabetes mellitus. Hyperuricaemia is associated with  insulin resistance and the metabolic syndrome.   • Glucose-6-phosphatase deficiency or von Gierke’s  disease (see Chapter 27). The tendency to  hyperuricaemia in these patients may be directly  related to the inability to convert glucose-6-  phosphate (G6P) to glucose. More G6P is available  for metabolism through intracellular pathways,  including:   - the pentose phosphate pathway, thus increasing  ribose phosphate (phosphoribose) synthesis; this  may accelerate the first step in purine synthesis,  with consequent urate overproduction,   - glycolysis, thus increasing lactic acid production;  lactic acid may reduce renal urate excretion.   • Reducing dietary purine intake, for example red  meats. This treatment is rarely effective by itself.   • An acute attack of gout can be treated with a non-  steroidal anti-inflammatory drug (NSAID) or  sometimes steroids.   • Colchicine, which has an anti-inflammatory effect  and inhibits neutrophil activation, can be used  in acute gouty arthritis but does not affect urate  metabolism. It is sometimes used if NSAIDs are  contraindicated.   • Reducing urate production by using drugs that  inhibit xanthine oxidase activity, such as allopurinol  (hydroxypyrazolopyrimidine), which is structurally  similar to hypoxanthine, so acting as a competitive  inhibitor of the enzyme. De novo synthesis may  also be decreased by this drug. However, allopurinol  may worsen an acute attack of gout. Initiation of  allopurinol should be made a few weeks after the  acute attack under the cover of an NSAID, assuming  that it is not contraindicated. Febuxostat is a non-  competitive inhibitor of xanthine oxidase and may  have a place in those intolerant to allopurinol.   • Increasing the renal excretion of urate with uricosuric  drugs, such as probenecid or sulfinpyrazone. These  drugs maybe effective if renal function is normal, but  are less so if there is renal glomerular dysfunction.  Fluid intake is usually kept high. Low doses of most  uricosuric drugs reduce urate secretion; they are  rarely used unless allopurinol is contraindicated.   • A careful drug history is important, as various drugs  (Box 20.1), such as thiazide diuretics, can evoke  hyperuricaemia, as well as a high alcohol intake.   • A family history may reveal a primary hereditary  form of gout or hyperuricaemia.   • Check renal function with plasma urea and  creatinine, as renal impairment is a common cause  of hyperuricaemia. A spot urinary urate to creatinine  concentration ratio may help to distinguish  hyperuricaemia due to reduced renal excretion from  renal impairment secondary to hyperuricaemia.   • Consider secondary conditions with high  cell turnover, such as tumours, psoriasis and  polycythaemia (see Box 20.1).   • Hyperuricaemia can also be seen in a metabolic  acidosis due to reduced renal urate excretion (see  Chapter 4).   • There is an association between hyperuricaemia and  the metabolic syndrome with hypertriglyceridaemia,  obesity and insulin resistance.   • A 24-h urine analysis for urate excretion on and off a  low-purine diet may give an indication of whether the  patient is an overproducer or underexcretor of urate.   Drugs: e.g. thiazides, nicotinic acid, warfarin,  ciclosporin, pyrazinamide, didanosineand ethambutol  Poisoning by other agents, such as alcohol and lead  Metabolic syndrome associated with insulin  resistance and hypertriglyceridaemia, obesity and  hypertension  Impaired urate excretion   Acute kidney injury or chronic kidney disease  Metabolic acidosis  High cell turnover  Haemolytic anaemia  Myeloproliferative disease  Malignancies  Psoriasis   • Do not forget the rare causes of hyperuricaemia, for  example Lesch-Nyman syndrome or von Gierke’s  disease.   Pseudogout is not a disorder of purine metabolism  although the clinical presentation is similar to that  of gout. Calcium pyrophosphate precipitates in joint  cavities, and calcification of cartilages is demonstrable  radiologically (chondrocalcinosis). The plasma urate  concentration is usually normal. The crystals of  calcium pyrophosphate may be identified in joint  fluid using a polarizing microscope, when positive  bifringence is observed. Patients may present with acute  pseudogout arthopathy or chronic chondrocalcinosis.  Pseudogout and chondrocalcinosis are associated  with hyperparathyroidism, hypophosphataemia,  hypothyroidism, haemochromatosis, renal  osteodystrophy, Wilson’s disease and acromegaly.   This is rare unless it is the result of the treatment of  hyperuricaemia with, for example, allopurinol or  probenecid. It is associated with proximal renal tubular  damage, in which the reabsorption of urate is reduced,  and can be seen in Fanconi’s syndrome. Hypouricaemia  is also a finding in some patients receiving parenteral  nutrition and in pregnancy, the syndrome of   inappropriate antidiuretic hormone (SIADH, see  Chapter 2) and type 1 diabetes mellitus.   This is a very rare inborn error of purine metabolism,  inherited as an autosomal recessive disorder, in which  there is a deficiency of xanthine oxidase in the liver.   Purine breakdown stops at the xanthine-hypoxanthine  stage, and plasma and urinary urate concentrations  are very low. Increased xanthine excretion may lead to  the formation of xanthine stones; this does not occur  during the treatment of gout with xanthine oxidase  inhibitors, perhaps because the drugs also inhibit  purine synthesis.   • Urate is formed as a product of purine metabolism  and is renally excreted.   • Gout can be primary or secondary, and the  clinical features include acute arthritis leading to  chronic arthropathy, gouty tophi, renal calculi and  obstructive uropathy.   • Allopurinol, a xanthine oxidase inhibitor, can  be used clinically to lower plasma uric acid  concentrations.   • Hypouricaemia is usually not of major clinical  significance in itself, although factors associated  with it include SIADH, pregnancy, Fanconi’s  syndrome and xanthinuria.   Abnormalities of haem metabolism are important  clinically. They include the porphyrias, which are  rare, and disorders involving haem iron, such as iron  deficiency, which are common in clinical practice.   Most body tissues synthesize haem. In bone marrow it  is incorporated into haemoglobin, an iron- containing  pigment that carries oxygen from the lungs to tissues,  and in muscle into myoglobin, which also binds  oxygen. In other cells it is used for the synthesis of  cytochromes and related compounds. The former are  components of the electron transport chain, which is  involved in oxidative phosphorylation. Quantitatively  the liver is the major non-erythropoietic haem-  producing organ. All haem pigments contain iron;  the oxygen-carrying ability of the haem molecule  depends on the presence of ferrous iron (Fe 2+ ), the  form normally present in both haemoglobin and  oxyhaemoglobin.   Red blood cells are broken down by the  reticuloendothelial system, predominantly in the  spleen. Released haemoglobin is split into the  peptide chain, globin, which enters the general  protein pool, and haem. The haem ring is split, a  process catalysed by haem oxidase, to form a linear  molecule, biliverdin. Released iron is reused and  biliverdin is reduced to lipid-soluble bilirubin. The  metabolism of bilirubin is discussed in Chapter 17  in the context of jaundice.   The main steps in the synthesis of haem are outlined  below and in Figure 21.1.   • 5-Aminolaevulinic acid (ALA) is formed by  condensation of glycine and succinate. The reaction  requires pyridoxal phosphate and is catalysed by  ALA synthase. This is the rate-limiting step in the   • Two molecules of ALA condense to form a  monopyrrole, porphobilinogen (PBG).   • Four molecules of PBG combine to form a  tetrapyrrole ring, uroporphyrinogen (Fig. 21.2). Two  isomers are formed, I and III. The major pathway  involves the III isomer.   • Haem is formed by the successive production of  coproporphyrinogen and protoporphyrin, followed  by incorporation of Fe 2+ into the centre of the ring.   • Haemoglobin consists of four haem molecules,  covalently linked to four (two pairs of) polypeptide  chains.   Figure 21.2 Porphobilinogen, the tetrapyrrole uroporphyrinogen III, which incorporates four porphobilinogen units  and haemoglobin. Uroporphyrinogen I differs only in the order of the side chains on one of the rings. Side chains:  A, acetate; M, methyl; P, propionate; V, vinyl.   The porphyrin precursors ALA, PBG and  uroporphyrinogen are water soluble and appear in the  urine. They are colourless, but PBG may spontaneously  oxidize to form uroporphyrin when exposed to air and  light.   Porphyrinogens also oxidize spontaneously to  the corresponding porphyrins, which are dark red  and fluoresce in ultraviolet light. A urine specimen  containing large amounts of porphyrinogens or their  precursors will gradually darken if left standing.  Protoporphyrin is excreted in bile and appears in the  faeces, whereas coproporphyrin(ogen) may be excreted  by either route.   Haemoglobin and related compounds  When oxygen is incorporated into haemoglobin to  form oxyhaemoglobin, the spatial arrangement of the  haem complexes is altered in such a way as to facilitate  further oxygen uptake. Other compounds related to  haemoglobin may sometimes be formed and some of  these may hinder the oxygen-carrying capacity.   Carboxyhaemoglobin is cherry red in colour and is  formed when carbon monoxide binds to haemoglobin or  displaces oxygen from oxyhaemoglobin; haemoglobin  has a greater affinity for carbon monoxide than for  oxygen. This occurs in carbon monoxide poisoning,  which can be lethal. Once carbon monoxide is removed  from inspired air, oxyhaemoglobin is reformed.   Consciousness is not lost until the carboxy form has  replaced about half the oxyhaemoglobin. (See oximetry  and blood gases in Chapter 4.)   Methaemoglobin is haemoglobin in which iron is in the  ferric (Fe 3+ ) form (haemin); therefore, it cannot carry  oxygen. It is brown and is normally present in very low  plasma concentrations; drugs such as sulphonamides  or nitrites/nitrates may increase methaemoglobin. The  symptoms of methaemoglobinaemia are due to hypoxia,  which causes cyanosis and an increased respiratory rate  and, if methaemoglobin is greater than 70 per cent of  the total haemoglobin, can be fatal. Methemoglobin  may cause a pulse oximeter to read about 85 per cent  regardless of the actual amount of oxygen saturation.  Methaemoglobinaemia is associated with glucose-  6-phosphate dehydrogenase (G6PD) deficiency (see  Chapter 27).   Methaemalbumin is also brown. It is formed when haem  combines with plasma albumin in conditions such as  severe intravascular haemolysis or acute haemorrhagic  pancreatitis when haemoglobin has been converted  to haemin in the abdominal cavity and absorbed.  Methaemalbumin occurs when the haemoglobin-  binding capacity of haptoglobin has been exceeded.   Sulphaemoglobin is similar to methaemoglobin but  contains sulphur; unlike methaemoglobin, it cannot be  reconverted to haemoglobin in vivo. It remains in intact   red blood cells. Methaemoglobinaemia-producing  drugs cause sulphaemoglobinaemia if hydrogen  sulphide is present, usually in the gut.   Myoglobin, a haem-protein complex, is normally  present in muscle. Plasma concentrations may rise if  skeletal muscle, e.g. rhabdomyolysis, or myocardial  muscle cells, e.g. myocardial infarction, are damaged.  Being of low molecular weight, it is rapidly cleared by  the kidneys.   Distribution of iron in the body  About 50-70 mmol (3-4 g) of iron are distributed  among body compartments. There is considerable  interchange of iron between stores and plasma. Free  iron is toxic. In normal subjects it is all protein bound:  in plasma it is bound to transferrin, in the storage  pools to protein in ferritin and haemosiderin, and  in erythrocytes it is incorporated into haemoglobin.  About 70 per cent of the total iron is circulating, largely  in erythrocyte haemoglobin. However, there are smaller  amounts in muscle myoglobin and iron-containing  enzymes and cytochromes.   Up to 25 per cent of the body iron is stored in the  reticuloendothelial system, in the liver, spleen and  bone marrow; bone marrow iron is drawn on for  haemoglobin synthesis. Iron is stored in cells in the  cytosol as ferritin or in the lyosomes as haemosiderin.  Ferritin iron is more easily released from protein than  that in haemosiderin. Haemosiderin can be seen by  light microscopy in unstained tissue preparations.  Ferritin and haemosiderin, but not haem iron, stain  with potassium ferrocyanide (Prussian blue reaction),  and this staining characteristic may be used to assess  the size of iron stores.   Iron deficiency becomes haematologically evident  only when no stainable iron is detectable in the  reticuloendothelial cells in bone marrow films. Iron  overload is likely when, because reticuloendothelial  storage capacity is exceeded, stainable iron is  demonstrable in parenchymal cells in liver biopsy  specimens. This is the storage pool.   Only about 50-70 mmol (3-4 mg), or about  0.1 per cent of the total body iron, is circulating  in plasma, all bound to transferrin; this fraction is  measured in plasma iron assays. This is the transit pool.  Iron can cross cell membranes only by active transport  in the Fe 2+ form; it is in this reduced state in both   oxyhaemoglobin and ‘reduced’ haemoglobin. It is in  the Fe 3+ form in ferritin and haemosiderin and when  bound to transferrin.   Body iron content control depends on absorption by an  active process in the duodenum: free Fe 2+ via the divalent  metal transporter 1 (DMT-1) and haem-bound iron via  the haem carrier protein 1. Within the enterocyte, some  of the iron combines with the protein apoferritin to  form ferritin. Free iron is exported from the enterocyte  via ferroportin into the circulation where it binds with  transferrin for transport and storage. Transferrin-bound  iron then enters target cells via receptor-mediated  endocytosis. Hepcidin is the main iron regulating  protein, and inhibits ferroportin. Concentrations of  hepcidin increase in iron overload and reduce in iron  deficiency. The human haemochromatosis protein  (HFE) regulates the binding of transferrin to the  transferrin receptor as well as regulating hepcidin.   Normally, about 18 pmol (1 mg) of iron is absorbed  each day and this just replaces loss. This amounts  to about 10 per cent of the iron ingested in the diet,  although the proportion depends to some extent on the  type of food. Once in the body, iron is in a virtually  closed system (Fig. 21.3).   Iron absorption seems to be influenced by any or all  of the following factors:   Most normal women taking an adequate diet  probably absorb slightly more iron than men and so  replace their higher losses in menstrual blood and  during pregnancy.   Iron requirements for growth during childhood and  adolescence are similar to, or slightly higher than, those  of menstruating women and can be met by increased  absorption from a normal diet.   This is poorly controlled, as loss from the body may  depend on the ferritin iron content of cells lost by  desquamation, mostly into the intestinal tract and  from the skin. The total daily loss by these routes is  about 18 pmol (1 mg). Urinary excretion is minimal, as  circulating iron is protein bound and not water soluble.   Normal iron loss is so small, and normal iron stores  are so large, that it would take about 3 years to become  iron deficient on a completely iron-free diet. Of course,  this period is much shorter if there is any blood loss,  such as menstruation.   Iron is transported in the plasma in the ferric form,  attached to the specific binding protein transferrin  at a concentration of about 18pmol/L. Transferrin is  normally capable of binding about 54 pmol/L of iron  and is therefore about one-third saturated. Transferrin-  bound iron is carried to stores and to bone marrow  cells and in the latter some iron passes directly into  developing erythrocytes to form haemoglobin.   The plasma iron concentration is likely to be a poor  index of the total body content because only a very  small proportion is in this compartment; it has no  function, except as a protein-bound transport fraction.  Plasma iron concentration is very variable, even under  physiological conditions.   The causes of physiological changes in plasma iron  concentrations are not well understood, but alterations  can be very rapid and almost certainly represent shifts  between plasma and stores, not changes in total body   iron. The following factors are known to affect plasma  concentrations within a population:   • Sex and age differences Plasma iron concentrations,  like those of haemoglobin and the erythrocyte  count, are higher in men than in women, probably  for hormonal reasons. The difference is first evident  at puberty, before significant menstrual loss has  occurred, and disappears at the menopause.  Androgens tend to increase the plasma iron  concentration and oestrogens to lower it.   • Pregnancy and oral contraceptives In the first few  weeks of pregnancy, the plasma iron may rise to  concentrations similar to those found in men. A similar  rise occurs in women taking some oral contraceptives.   Iron concentrations can vary within an individual  by up to 100 per cent or more. This can be due to the  following:   • Random variations Day-to-day variations may be as  much as three-fold and usually overshadow cyclical  changes. They may be associated with physical or  mental stress or diet, but usually no cause can be  found.   • Circadian (diurnal) rhythm The plasma iron  concentration is higher in the morning than in the  evening. If subjects are kept awake at night, this  difference is less marked; it is reversed in night  workers.   • Monthly variations in women The plasma iron may  reach very low concentrations just before or during  the menstrual period. The reduction is probably due  to hormonal factors rather than blood loss.   Iron deficiency and iron overload usually cause low  and high plasma iron concentrations respectively. Iron  deficiency is associated with a hypochromic, microcytic  anaemia and with reduced amounts of stainable  bone marrow iron. Plasma ferritin concentrations are  usually, but not always, low. Iron overload is associated  with increased amounts of stainable iron in liver biopsy  specimens, and plasma ferritin concentrations are high.   Plasma iron is of little value in the diagnosis of iron  deficiency not least because other pathological factors  may affect plasma iron concentrations including the  following:   • Any acute or chronic illness (even a bad cold) causing a  fall in plasma iron concentration Chronic conditions  such as malignancy, renal disease, rheumatoid  arthritis and chronic infections are often associated  with normochromic, normocytic anaemia. Iron  stores and plasma ferritin concentrations are normal  or even increased; the anaemia does not respond to  iron therapy. Iron deficiency may be superimposed  on the anaemia of chronic illness, especially if drugs  are being taken that cause gastrointestinal bleeding;  plasma ferritin concentrations are then variable. The  finding of hypochromic erythrocytes is the most  sensitive index of this complication. Tow plasma  iron concentrations occur whether or not there is  any iron deficiency.   • Disorders in which the marrow cannot use iron, either  because it is hypoplastic or because some other  essential erythropoietic factor, such as vitamin B p  or folate, is deficient; plasma iron concentrations  are often high. Blood and marrow films may show  a typical picture, but, for example in pyridoxine-  responsive anaemia and in thalassaemia, the findings  in the blood film may resemble those of iron  deficiency; in the last two conditions the presence of  stainable marrow iron stores excludes the diagnosis  of iron deficiency.   • Haemolytic anaemia The plasma iron concentration  may be high during a haemolytic episode, as iron,  liberated from the destroyed erythrocytes, enters the  plasma; it is usually normal during the quiescent  periods when the iron enters the reticuloendothelial   system. Marrow iron stores and plasma ferritin  concentrations are usually increased in chronic  haemolytic conditions.   • Acute liver disease Disruption of hepatocytes may  release ferritin iron into the bloodstream and cause  a transient rise in the plasma iron concentration.  Cirrhosis may be associated with a similar finding,  perhaps due to increased iron absorption and intake.   Transferrin and total iron-binding capacity  Plasma iron concentrations alone rarely give  information about the state of iron stores. In rare  situations in which doubt remains after haematological  investigation, diagnostic precision may sometimes be  improved by measuring both the plasma transferrin  and the iron concentrations.   Plasma transferrin can be directly assayed or  measured indirectly by adding an excess of inorganic  iron to the plasma, any not bound to protein being  removed, usually with an exchange resin. The  concentration of iron remaining is assayed and the result  expressed as the total iron-binding capacity (TIBC).  Unsaturated iron-binding capacity (UIBC) is the TIBC  minus plasma iron concentration. This is usually a  valid approximation of the transferrin concentration.  In rare circumstances, of which the most common is  severe liver disease, plasma ferritin concentrations are  high enough to bind significant amounts of iron, and  the results of iron-binding capacity measurements are  then misleading as an assessment of the transferrin  concentration.   The plasma transferrin concentration is less labile than  that of iron. However, it rises:   • after about the 28th week of pregnancy even if iron  stores are normal,   • fall in those chronic illnesses associated with low  plasma iron concentrations,   • may be very lowin the nephrotic syndrome, associated  with a low plasma iron concentration, because the  relatively low-molecular-weight transferrin is lost in  the urine together with iron.   Thus the low plasma iron concentration of  uncomplicated iron deficiency is associated with a  high transferrin concentration and TIBC; that of non-  iron deficiency is associated with low concentrations  although plasma ferritin is the preferred investigation  for iron deficiency. If iron deficiency coexists with the  anaemia of chronic illness, the opposing effects of the  two conditions on the transferrin concentration make  it difficult to interpret transferrin, as well as plasma  iron, concentrations.   Circulating ferritin is usually in equilibrium with  that in stores. However, it is an ‘acute-phase’ protein  and its synthesis is increased in many inflammatory  conditions. The normal plasma ferritin concentration  is about lOOpg/L. A plasma ferritin concentration  below about lOpg/L suggests iron deficiency. Results  can be misleading if there is coexistent inflammatory  disease as accelerated synthesis may lead to normal  or even high plasma concentrations despite very low  iron stores. In this situation, the results of plasma iron  and transferrin assays are also difficult to interpret;  haematological parameters remain the most reliable  diagnostic indicators of iron deficiency, and possibly  also assay of the soluble transferrin receptor.   High concentrations of plasma ferritin usually occur in  significant iron overload, but may also be due to:   Thus the finding of a normal or low plasma ferritin  concentration almost certainly excludes the diagnosis  of iron overload, but a high one does not necessarily  confirm it.   The laboratory findings in conditions that may affect  plasma iron concentrations are summarized in Table  21.1.   Cells express transferrin receptors (TfRs) on their  surface. Plasma or so-called soluble transferrin binds  to TfR, preferring the diferric transferrin form.  The TfR-transferrin complex is internalized into  endosomes, where the iron is released into the cytosol.  The transferrin bound to the TfR returns to the cell  surface, where the apotransferrin dissociates and  another diferric transferrin can bind. It can therefore  be seen that iron uptake into cells is dependent on the  number of cell surface TfRs and the concentration and  percentage saturation of transferrin.   Plasma TfR concentrations reflect total cellular TfR  levels, which are increased in iron deficiency as TfR is  up-regulated due to the cell’s requirements for iron,  but, unlike ferritin, are not increased as a result of the   acute-phase response of inflammation. Plasma TfR  concentrations are also directly related to the rate of  erythropoiesis and to increased erythrocyte turnover,  such as occurs in haemolytic anaemia.   As the body iron content is determined by control  of absorption, rather than excretion, parenteral  iron therapy (which bypasses absorption) and  repeated transfusions of blood [which contains  about 4.5 mmol (250 mg) of iron per unit] may cause  iron overload. In anaemias other than those due to  iron deficiency, stores are normal or even increased.  Proven iron deficiency usually requires iron therapy  and the oral route is preferable, as anaphylaxis may  occur with parenteral administration. Repeated blood  transfusions may be needed to correct severe non-  iron deficiency anaemia, for example if the marrow  is hypoplastic, but the danger of overload should be  remembered.   Iron absorption is stimulated by anaemia even if  iron stores are increased. The treatment of non-iron  deficiency anaemia with oral iron supplements is not  only ineffective, but can also lead to iron overload; this  is especially likely in haemolytic anaemia, in which the  iron released from destroyed erythrocytes remains in  the body.   The only route of iron loss is cell desquamation. Iron  absorbed from the gastrointestinal tract or administered  parenterally in excess of daily loss accumulates in body  stores. If such a ‘positive balance’ is maintained over  long periods, iron stores may exceed 350 mmol (20 g),  that is, about five times the normal amount.   - anaemia with increased, but ineffective  erythropoiesis, for example sideroblastic  anaemia, thalassaemia, myelodysplasia,   A very rare cause of iron overload is an inherited  deficiency of transferrin.   A 46-year-old man was investigated in the hepatology  clinic because of abnormal liver function tests. He  was known to have type 2 diabetes mellitus. The  results of some of his biochemistry tests prior to liver  biopsy were as follows:   The patient was found to have haemochromatosis.  This was confirmed by studies of the HFE gene,  which showed him to be homozygous for the C282Y  mutation, and iron staining on liver biopsy. His  diabetes mellitus may also have been secondary  to this, as were the abnormal liver function tests.  Elevated plasma ferritin concentrations can be  seen in liver disease, haemolysis, alcohol excess and  conditions in which there is an acute-phase response.  However, the high iron saturation of more than  45 per cent suggests haemochromatosis.   The effect of the accumulated iron depends on its  distribution in the body. This, in turn, is influenced  partly by the route of entry. Two main patterns are seen  at postmortem or in biopsy specimens.   Parenchymal iron overload occurs in  haemochromatosis and in patients with ineffective  erythropoiesis. Iron accumulates in the parenchymal  cells of the liver, pancreas, heart and other organs.  There is usually associated functional disturbance or  tissue damage.   Reticuloendothelial iron overload is seen after  excessive parenteral administration of iron or multiple  blood transfusions. The iron accumulates initially in  the reticuloendothelial cells of the liver, spleen and  bone marrow.   In dietary iron overload, both hepatic  reticuloendothelial and parenchymal overload may  occur, associated with scurvy and osteoporosis.  Whatever the cause of massive iron overload, there may  be parenchymal accumulation and tissue damage.   Haemosiderosis is a histological definition. An  increase in iron stores as haemosiderin can be seen. It  does not necessarily mean that there is an increase in  total body iron; for example, in many types of anaemia  there is reduced haemoglobin iron but increased storage  iron. Haemochromatosis describes the clinical disorder  due to parenchymal iron-induced damage.   Syndromes of iron overload  Hereditary or primary haemochromatosis  Hereditary haemochromatosis usually has an  autosomal recessive mode of inheritance and a defect  of the haemochromatosis gene ( HFE ). Approximately  1 in 10 people in Western societies are carriers and  about 1 in 1000 are homozygous. The gene for this  disorder is closely associated with the human leucocyte  antigen (HLA) gene locus on chromosome 6. Some  forms of haemochromatosis result in low hepcidin  concentrations. Factors such as alcohol abuse may hasten  the accumulation of iron and the development of liver  damage. Males are more likely to manifest the condition,  as menstruation in females lowers tissue iron stores.   There is increased intestinal absorption of iron  over many years, which produces large iron stores in  the tissues, such as the liver, pancreas, joints, heart and  gonads. It presents, usually in middle age, as cirrhosis  of the liver, sometimes with diabetes mellitus, joint  pains, cardiomyopathy, hypogonadism and greyish  skin pigmentation due to melanin, not iron. It has been  referred to as ‘bronzed diabetes’, although such skin  features tend to occur late.   Examination of liver biopsy specimens maybe useful,  giving an idea of disease severity and showing increased  iron content when stained by Perl’s blue staining,  although magnetic resonance imaging (MRI) is also  able to determine iron content non-invasively. There  is an association with hepatocellular carcinoma, which  can be screened for by assay of plasma a-fetoprotein  (AFP).   The diagnosis may be made on the basis of the  measurement of plasma ferritin, which is often greater  than 1000 pg/L. Also plasma iron concentration  and TIBC, with a saturation of greater than about  45 per cent, are indicative of haemochromatosis and  may increase before plasma ferritin concentration does.   The relatives of patients with proven hereditary  haemochromatosis should be investigated. Genetic  tests are now available, with a majority of cases of  haemochromatosis usually being due to the C282Y  or H63D HFE gene mutations. There are rarer non-  HFE forms of haemochromatosis associated with gene  defects of hepcidin, ferroportin, the transferrin receptor  and DMT-1.   Treatment is often by venesection, aiming to reduce  the plasma ferritin to less than 100 pg/L. Each 500 mL  unit of blood removes about 250 mg of iron from the  body.   Several types of anaemia may be associated with iron  overload. In some, such as hypoplastic anaemia and the  anaemia of chronic kidney disease, the cause is multiple  blood transfusions; the iron initially accumulates in the  reticuloendothelial system. If overload is massive (often  over 100 units of blood), deposition may occur in  parenchymal cells, with the development of secondary  haemochromatosis.   In anaemias characterized by erythroid marrow  hyperplasia but with ineffective erythropoiesis, such  as thalassaemia major and sideroblastic anaemia, there  is, in addition, increased absorption of iron. Secondary  haemochromatosis develops at a lower transfusion load  than in hypoplastic anaemia.   This can obviously not be treated by venesection. The  tendency for transfusion to aggravate iron overload  further can be minimized by giving the iron-chelating  agent desferrioxamine each time; this can be excreted in  the urine with any non-haemoglobin iron.   Increased iron absorption due to excessive intake is rare.  One well-described form is, however, relatively common  in the rural black population of southern Africa. The  source is beer brewed in iron containers. Usually, the  excess is confined to the reticuloendothelial system and  the liver (both portal tracts and parenchymal cells), and  there is no tissue damage. In a small number of cases,  deposition in the parenchymal cells of other organs  occurs and the clinical picture may resemble that of  primary haemochromatosis; it may be distinguished by  the high concentration of iron in the reticuloendothelial  system seen in the bone marrow.   Scurvy and osteoporosis may occur in this form  of iron overload. The ascorbate deficiency may be  due to its irreversible oxidation in the presence of  excessive amounts of iron, and osteoporosis sometimes  accompanies scurvy. Ascorbate deficiency also  interferes with normal mobilization of iron from the  reticuloendothelial cells; plasma iron concentrations  may be low and the response to chelating agents poor,  despite iron overload.   There are many causes of anaemia, which may be  due either to iron deficiency or to a variety of other  conditions, sometimes associated with high iron  stores. The subject of the diagnosis of anaemia, such  as haemolytic or pernicious anaemia, is covered more  fully in textbooks of haematology. Blood haemoglobin  reflects the major metabolic pool of iron, ferritin the  storage pool and transferrin the transit pool. The  clinical impression of anaemia should be confirmed  by blood haemoglobin estimation. Iron deficiency can,  however, exist with haemoglobin concentrations within  the reference range.   The mean corpuscular volume (MCV) and mean  corpuscular haemoglobin (MCH) should be checked  and often a blood film examined. Iron deficiency  anaemia is microcytic and hypochromic in type, and  these findings may be evident before the haemoglobin  concentration has fallen below the reference range.  Normochromic, normocytic anaemia is a non-specific  finding, usually associated with other chronic disease; it  is associated with iron deficiency only if there has been  very recent blood loss.   In most cases of anaemia, consideration of these  findings together with the clinical picture may elucidate  the cause. Anaemias such as those due to sickle cell  disease, thalassaemia or of the sideroblastic type,  although rarer, are most likely to confuse the picture,  since they too are hypochromic but are not due to iron  deficiency. A low plasma ferritin concentration (less  than 10 pg/L) is indicative of iron deficiency due to low  iron stores.   One major diagnostic dilemma is to separate iron  deficiency anaemia from anaemia of chronic disorders,  for example carcinoma or inflammatory disease. In  the latter there is often an acute-phase response, which  can raise plasma ferritin concentrations, which are  influenced by inflammation. This can, therefore/mask’ a   low plasma ferritin concentration due to iron deficiency.  A plasma ferritin concentration more than 200 pg/L  under these circumstances probably rules out iron  deficiency anaemia, but values more than 10 pg/L but  less than 200 pg/L are equivocal and do not distinguish  between iron deficiency anaemia and anaemia of  chronic disorders. Under these circumstances, plasma-  soluble TfR assay may be useful; this should be raised in  iron deficiency but not in anaemia of chronic disorders.   An unequivocally low plasma ferritin concentration  confirms iron deficiency, but a normal or high one  should not be assumed to exclude it. A bone marrow  film for iron staining may be needed to confirm the  diagnosis of iron deficiency in ambiguous cases. If a  diagnosis of iron deficiency is made, it is essential to rule  out blood loss and its source. Particularly important  is to exclude gastrointestinal malignancy and also  gynaecological pathology. Faecal occult blood samples  may show the presence of gastrointestinal blood loss,  which may necessitate prompt endoscopic investigation  (see Chapter 16). A patient with proven iron deficiency  who shows no response to oral iron treatment within  a few weeks may not be taking the tablets. If iron has  been taken, malabsorption (usually as part of a general  absorption defect) is a possible explanation of a poor  response.   All of these should be measured. The plasma iron  concentration is almost invariably high in primary  haemochromatosis, often more than 36 pmol/L.  This is associated with a reduced plasma transferrin  concentration and TIBC, and the percentage  saturation is usually greater than about 45 per cent,  and may be nearer 90-100 per cent. In the presence  of infection or malignancy, however, the plasma iron  concentration and percentage saturation may be lower  than expected; the plasma transferrin concentration  remains low.   Plasma ferritin concentrations are high in  most patients with iron overload (whether  reticuloendothelial or parenchymal). It is rare in  hereditary haemochromatosis to have a normal plasma  ferritin concentration, although this may occur in the  early stages, when the iron saturation may be more  useful diagnostically. Remember that plasma ferritin  can be elevated in hepatic disease and high alcohol   A 64-year-old woman saw her general practitioner  because of weight loss, tiredness and change in bowel  habit (the recent development of loose stools). The  following relevant laboratory results were returned:   Mean corpuscular haemoglobin (MCV) 70 fL (80-95)  Mean corpuscular volume (MCH) 23 pg (28-34)   The hypochromic, microcytic anaemia (suggested by  the low MCH and MCV respectively) and also low  plasma ferritin and iron concentrations all point to  an iron deficiency. The patient was later shown by  colonoscopy to have a carcinoma of her descending  colon. Iron deficiency should prompt investigation  as to why this is present, and bleeding needs to  be excluded. Although not routinely needed, the  plasma transferrin receptor concentration would be  expected to be increased in iron deficiency.   intake. There is also a rare familial hyperferritinaemia  due to a defect in ferritin synthesis that should not be  misdiagnosed as haemochromatosis.   If all these values are normal, it is unlikely that the  patient has iron overload. If there is doubt, particularly  in the face of a positive family history, genetic studies of  the HFE gene may be indicated.   Liver biopsy specimens contain large amounts of  stainable iron, which may be mainly in parenchymal or  mainly in reticuloendothelial cells. If haemochromatosis  is confirmed, liver function tests are necessary, as is   exclusion of diabetes mellitus or impaired glucose  regulation and other clinical features of iron overload  such as pituitary or gonadal involvement. Cirrhosis may  progress to hepatocellular carcinoma, which should be  looked out for.   This is usually normal in haemochromatosis, in which  overload is predominantly parenchymal, but may  be greatly increased in reticuloendothelial overload.  A similar loading of the reticuloendothelial cells is  found when there is deficient use of marrow iron for  haemoglobin synthesis, as in many haematological,  neoplastic and chronic inflammatory diseases.   The blood relatives of patients found to have  haemochromatosis should be investigated. Estimations  of plasma iron and percentage saturation of transferrin  are the most sensitive tests and may change before  ferritin levels rise if iron stores are not significantly  increased. Genetic studies looking at HFE gene  mutations are now being used.   The porphyrias are a rare group of disorders of haem  synthesis, resulting from a deficiency of one of the  enzymes on the haem synthetic pathway (Fig. 21.4).  Haem production is impaired, but reduced feedback  inhibition of ALA synthase (the rate-limiting step)  may maintain adequate haem levels at the expense of  overproduction of porphyrins or their precursors.   Porphyrias can be classified into those that  present with acute porphyric attacks and those with   Figure 21 .4 Sites of enzyme deficiencies in (1) acute  intermittent porphyria, (2) hereditary coproporphyria,  (3) variegate porphyria. ALA, 5-aminolaevulinic acid;  COPRO, coproporphyrinogen; PBG, porphobilinogen;  URO, uroporphyrinogen; PROTO, protoporphyrinogen.   - porphyria cutanea tarda, which can be either  genetic or acquired,   The major clinical and biochemical features are  outlined in Table 21.2 and discussed briefly below and  correlate well with the biochemical abnormalities.   A 17-year-old woman was admitted under the  care of the general surgeons because of abdominal  pain, which she had been experiencing with  varying severity over the preceding year. She  underwent a laparotomy, although no abdominal  abnormality was found. However, post-operatively  her blood pressure rose from 128/70 mmHg to  170/120 mmHg. An acute porphyria was considered  and the following porphyria screening results were  obtained:   Urinary porphobilinogen (PBG) 213pmol/L (0-10)  Urinary total porphyrins 6400nmol/T (20-330)  Faecal porphyrins 1 10 nmol/g dry weight (10-210)   Abdominal pain and hypertension can be presenting  features of an acute porphyria. Further investigation,  including family studies and genetic tests, confirmed  the diagnosis of acute intermittent porphyria.  Note that the anaesthetic agents given for the  operation worsened the acute attack. Indeed, certain  anaesthetic drugs, such as thiopental, can induce  5-aminolaevulinic acid synthase activity, thereby  increasing flux through the porphyrin pathways.  The lack of skin manifestations and normal  faecal porphyrins support the diagnosis of acute  intermittent porphyria.   Porphobilinogen synthase deficiency is an autosomal  recessive condition; PBG concentration is not raised,  although that of ALA is, and the condition is very  rare. The other three acute porphyrias listed above  are autosomal dominantly inherited disorders and  have latent and acute phases. The symptoms and  biochemical abnormalities of the latent phases differ  and reflect the nature of the enzyme defect. In the acute  phases, however, the biochemical and clinical pictures  characteristic of excessive ALA and PBG production  associated with neurological and abdominal symptoms  develop. The similarities and differences are best  explained by referring to a simplified scheme of the  haem synthetic pathway (see Fig. 21.1).   The enzyme defect tends to reduce haem levels, which  in turn increase ALA synthase activity by a decrease  in negative feedback inhibition. Increased ALA  synthase activity has been demonstrated in all the  porphyrias. Haem levels are maintained at the expense  of the accumulation and excretion of the substance  immediately before the block.   In acute intermittent porphyria there is increased  urinary ALA and PBG excretion, although it may not  be detectable in all patients; the latent phase is usually  asymptomatic. It is due to PBG deaminase deficiency.   In hereditary coproporphyria there is increased  faecal coproporphyrin excretion; the increase in the  concentration of porphyrins may produce skin lesions,  but less commonly than in variegate porphyria. It is due  to coproporphyrinogen oxidase deficiency.   In variegate porphyria there is increased faecal  protoporphyrin excretion; the increase in the  concentration of porphyrins may produce skin  lesions. There is no increase in erythrocyte porphyrins;  however, plasma emission fluorescence shows a peak  of 624-626 nm when stimulated at 405 nm, while the  other acute porphyrias show peaks at 615nm. It is due  to protoporphyrinogen oxidase deficiency.   It is essential to investigate the close blood relatives of  any patient with porphyria. Screening tests for excess  urinary PBG and ALA are inadequate to diagnose latent  acute intermittent porphyria, and even quantitative  estimation may fail to detect all carriers. The activity of  the relevant enzymes can be measured in erythrocytes,  and genetic tests may be useful.   AIP, acute intermittent porphyria; CEP, congenital erythropoietic porphyria; HC, hereditary coproporphyria; PCT, porphyria cutanea tarda;  PP, protoporphyria; VP, variegate porphyria.   Acute disturbances such as peripheral neuropathy or  abdominal pain may occur in an acute attack, in which  the precursors ALA and PBG are produced in excess.  These features may be due to a direct toxic effect of  ALA or PBG. Convulsions may occur in acute attacks,  sometimes associated with hyponatraemia; this may  be due to the syndrome of inappropriate antidiuretic  hormone (see Chapter 2).   5-Aminolaevulinate synthase activity may be  further increased by a number of drugs, particularly  barbiturates, estrogens, sulphonamides, phenytoin,  halothane and griseofulvin. So many drugs have been  implicated that it is probably safer to prescribe from a  ‘White List’ rather than excluding particular drugs. Also  important are alcohol and smoking and acute illness  such as infection (this may be a direct effect or may be  due to an increased demand for haem).   Acute attacks occur only in a small proportion of  patients exposed to the provoking agents. They are more  common in premenstrual or pregnant women, and in  women rather than in men, and usually occur only after  puberty. Colicky abdominal pain, due to involvement  of the autonomic nervous system, and neurological  symptoms ranging from peripheral neuropathy to  quadriplegia are usually the presenting features. Death  may result from respiratory paralysis or blood pressure  instability due to autonomic neuropathy.   The acute attack closely resembles serious acute  intra-abdominal conditions and, if the diagnosis is not  made, the patient may be subjected to surgery with the  use of a barbiturate anaesthetic; this and the stress of  operation may aggravate the condition.   The acute attack is marked by an increase in ALA and  PBG production. In acute intermittent porphyria, this  increase is due to the block imposed by an inherited  deficiency of PBG deaminase; in hepatic coproporphyria  and variegate porphyria, this enzyme becomes rate  limiting and is unable to respond normally to the   increased demand. The increase in urinary ALA and  PBG concentrations is the hallmark of the acute porphyric  attack and, in hereditary coproporphyria and variegate  porphyria, is superimposed on all the other biochemical  abnormalities. The accelerated activity of the pathway  and the spontaneous conversion of the precursors  to porphyrin lead to increased urinary porphyrin  excretion. About 1 per cent of acute attacks are fatal. It  is important to stop precipitating drugs and not to use  medications that could evoke an attack. Convulsions  may be treated with gabapentin or vigabatrin. Haem  arginate, given by slow intravenous infusion, can result  in a reduction in ALA and PBG concentrations and  thus reduce some of the features of the acute attack, but  not usually the neuropathy.   The cutaneous porphyrias include porphyria cutanea  tarda, congenital erythropoietic porphyria and  protoporphyria.   This is the most common porphyria. In patients with  porphyria cutanea tarda, the skin is unduly sensitive to  minor trauma, particularly in sun-exposed areas; the  most common presenting feature is blistering on the  backs of the hands. Less commonly, the lesions appear  on the face. Increased facial hair and hyperpigmentation  occur in chronic cases. Acute attacks do not occur, but  this type of porphyria is associated with hepatic damage.   The basic defect is an inability to convert  uroporphyrinogen to coproporphyrinogen due to a  deficiency of uroporphyrinogen decarboxylase. There  are two types of porphyria cutanea tarda:   • It may be an autosomal dominantly inherited disorder.  The familial form is rare, although environmental  factors are also important.   • Most cases are acquired. Factors that produce clinical  disease, possibly by aggravating an underlying genetic   deficiency, include alcohol abuse, iron overload or  high-dose estrogen therapy. Symptoms improve when  the offending substance is withdrawn. Some liver  toxins such as hexachlorobenzene directly inhibit the  activity of the enzyme. There is an association with  hepatitis C. Iron overload can also be present and  haemochromatosis needs to be excluded.   The impaired conversion leads to an accumulation  of uroporphyrinogen and porphyrins intermediate  between it and coproporphyrinogen. These deposit  in the skin and are excreted in the urine in increased  amounts. Faecal porphyrins are not increased but the  abnormal pattern of intermediate porphyrins may  be detected by chromatography; this finding is of  diagnostic value. It is important not to confuse this  disorder with the coproporphyrinuria of liver disease  (see below).   Two rare inherited disorders are associated with the  accumulation of porphyrins in erythrocytes. Acute  porphyric attacks do not occur and ALA and PBG  excretion are normal.   Congenital erythropoietic porphyria is inherited  as an autosomal recessive characteristic due to  uroporphyrinogen III synthase deficiency. Usually, blood  erythrocyte and plasma uroporphyrin I concentrations  are very high from infancy onwards and there is severe  photosensitivity. Porphyrins are also deposited in bones  and teeth, which fluoresce in ultraviolet light; the teeth  may be brownish-pink in colour. Hirsutism, especially  of the face, also occurs and there is haemolytic anaemia.  Urinary porphyrin concentrations are grossly increased,  although faecal porphyrin levels are less so.   This is an autosomal dominantly inherited disorder due  to ferrochelatase deficiency in which protoporphyrin  concentrations are increased in erythrocytes and faeces.  There is mild photosensitivity, and hepatocellular  damage may lead to liver failure.   These two conditions tend to give rise to  photosensitivity, whereas there is increased skin fragility  in the other porphyrias that involve the skin. The  erythropoietic porphyrias can be treated by sunlight  avoidance, for example by using skin sunblocks. In  porphyria cutanea tarda, venesection may be used to  reduce iron stores and oral chloroquine to increase the   excretion of porphyrins. Carotene treatment may be  useful in erythropoietic porphyria.   Porphyria is not the only cause of disordered porphyrin  metabolism, and positive screening tests must be  confirmed by quantitative analysis, with identification  of the porphyrin. Other causes must be considered.   Lead poisoning inhibits several of the enzymes  involved in haem synthesis, including PBG synthase  and ferrochelatase, and eventually causes anaemia. The  urine contains increased amounts of ALA (an early  and sensitive test), and coproporphyrin. Some of the  symptoms of lead poisoning, such as abdominal pain  and peripheral neuropathy, are similar to those of the  acute porphyric attack, and may cause difficulty in  diagnosis. However, the excretion of PBG is not usually  increased. Zinc protoporphyrin concentration rises  with increased lead exposure, although the method of  choice for assessing exposure to inorganic lead is blood  lead concentration. Other features of lead poisoning  include lead staining of the teeth and basophilic  stippling of red blood cells.   This may increase urinary coproporphyrin levels,  possibly because of decreased biliary excretion. It is  probably the most common cause of porphyrinuria.  Occasionally there is mild photosensitivity (in  porphyria cutanea tarda, the more severe skin lesions  are due to uroporphyrin excess).   These lesions may produce raised levels of faecal  porphyrin by degradation of haemoglobin. If there is  bleeding from the lower part of the tract, the blood  reaches the rectum before there is time for conversion;  this may be of help in locating the approximate site of  bleeding.   The laboratory should be notified and a check made  of which type of samples is required. Porphyrins   are relatively stable, but samples must be protected  from light. Porphobilinogen rapidly polymerizes to  uroporphyrins and consequently a random fresh  urine sample is more suitable than a 24-h collection.  All samples should be analysed as soon as possible  after collection. Samples should not be sent simply  requesting a ‘porphyrin screen’; an indication should  be given of which type of porphyria is suspected, giving  the relevant clinical details, so that the laboratory can  select the appropriate tests.   Generally, urine, faeces and blood (both plasma and  erythrocytes) are needed for complete analysis. The  technique of plasma fluorescence has recently proved  useful to distinguish some of the porphyrias (Table  21.3). Specialist laboratories can carry out enzyme  assays and genetic tests.   Fresh urine should be tested immediately for PBG  using Ehrlich’s reagent. If PBG is not present, it is highly  unlikely that the patient is suffering from an acute  porphyric attack unless he or she has the rare PBG  synthase deficiency, in which case ALA concentration  would be expected to be raised. A patient with a history  of repeated attacks of abdominal pain or neurological  symptoms may have acute intermittent porphyria,  variegate porphyria or hereditary coproporphyria.   If an acute porphyria is confirmed, the concentration  of porphyrins in urine and in a random sample of faeces  should be measured. Raised values suggest variegate  porphyria (protoporphyrin and coproporphyrin) or  hereditary coproporphyria (coproporphyrin). Acute  intermittent porphyria does not normally show   Patients with acute intermittent porphyria who  have once had an acute attack may continue to excrete  increased amounts of PBG for many months. This  condition does not show skin lesions, unlike the other  acute porphyrias. Plasma fluorescence may reveal  different emission peaks when stimulated at 405 nm,  for example variegate porphyria may show a peak at  625 nm when stimulated at 405 nm, whereas the other  acute porphyrias show a peak at 615 nm.   It is possible to assay enzymes of haem metabolism in  red blood cells. For example, decreased PBG deaminase  is found in patients with acute intermittent porphyria.  There is often overlap in levels between affected and  unaffected people but, within a family, carriers can be  shown to have levels about half those of unaffected  members. This test may also detect affected children.  However, the enzyme activity is related to the mean  age of the red blood cells; this assay is not suitable  for children under the age of about 9 months or for  individuals with haemolytic disorders. Negative urine  and faeces porphyrin tests do not exclude the diagnosis  of latent porphyria, and in these cases enzyme and  genetic tests may be useful.   Skin lesions may occur in any type of porphyria  other than acute intermittent porphyria. Blood,  urine and faeces should be sent for testing. Increased  concentrations of erythrocyte porphyrins suggest  protoporphyria or congenital erythropoietic porphyria    +, present; absent; AIP, acute intermittent porphyria; CEP, congenital erythropoietic porphyria; Copro, coproporphyrinogen I or  III; HC, hereditary coproporphyria; Isocopro, isocoproporphyrinogen; PBG, porphobilinogen; PCT, porphyria cutanea tarda; POR,  porphyrin; PP, protoporphyria; Proto, protoporphyrinogen; Urohepta, uroheptaporphyrinogen; Uro, uroporphyrinogen; VP, variegate  porphyria; Zn, zinc.   a Plasma fluorescence emission wavelength peak when stimulated at 405 nm.   (very rare). High concentrations may also occur in iron  deficiency anaemia and lead poisoning.   Increased concentrations of urinary porphyrins  suggest porphyria cutanea tarda or congenital  erythropoietic porphyria. The increased uroporphyrin  excretion in these conditions must be distinguished  from the coproporphyrinuria of liver disease.  Increased concentrations of faecal porphyrins occur  in protoporphyria, variegate porphyria and hereditary  coproporphyria. These may be distinguished by  chromatographic separation of porphyrins; this will  also demonstrate the abnormal pattern of porphyria  cutanea tarda.   Protoporphyria shows a unique plasma fluorescence  emission peak of 632 nm when stimulated at 405 nm.  Enzyme and genetic tests may be useful.   If one of the porphyrias is diagnosed, it is essential to  investigate blood relatives. Those found to have the  condition must be counselled regarding the medications  that may precipitate an attack. King George III was  thought to have an acute porphyria, although it has not  been proven (Fig. 21.5).   Carriers of variegate porphyria and hereditary  coproporphyria may be identified after puberty by the  demonstration of clearly increased faecal porphyrin  excretion. Normal excretion before puberty does not  exclude the diagnosis. Some of the porphyrias may be  diagnosed by enzyme determination, for example acute  intermittent porphyria is detected by measuring red   cell PBG deaminase activity. Genetic studies are now  possible in specialist laboratories to investigate the  porphyrias.    Figure 21.5 King George III was thought to have an  acute porphyria, although it has not been categorically  proven. © Bettman/CORBIS.   • Haemoglobin contains most (70 per cent) of  the body’s iron. Iron is mandatory for normal  haemopoiesis.   • Ferritin is an intracellular iron-binding protein.  Iron is also absorbed in the small intestine and  bound to transferrin within the circulation. It is  stored bound to ferritin.   • Iron deficiency is common and poor iron intake  or blood loss needs to be excluded. A low plasma  ferritin concentration suggests iron deficiency.   • Unbound iron is toxic to the body. Haemochroma-  tosis is an autosomal recessive condition of chronic  iron overload. This is associated with liver disease,   The haem moiety of haemoglobin contains  tetrapyrrole rings synthesized from porphyrinogen  precursors. Disorders of the haem synthetic  pathways can lead to porphyria. Most of the  porphyrias are autosomal dominant.   The porphyrias can be divided into (a) the acute  group, which can present with acute abdomen,  neuropsychiatric symptoms and hypertension, and  (b) cutaneous (non-acute) forms that may present  with skin symptoms.   The clinical biochemistry laboratory often plays a  central role in the diagnosis and management of acute  myocardial infarction. A number of cardiovascular  risk factors have been described, some of which can be  assayed in the biochemistry laboratory, and these are  discussed in this chapter.   One of the largest killers in Western urbanized societies is  acute myocardial infarction. Its diagnosis is usually made  on the clinical presentation and electrocardiographic   A 46-year-old man was admitted late at night  (23.30 h) to casualty by ambulance because of  a tight central chest pain that had started at  03.00 h that morning. He had delayed calling for  medical assistance because he thought it was only  indigestion. In the ambulance, the paramedics gave  him an intramuscular injection of diamorphine for  pain relief. The ECG was normal.   The normal plasma troponin T makes a diagnosis of  acute myocardial infarction unlikely, as this would  be expected to be elevated in view of when the chest  pain started. The elevated plasma CK concentration  is not specific for cardiac damage and may have been  raised as a result of muscle damage secondary to  the intramuscular injection: beware of interpreting  elevated plasma CK concentrations in the presence  of intramuscular injections. A CK-MB concentration  would be less likely than total CK concentration to  be elevated in skeletal muscle damage, as CK-MB is  predominantly cardiac in origin.   findings and confirmed by the characteristic changes in  plasma enzyme activities or troponin levels. Myocardial  infarction can be defined as a typical rise and/or fall  of cardiac biomarkers (preferably troponin) with at  least one value above the 99th percentile of the upper  reference limit together with evidence of ischaemia  with at least one of the following: symptoms of  ischaemia, electrocardiogram (ECG) changes (new ST  changes or new left bundle branch block), development  of ECG pathological Q waves or imaging evidence of  myocardial ischaemia. (Fig. 22.1). This classification   A 66-year-old man was sent by his general  practitioner to casualty because of tight chest pain  that had occurred 3 days previously. The pain had  largely resolved after 6 h, but he was left feeling weak  and breathless, which worsened over a few days,  causing him eventually to seek medical attention.  The following laboratory test results were found:   An electrocardiogram showed changes suggestive of  a myocardial infarction in the lateral leads V4-V6.   The plasma CK activity has returned to normal  because of the time delay since myocardial infarction,  while the plasma troponin T concentration still  remains elevated. Plasma CK usually starts to rise  4-6 h after a myocardial infarction and to normalize  after a couple of days. Conversely, plasma troponin  T starts to rise at 4-6 h post infarct and remains  elevated for as long as about 10 days. Troponins are  thus useful cardiac markers in both the early hours  and a few days later.   Figure 22.1 ECG changes; showing ST depression as  in ischaemic angina pectoris and ST elevation after a  myocardial infarction. Adapted with kind permission  from Kinirons M and Ellis H. French’s Index of  Differential Diagnosis, 15th edition. London: Hodder  Arnold, 2011.   also recognizes that patients previously classified as  having unstable angina or minor myocardial injury are  now reclassified as having non-ST segment elevation  myocardial infarction (NSTEMI).   It is useful to note that acute myocardial infarction  and NSTEMI have a common pathophysiological  pathway, namely acute coronary artery plaque rupture,  followed by a series of events resulting in thrombosis  formation. The outcome is dependent on the state  of the collateral circulation and the severity of artery  occlusion (Fig. 22.2). In other words, clinically we no  longer think in terms of the presence or absence of an  acute myocardial infarction, but instead of a spectrum  of disease ranging from angina pectoris through to  acute myocardial infarction; this stratification is based  upon cardiac markers reflecting ischaemic damage.  Acute coronary syndrome is the term used to embrace  ST segment elevation myocardial infarction (STEMI),  as well as NSTEMI and unstable angina pectoris   It is also particularly important to diagnose acute  myocardial infarction promptly, as thrombolytic  therapy may be indicated to try to dissolve the thrombus  in situ or angioplasty. The earlier this therapy is given,  the better the prognosis, as ‘minutes are myocardium’.  Thus, biochemical markers need to be specific, sensitive  and change rapidly to optimize the diagnosis and  treatment of acute myocardial infarction.   Troponins are muscle-regulatory proteins present in  skeletal and cardiac muscle. Three troponins have  been reported, namely troponin C (TnC), troponin I  (Tnl) and troponin T (TnT). There are no structural    Figure 22.2 Coronary arteriogram showing critical right  coronary artery stenosis (arrowed). Reproduced with  kind permission from Browse NL, Black J, Burnand  KG, Corbett SA and Thomas WEG (eds). Browse’s  Introduction to the Investigation and Management of  Surgical Disease. London: Hodder Arnold, 2010.   differences between cardiac and skeletal muscle TnC.  However, the cardiac and skeletal forms of Tnl and TnT  are structurally different and can be distinguished by  immunological assays.   Troponin I and TnT appear in the plasma 4-8 h  or earlier with high- sensitivity troponin assays after  symptoms of acute myocardial infarction, and are  best measured 12 h after the start of chest pain. They  are therefore not early markers of acute myocardial  infarction, but they do stay elevated for about 7-10  days in plasma, which makes them useful in the late  presentation of chest pain.   Troponin T may be elevated in patients with  chronic kidney disease and thus may not be so cardio-  specific. However, Tnl can be extensively modified by  proteolysis after release into the plasma. This means  that measurements may give varying results as different  antigenic epitopes are revealed, depending upon the  commercial supplier of the assay. There is no universal  agreement as to which troponin assay, i.e. Tnl or TnT,  is best.   An increased Tnl or TnT concentration is a sensitive  marker of occult myocardial damage even in non-  ischaemic conditions. Plasma troponin concentrations  are increased in subarachnoid haemorrhage (due to  vasoactive peptide release affecting the myocardium),   hypertension, tachyarrhythmias, cardiac surgery, sepsis,  congestive cardiac failure, pulmonary embolism and  hypothyroidism. It should be remembered that the  universal definition of an acute myocardial infarction  requires not only a change in troponin but also clinical  or ECG evidence.   Research has shown that troponins allow a subset  of patients with rest-unstable angina to be classified  despite normal plasma CK-MB concentrations. Those  with raised plasma cardiac troponin concentrations  were found to be more likely to have an acute  myocardial infarction or die within 6 months of  the event. In other words, troponins allow risk  stratification of patients with acute coronary  syndrome. In another study it was found that, unlike  CK-MB, cardiac troponins have prognostic value  after coronary thrombolysis therapy, with troponin  concentrations on admission correlating with 6 -week  mortality. Low-molecular-weight heparin or platelet-  receptor blockers (glycoprotein GIIB/IIIA inhibitors)  reduce cardiac events in non-Q-wave coronary  syndromes with raised concentrations of cardiac  troponins compared with those without troponin  concentration elevation. There are also new sensitivity  troponin assays which may allow earlier diagnosis and  better risk stratification.   In view of these findings, cardiac troponins have  been recommended as the biochemical cardiac marker  of choice. However, sometimes a panel of cardiac  markers may be useful in making the diagnosis of  acute myocardial infarction, for example myoglobin  rises early, thus alerting the clinician to possible cardiac  problems, while the later rise of troponin allows  greater specificity. Other cardiac markers that are  being studied include ischaemia-modified albumin,  pro-brain natriuretic peptide (pro-BNP) and glycogen  phosphorylase isoenzymes BB.   As mentioned in Chapter 21, myoglobin is a low-  molecular-weight haem-containing protein found  in both skeletal and cardiac muscle. Because of its  low molecular weight, it is rapidly released from the  myocardium upon damage, and a typical rise occurs  within 2-4 h after the onset of acute myocardial  infarction. This is useful for the early diagnosis of acute  myocardial infarction, as this rise is generally earlier  than that of the other currently used cardiac markers.  Unfortunately, myoglobin is not cardiac specific, being  also found in skeletal muscle, and thus is less useful   in the diagnosis of acute myocardial infarction unless  used in conjunction with other markers.   The plasma enzyme estimation of greatest value  is that of CK (see also enzymology, Chapter 18),  although this has been largely superseded by the  assay of cardiac troponins. At one time, the enzymes  lactate dehydrogenase (LDH) or hydroxybutyrate  dehydrogenase (HBD) and aspartate aminotransferase  (AST) were used diagnostically, although they are now  rarely assayed in the diagnosis of acute myocardial  infarction or NSTEMI.   An approximate guide to the sequence of changes  after acute myocardial infarction is given in Table 22.1  (see also Fig. 22.3).   All plasma enzyme activities (including that of CK-  MB) maybe normal until at least 4h after the onset of  chest pain due to a myocardial infarction; blood should  not be taken for enzyme assay until this time has elapsed. If  the initial plasma CK activity is normal, a second sample  should be taken about 4-6 h later. A rise in the plasma  CK activity supports the diagnosis of an infarction. The  simultaneous measurement of plasma CK-MB activity,  which is shown to exceed about 5 per cent of the total  CK activity, may occasionally help in early diagnosis; a  raised plasma CK-MB activity or concentration alone is  not diagnostic of an infarction.   Most of the CK released after a myocardial infarction  is, in fact, the MM isoenzyme (CK-MM), which is  found in both skeletal and myocardial muscle and has a  longer half-life than the MB fraction. After about 24 h,  the finding of a high MM and undetectable MB does  not exclude myocardial damage as a cause of high total  CK activities; by this time the plasma HBD activity is  usually raised.   Table 22.1 The time sequence of changes in plasma  cardiac markers after acute myocardial infarction   AST, aspartate transaminase; CK, creatine kinase; HBD,  hydroxybutyrate dehydrogenase; LDH, lactate dehydrogenase.   Figure 22.3 Plasma cardiac markers post-acute  myocardial infarction. AST, aspartate transaminase;  CK, creatine kinase; LDH, lactate dehydrogenase.  Reproduced with kind permission from Candlish  JK and Crook M. Notes on Clinical Biochemistry.  Singapore: World Scientific Publishing, 1993.   A raised plasma total CK activity, due entirely to  CK-MM, may follow recent intramuscular injection,  exercise, surgery or various skeletal muscular disorders  (see Chapter 18). Plasma enzyme activities are raised  in about 95 per cent of cases of myocardial infarction  and are sometimes very high. The degree of rise is a  crude indicator of the size of the myocardial infarct,  but is of limited prognostic value. The prognosis often  depends more on the site than on the size of the infarct.  A second rise of plasma enzyme activities after their  return to normal may indicate extension of the damage.  A prolonged rise in plasma CK may suggest a cardiac  ventricular aneurysm. These plasma enzyme activities  do not usually rise significantly after an episode of  angina pectoris without infarction; however, troponins  rise in acute coronary syndrome but not usually in  stable angina pectoris.   The sequence of changes in plasma AST activity  after myocardial infarction are similar to those of CK  (see Table 22.1), although it rises significantly less  with respect to the upper reference limit. Even a small  myocardial infarct does cause some hepatic congestion  due to right-sided heart dysfunction, and this may  contribute to the rise of plasma AST activity. This is  rarely a diagnostic problem because the increase in  plasma AST activity following an infarction is usually  much greater. If there is primary hepatic dysfunction,  congestive cardiac failure without infarction or   pulmonary embolism (which, by impairing pulmonary  blood flow, usually causes some hepatic congestion),  plasma AST rises whereas LDHj (HBD), but not total  LDH, activity usually remains normal.   Many laboratory assays rely on measuring the  enzyme activity of CK. However, it has been suggested  that greater diagnostic accuracy may be obtained by  measuring CK-MB mass by immunological assays  instead of assaying the activity of the enzyme, although  these assays may be more expensive and may be less  readily available than activity measurements.   Creatine kinase isoenzyme MB (CK-MB) also  exists as two isoforms, namely CK-MBj and CK-  MB,. The latter is predominantly released from the  myocardium. Creatine kinase-MB that is routinely  assayed reflects the sum of the two isoforms. Normally  CK-MB 1 predominates in plasma, but after an acute  myocardial infarction this is reversed. The assay for  CK-MB isoforms is, however, complicated and requires  specialized technology.   Chronic heart failure can be defined as a clinical  condition associated with symptoms and signs of left  or right ventricular dysfunction. The diagnosis is often  made clinically on the basis of the presence of dyspnoea,  fatigue, signs of fluid overload, for example pulmonary  crepitations, peripheral oedema and raised jugular  venous pressure. However, sometimes the diagnosis  can be difficult, and further investigations are necessary  if available. Echocardiography requires experienced  personnel and can measure cardiac ejection fraction,  which may be reduced in chronic heart failure.   There are three major natriuretic peptides (NPs),  namely atrial natriuretic peptide (ANP), brain  natriuretic peptide (BNP) and C-type natriuretic  peptide (CNP). Atrial natriuretic peptide is produced  in the atria and is released upon increased atrial wall  tension, such as occurs with increased intravascular  volume. Brain natriuretic peptide is found in brain and  cardiac ventricles and its concentration is increased in the  plasma in cardiac failure and ventricular hypertrophy.  C-type natriuretic peptide exists in a number of tissues,  including the brain, kidney and endothelial cells. There  are also NP receptors: the A-type receptors bind ANP  and BNP; the B type binds CNP; and the C receptor  binds all NPs and is involved in their clearance. Both  ANP and BNP produce natriuresis (see Chapter 2)  and increase venous capacitance as well as reducing   extracellular fluid volume; CNP is a potent vasodilator  and probably has a more local tissue action rather than  working as a cardiac hormone.   It has been recommended by the National Institute  for Health and Clinical Excellence (NICE) that plasma  BNP and pro-BNP may be useful markers of cardiac  failure, as a normal plasma concentration virtually rules  out the condition. Also, plasma BNP has prognostic  value in patients with acute coronary syndrome and  cardiac failure, with those with the highest levels having  a greater mortality and morbidity. Plasma BNP is a  marker of both right and left ventricular dysfunction,  although it is non-specific and can also be raised  independently in obesity and renal disease.   The diagnosis of deep vein thrombosis (DVT), for  example in leg or pelvic veins, can be difficult clinically,  but it is important not to miss this condition as it may  lead to a fatal pulmonary embolus. D-dimers are released  into the plasma upon thrombosis formation when fib-  rinogen is converted to fibrin. Raised plasma D-dimer  concentration is, therefore, a useful marker of the  presence of a thrombus and a useful tool in the diagnosis  of a DVT. A raised plasma D-dimer concentration  may lead the clinician to request ultrasound or other  imaging techniques to look for a DVT or pulmonary  embolus. A raised plasma fibrinogen concentration is  a risk factor for cardiovascular disease and thrombosis.   Cardiovascular disease, including coronary heart disease  and myocardial infarction, is responsible for the major  burden of mortality in urbanized societies and is more  common in males, the elderly and those with a positive  family history of premature coronary heart disease  (for example below the age of 60 years). In addition,  certain ethnic groups are more at risk of coronary heart  disease, such as some South Asian groups.   There are probably at least 200 cardiovascular risk  factors, although the major ones are abnormal lipids  [high plasma cholesterol and triglycerides and low high-  density lipoprotein (HDL) cholesterol], hypertension,  smoking, diabetes mellitus, obesity and a family history  of cardiovascular disease. These constitute the so-called  ‘classic’ risk factors.   Various recent studies have shown other cardiovascular  risk factors, one of which is homocysteine, a sulphur-  containing amino acid. It has been known for a  while now that patients with homocystinuria, who  have a deficiency of cystathionine [3-synthase (an  autosomal recessive condition with an incidence of 1  in 200 000), have a thrombotic tendency (see Chapters  15 and 27). This has led researchers to look at the part  homocysteine may play in cardiovascular disease. There  is an association between raised plasma homocysteine  concentrations and cardiovascular disease, in that  individuals with moderate to severe concentration  elevations may have an increased cardiovascular risk.   Homocysteine is derived from methionine by  demethylation. It can also be metabolized to cysteine  by transulphuration, or remethylated using betaine  or methyltetrahydrofolate. Thus elevated plasma  homocysteine can be due to deficiencies of vitamin  B 12 , folate or vitamin B ( . There is also evidence that a  mutation of the methylene tetrahydrofolate reductase  (. MTHFR ) gene (single base-pair substitution C677T)  results in elevated plasma homocysteine levels (see  Chapter 15).   Plasma homocysteine concentrations above  lOpmol/L confer increased cardiovascular risk.  Concentrations of homocysteine increase with age,  impaired renal function, hypothyroidism, psoriasis and  certain drugs such as methotrexate, theophylline and  levodopa. Treatment with folate and/or vitamin B 12 and  B g can lower plasma homocysteine concentrations, but   Research has shown that atherosclerosis involves  inflammatory processes. Macrophages, monocytes and  T lymphocytes are found in the atheromatous plaques,  as are raised concentrations of cytokines with acute-  phase reactants. Cytokines such as interleukin 6 (IL-6)  increase the hepatic synthesis of acute-phase proteins,  including C-reactive protein (CRP, see Chapter 19).   Plasma CRP concentration may reflect the likelihood  of the atheromatous plaque rupturing. High-sensitivity  CRP assays (hs-CRP) have recently been developed,  and a positive association between future coronary  events and plasma hs-CRP concentration has been  found. Plasma hs-CRP may also have prognostic value  in patients with acute coronary syndrome, with those  with an hs-CRP of more than 3mg/L at onset being  more likely to suffer acute myocardial infarction or  cardiovascular death. Desirable values are less than  1 mg/L, with those at intermediate cardiovascular risk  having values 1-3 mg/L. In terms of cardiovascular  risk event prediction, plasma hs-CRP may be stronger  than the plasma total cholesterol to HDL ratio (see  Chapter 13). However, currently in the UK hs-CRP  is not recommended for patient risk stratification or  treatment decisions.   Hypertension can be defined as sustained systolic blood  pressure of more than 140 mmHg and/or diastolic blood  pressure more than 90 mmHg. It is a major risk factor   for coronary heart disease and stroke. In most cases the  cause is unknown and it is called essential hypertension.  There are many causes of hypertension, including  obesity, steroid use, insulin resistance and high alcohol  intake. Rarer secondary causes include renal disease  such as polycystic disease, scleroderma, pyelonephritis  and renal artery stenosis (Table 22.3). Endocrine causes  include phaeochromocytoma (Chapter 24), Cushing’s  syndrome (Chapter 8), Conn’s syndrome (Chapter  8), acromegaly (Chapter 7) and hyperparathyroidism  (Chapter 6). Also consider aortic coarctation and, if the  patient is pregnant, pre-eclampsia (Chapter 10).   Clinical biochemistry tests have an important part to  play in the management of hypertension. First, they have  a role in diagnosis. The biochemical investigation of  hyperparathyroidism, acromegaly, Cushing’s syndrome,  primary hyperaldosteronism (Conn’s syndrome) and  phaeochromocytoma, and of other rare conditions such  as Liddle’s syndrome and Gordon’s syndrome, which  may present with hypokalaemia or hyperkalaemia  respectively, are covered in other chapters of this book.   Biochemical testing also has a role in assessing  renal function (see Chapter 3) and in monitoring  antihypertensive therapy. Hypokalaemia can develop in  patients treated with thiazide diuretics (non-potassium  sparing), as can hyperuricaemia, hypercalcaemia,  hyperglycaemia and hyponatraemia.   Angiotensin-converting enzyme (ACE) inhibitors  and angiotensin II receptor antagonists (ARAs) or  blockers (ARBs) are also used to treat hypertension  and may cause hyperkalaemia and a rise in plasma  creatinine, particularly in patients with renal artery  stenosis. In renal artery stenosis, glomerular filtration   Table 22.3 Some secondary causes of hypertension and initial biochemical investigations  that may be useful   GH, growth hormone; HMMA, 4-hydroxy-3-methoxymandelic acid; IGF, insulin-like growth factor; PTH, parathyroid  hormone; VMA, vanillyl mandelic acid.   is maintained by angiotensin II and these drugs can  thus reduce the glomerular filtration rate, with a  concomitant impairment in renal function, for example  a 10-20 per cent rise in plasma creatinine may occur. It   is for this reason that renal function should be closely  monitored within days of initiating an ACE inhibitor  or ARA and after an increase in dose. Plasma renin  concentration is usually raised in renal artery stenosis.   • Cardiovascular disease is one of the world’s biggest  killers. It has many risk factors, which can be  divided into those that are modifiable and those  that are not. Of the former, the major contributors  are smoking, diabetes mellitus, abnormal lipids,  hypertension and obesity.   • The diagnosis of acute myocardial infarction can  be difficult, but it is important to make a prompt  diagnosis, as intervention needs to be given early.  Plasma CK can be used, and exists as three major  isoenzymes (BB, MB and MM). Total plasma CK lacks  cardiac specificity, and the CK-MB isoenzyme is more  specific and either its activity or its mass amount can  be assayed. However, the troponins have essentially  taken over from CK for diagnostic purposes.   The troponins are more specific cardiac markers  than CK. Also, plasma troponin stays elevated for  longer than CK after an infarct. Troponins are  therefore useful for the early and late diagnosis of  myocardial infarction.   Plasma myoglobin becomes elevated within about  2-4 h of myocardial infarction but is not specific  for cardiac damage, as it is also found in skeletal  muscle.   Plasma natriuretic peptides may be elevated in  ventricular dysfunction and are therefore useful in  the diagnosis of cardiac failure.   Biochemistry investigations may be useful in the  management and investigation of hypertension.   This chapter looks at various important biological  fluids and how their biochemical analysis can be useful  clinically.   In adults, the total volume of cerebrospinal fluid (CSF)  is about 135 mL, produced at a rate of 500 mL/ day.  This is predominantly formed by plasma ultrafiltration  through the capillary walls of the choroid plexuses in  the brain’s lateral ventricles. These plexuses also actively  secrete small amounts of substances such as chloride.   The fluid passes from the lateral, through the  third and fourth ventricles, into the subarachnoid  space between the pia and subarachnoid mater, from  where much is reabsorbed into the circulation by  arachnoid villi. The remaining fluid flows through the  subarachnoid space, completely surrounding the brain  and spinal cord; thus it supports and protects these  structures against injury. Like lymph, CSF removes  waste products of metabolism.   Cerebrospinal fluid circulates very slowly, allowing  contact with cells of the central nervous system (CNS).  The uptake of glucose by these cells probably results in  lower concentrations relative to plasma. Concentrations  of analytes in the CSF should always be compared with  those in plasma because alterations in the latter are  reflected in the CSF even when CNS metabolism is  normal.   Flow is slowest, and therefore contact longest, in the  lower lumbar region, where the subarachnoid space  comes to an end; therefore, the composition of CSF  from lumbar puncture is different from that of cisternal  or ventricular puncture.   Examination of the cerebrospinal fluid  Although biochemical investigation of the CSF is  important, so is microbiological and cytological  examination. Textbooks of microbiology and cytology  should be consulted for further diagnostic details if  required.   A 1 7-year-old female student was admitted to hospital  with fever, neck stiffness, headache, photophobia and  a purpuric rash. She had the following cerebrospinal  fluid (CSF) laboratory results:   Cerebrospinal fluid microscopy showed elevated  leucocytes, and Gram staining showed gram-negative  Meningococcus.   A lumbar puncture to obtain CSF is potentially a  dangerous, invasive investigation. It carries the risk  of cerebral herniation, bleeding and infection. In this  case, meningitis was suspected, which was confirmed  by the CSF results. Note the raised protein and low  glucose concentrations relative to plasma due to  bacteria. The presence of bacteria is confirmed by  microscopy. Meningococcus can rapidly cause death,  and is associated with massive haemorrhagic adrenal  destruction, as in Waterhouse-Friderichsen syndrome.   Cerebrospinal fluid is usually collected by lumbar  puncture. This procedure may be dangerous if the  intracranial pressure is raised (potentially lethal  brainstem herniation through the foramen magnum  may occur) , and the clinician should therefore check that  there is no papilloedema before proceeding; sometimes  brain imaging, for example computerized tomography  (CT) scanning, may be indicated. Small-bore needles  may reduce the risk of post-lumbar puncture headache.   Usually a total of about 5mL of CSF should be  collected as 1-2 mL aliquots into sterile containers  and sent first for microbiological examinations; if   necessary, any remaining specimen can then be used  for biochemical analysis. If a CSF glucose concentration  is indicated, 0.5 mL should be collected into a fluoride  tube and promptly sent to the laboratory with a blood  sample taken at the same time. The CSF is potentially  highly infectious and must be handled and transported  with care.   Normal CSF is completely clear and colourless; slight  turbidity is most easily detected by visual comparison  with water.   Clotting occurs when there is excess fibrinogen in the  specimen, usually associated with a very high protein  concentration. This finding occurs with tuberculous  meningitis or with tumours of the CNS.   A bright red colour may result from damage to a blood  vessel during lumbar puncture (traumatic tap) or a  recent haemorrhage into the subarachnoid space.   If CSF is collected as three separate aliquots, blood  staining will be progressively less in the aliquots if  bleeding is due to the lumbar puncture itself, whereas  all aliquots would be expected to be bloody if there was  a subarachnoid bleed.   Xanthochromia is defined as a yellow coloration  of the CSF and results from altered haemoglobin, the  colour appearing several days after a subarachnoid  haemorrhage and, depending on the extent of the  bleeding, lasting for up to a week or more or jaundice  (which will be clinically obvious and may impart a  yellow colour to the CSF).   Visual appraisal of CSF is not sufficiently sensitive  to detect subtle degrees of xanthochromia for the  diagnosis of subarachnoid haemorrhage. In such  cases spectrophotometric examination of CSF is  important. Spectrophotometric analysis may reveal  an oxyhaemoglobin absorption peak of 413-415 nm;  bilirubin shows an additional peak at 450-460 nm. This  test is particularly useful in patients with subarachnoid  haemorrhage who have a negative brain CT scan.  The presence of methaemoglobin or bilirubin is  strongly suggestive of a subarachnoid haemorrhage, as  oxyhaemoglobin alone is not necessarily confirmatory  because it may simply reflect a ‘bloody’ CSF tap. The test  should be done at least 12 h after initiation of headache.   CSF bilirubin can, however, also be raised when serum  bilirubin is elevated.   Turbidity is usually due to infection or high CSF protein  content. It may also occur after haemorrhage.   The CSF glucose concentration is slightly lower than  that in plasma and, under normal circumstances, is  rarely less than 50 per cent of the plasma concentration.  Provided that CSF for glucose assay has been preserved  with fluoride, an abnormally low glucose concentration  occurs in the following:   • Hypoglycaemia The CSF glucose concentration  parallels that of plasma, although there is a delay  before changes in plasma glucose concentrations are  reflected in the CSF. Hypoglycaemia may cause coma  and low CSF glucose concentrations in the absence  of any primary cerebral abnormality (see Chapter  12). Both plasma and CSF concentrations must be  measured.   • Infection If there is an increased polymorphonuclear  leucocyte count or a bacterial infection, the CSF  glucose concentration may be very low because of  increased metabolism of glucose. The CSF glucose  concentration may be particularly low in pyogenic  meningitis and tuberculous meningitis; in viral  meningitis, it is often normal. The estimation of  CSF glucose does not reliably distinguish between  different forms of infective meningitis because the  result may be normal in any form.   • Widespread malignant infiltration of the meninges  may also be associated with low CSF glucose  concentrations.   The CSF protein concentration in the lumbar spine  is up to three times higher than that in the ventricles;  the normal lumbar concentration is below 0.4 g/L. In  newborn infants, because of the relatively high vascular  permeability, the CSF protein concentration is about  three times that of the adult.   Changes in concentration of, for example,  immunoglobulin G (IgG) do not necessarily indicate   cerebral disease, but may simply reflect changes in  plasma (normally 80 per cent of total CSF protein has  transferred across from the plasma). Therefore the  results of assays can only be interpreted if those of the  two fluids are compared.   Cerebral disease may change the total concentration  of CSF protein and the proportions of its constituents,  for two reasons:   • The vascular and meningeal permeabilities can  increase, allowing more protein to enter the CSF.   • Proteins (immunoglobulins) may be synthesized  within the cerebrospinal canal by inflammatory or  other invading cells.   Measurement of CSF total protein is a relatively  insensitive test for the diagnosis of cerebral disease,  because early changes in the concentration of a specific  protein do not always cause a detectable rise in the total  protein concentration.   The CSF total protein concentration may be  increased in the following situations.   • In the presence of blood, due to haemoglobin and  plasma proteins.   • In the presence of pus, due to cell protein and to  exudation from inflamed surfaces.   • In non-purulent inflammation of cerebral tissue,  when there may be a definite rise in total protein  concentration despite the absence of detectable cells  in the CSF. Cells may also be undetectable in some  cases of bacterial meningitis, particularly in children,  in immunocompromised patients, or if antibiotics  have been given before lumbar puncture.   • If there is blockage of the spinal canal which, by  impairing the flow of CSF distal to the block, allows  longer for equilibrium with the circulation and so  brings the composition of CSF slightly nearer to that  of plasma (Froin’s syndrome). Increased pressure in  the CSF may also increase protein. Such blockage  may be caused by:   • Where there is local synthesis of immunoglobulins by  plasma cells within the CSF.   In the presence of blood or pus, tests for individual CSF  proteins are not useful and may even be misleading. They   may, however, detect abnormalities when the total protein  concentration is equivocally raised or normal, and may  help to elucidate the cause of a high concentration.   Increased capillary permeability, with a similar  increase in the permeability of the blood-brain  barrier, may be demonstrated by finding relatively  high-molecular-weight proteins not normally present  in CSF. This non-specific pattern is associated with a  large number of inflammatory conditions, but may  sometimes facilitate diagnosis.   The identification of immunoglobulins synthesized  within the CSF, particularly IgG and IgA, may help  with the diagnosis of multiple sclerosis or other  demyelinating disorders. Abnormal immunoglobulin  synthesis may be detected by the following.   • The finding of a characteristic electrophoretic  pattern with multiple (‘oligoclonal’) bands in the  y-globulin region. Oligoclonal bands signify cerebral  disease only if they are found in the CSF and not  in the serum. Although such bands can be detected  in more than 90 per cent of patients with multiple  sclerosis, the finding is not specific for this condition.  Occasionally, intrathecal malignant B lymphocytes  produce a local monoclonal band.   • Comparison of the IgG and albumin CSF to plasma  ratio. Albumin has a lower molecular weight than IgG  and its concentration increases disproportionately  in CSF if increased vascular permeability due to   A 43-year-old man attended the ear, nose and throat  (ENT) department because of a nasal discharge. He  had had brain surgery 3 years previously following a  head injury. Although he had initially been treated  as having allergic rhinitis, the ENT consultant sent  some of the nasal fluid to the clinical biochemistry  laboratory for tau protein analysis. The results  returned showed the presence of tau protein in his  nasal fluid.   Nasal fluid should not normally contain tau  (asialotransferrin) protein, as this is usually found  in cerebrospinal fluid (CSF). Therefore, the findings  suggest that the nasal discharge contained CSF. The  previous head injury had resulted in a dura tear,  allowing CSF to leak from the nose.   inflammation is the cause of the high protein  concentration. In this case the CSF to plasma ratio  of IgG to albumin will be low or, if permeability is so  increased that the two proteins diffuse at almost the  same rate, normal. In conditions such as multiple  sclerosis in which IgG has been synthesized within the  CSF, the ratio is high. This method is less sensitive than  the detection of oligoclonal bands (see Chapter 19).   Increased CSF immunoglobulin synthesis, with  oligoclonal bands, may be found in:   As in any ultrafiltrate, the concentration of high-  molecular-weight CSF proteins is very much lower  than in plasma. Also, electrophoresis shows that the  proportions of individual proteins are slightly different  in CSF from those in serum. For example, in the CSF, the  concentration of pre-albumin is higher and of y-globulin  lower relative to other proteins than in plasma. The tau  protein, detectable in the (3 — y region in CSF, is a variant  of transferrin (asialotransferrin), which cannot be  absorbed as the ultrafiltrate passes through the choroid  plexus; this modified form cannot be reabsorbed into the  circulation and therefore is not found in plasma.   In plasma, the asialotransferrin concentration is very  low, as desialylated glycoproteins are rapidly removed  from the circulation by the hepatic asialoglycoprotein  receptor. A nasal secretion (rhinorrhoea) containing  tau protein is suggestive of the source being CSF, as  normal nasal secretions do not contain tau protein.   C-reactive protein and lactate have also been used in  certain circumstances to indicate bacterial infection.   However, these tests are not specific, as both can be  elevated in other conditions. Microbiological tests are  often more useful if infection is suspected.   • Heavily bloodstained (assuming a non-traumatic  lumbar puncture) in three consecutive specimens:  there has probably been a cerebral haemorrhage.   • Send for microbiological examination and for  estimation for glucose and protein concentrations.   • Xanthochromic: in addition to the above tests, send  the specimen for spectrophotometric examination.   • If indicated, intrathecal immunoglobulin synthesis  may be confirmed, either using the CSF to plasma  ratio of IgG to albumin or by the detection of  multiple (oligoclonal) bands in the CSF.   This is a plasma ultrafiltrate; there is usually less than  10 mL of this fluid in each pleural cavity. If the rate of  removal is less than the rate of formation, pleural fluid  will accumulate. Decreased removal may be due to  decreased pleural space pressure, for example bronchial  obstruction, or impaired lymphatic drainage, for  example neoplasms (Fig 23.1). Pleural fluid production   A 69-year-old male smoker presented to the chest  clinic with shortness of breath, haemoptysis, cough  and weight loss. A chest radiograph revealed a left-  sided pleural effusion and upper zone ‘shadow’. The  effusion was ‘tapped’ and the following results were  obtained:   These biochemical tests suggest a pleural exudate.  Note the raised pleural fluid LDH and considerably  raised pleural fluid protein (more than 30 g/L)  concentrations. The cytology showed malignant  cells. A lung carcinoma was found on bronchoscopy.  Exudate pleural fluid is associated with malignant  lung disease.   is increased if there is decreased colloid osmotic  pressure (for example hypoproteinaemia), increased  capillary vessel permeability (for example infection) or  hydrostatic pressure elevation (as in cardiac failure).   Pleural effusion fluid can be divided into exudates  and transudates (Box 23.1).   Pleural fluid is often ‘tapped’ with a needle  (thoracentesis) and samples sent to the laboratory  for analysis. A pleural fluid protein concentration of  greater than 30g/L is suggestive of an exudate. Raised  lactate dehydrogenase (LDH) activity in pleural fluid   Infective inflammatory, e.g. pneumonia ortuberculosis  Non-infective inflammatory, e.g. pulmonary  embolism, rheumatoid arthritis, drugs such as  amiodarone   is indicative of a malignancy or inflammatory process  and is more in keeping with an exudate.   Light and colleagues used these observations to  devise Light’s criteria for the identification of exudates,  for which both pleural and plasma analyses for protein  and LDH are needed.   A pleural fluid is defined as an exudate if any one of  the following three conditions is satisfied:   • pleural fluid LDH more than 0.6 the upper normal  reference range for plasma,   Other biochemical tests (in addition to  microbiological tests and cytology) on pleural fluid  may include the following:   • Amylase activity may be raised in pleural effusions  secondary to acute pancreatitis, although other  causes include some malignant effusions and  oesophageal rupture.   • A raised pleural triglyceride concentration, in  comparison with plasma, is suggestive of chylo-  thorax. This occurs when lymph or chyle leaks from  the thoracic duct into the pleural space.   • A low pleural fluid glucose concentration, below  1.2mmol/L, may be found in effusions due to  rheumatoid arthritis, although this may also be seen  with bacterial infections.   • Tumour markers such as carcinoembryonic antigen  ( CEA) , carbohydrate antigen ( CA) - 1 2 5, CA- 1 5 - 3 and  CA-19-9 may sometimes be used to help diagnose  malignant pleural effusions. However, cytological  examination may be more useful.   • Adenosine deaminase is released from activated  lymphocytes. A raised pleural fluid adenosine  deaminase activity is suggestive of tuberculosis.  This test is useful if organisms cannot be cultured.  Interferon-y can also be used as an alternative to  adenosine deaminase.   • A raised pro-brain natriuretic peptide (pro-BNP)  concentration in pleural fluid may suggest a cardiac  failure as a cause   • A pleural fluid pH < 7.2 is predictive of the need for  pleural fluid drainage if infected.   • This is fluid in the peritoneal cavity. Sometimes fluid  accumulation can be huge, occasionally exceeding  20 L, and may cause pressure symptoms such as   abdominal distension and breathing difficulties.  Ascitic fluid drainage (paracentesis) maybe necessary  to facilitate diagnosis and relieve symptoms.   • Like pleural fluid, ascites can be divided into  exudates and transudates. Similar tests to those  done for pleural fluid can be requested. However,  it has been suggested that, rather than classifying  into transudate or exudate, it may be more  useful to classify the ascitic fluid upon the basis  of the serum ascites albumin gradient (serum  albumin concentration minus ascites albumin   concentration). A high gradient of > 1 1 g/L suggests  portal hypertension, e.g. cirrhosis, Budd-Chiari  syndrome, constrictive pericarditis, kwashiorkor or  cardiac failure, and a gradient <11 g/L is indicative  of a non-portal hypertensive aetiology, e.g.  carcinoma, infection such as spontaneous bacterial  peritonitis, nephrotic syndrome or pancreatitis  (see Chapter 17).   • Meig’s syndrome is the triad of pleural effusion,  ascites and ovarian tumour (fibroma).   • In adults, the total volume of CSF is about  135 mL, produced at a rate of 500 mL/ day. This is  predominantly formed by plasma ultrafiltration  through the capillary walls of the choroid plexuses  in the brain’s lateral ventricles.   • Laboratory analysis of CSF can be useful in the  diagnosis of various diseases, including meningitis  and subarachnoid haemorrhage.   Pleural fluid can also be analysed in the laboratory  and divided into transudates (protein less than  30 g/L) and exudates (protein more than 30 g/L).  This may be useful when searching for the cause of  the effusion.   Laboratory tests, including the serum to ascites  albumin gradient, can also help in the diagnosis of  ascitic fluid accumulation.   This chapter looks at how clinical biochemistry tests  can be used to diagnose and monitor patients with  malignant disease. Malignant disease is one of the  world’s major ‘killers’, and it is therefore important to  know the basic principles of biochemical diagnosis and  monitoring in such patients.   Neoplastic cells of differentiated tissues sometimes  synthesize enough compounds not normally thought  of as coming from that tissue to be detectable in body  fluids. These substances fall into two principal groups:   • those that alter metabolism and thus may produce  clinical effects, some of which are hormonal  syndromes,   • those that, although biologically inactive, may be  analytically detectable in body fluids; these are  sometimes used as tumour markers.   Certain rare syndromes with endocrine or  neurotransmitter properties are associated with  neoplasia. These cells may share common cytological  characteristics, originating in the embryonic ectoblast.  Staining techniques have shown their ability for amine  precursor uptake and decarboxylation (APUD) with  the production of amines. Tumours of these cells  have therefore been called APUDomas (although  this older classification has been challenged). Some  secrete physiologically active amines, whereas others,  such as the pituitary and parathyroid glands and the  calcitonin-producing C cells of the thyroid, secrete  peptide hormones. Many occur in the gastrointestinal  tract and pancreas. Secreting tumours of tissues of the  sympathetic nervous system and the amine-secreting  carcinoid tumours are discussed in this chapter.   The sympathetic nervous tissue comprising the adrenal  medulla and sympathetic ganglia is derived from the   embryonic neural crest and is composed of two types of  cells,thechromaffincellsandthenervecells,bothofwhich  can synthesize active catecholamines (dihydroxylated  phenolic amines). Adrenaline (epinephrine) is almost  exclusively a product of the adrenal medulla, whereas  noradrenaline (norepinephrine) is predominantly  formed at sympathetic nerve endings.   A 34-year-old man was referred to the hypertension  clinic because of uncontrolled hypertension. His  blood pressure in the clinic was 186/104 mmHg,  despite treatment with bendroflumethiazide,  enalapril and felodipine. Some biochemistry tests  were requested, which gave the following results:   Phaeochromocytoma is the likely diagnosis, given  the raised urinary metanephrine concentrations.  This was confirmed by further tests, including a  radioisotope metaiodobenzylguanidine (MIBG)  scan and an adrenal MRI scan. The MEN syndrome  needs to be excluded, given the known association  with phaeochromocytoma. It is often wise to seek a  secondary cause of hypertension in young individuals  with hypertension, particularly those refractory to  treatment.   Metabolism of catecholamines  Adrenaline and noradrenaline are formed from the  amine precursor tyrosine via dihydroxyphenylalanine  and dihydroxyphenylethylamine (dopamine) and bear  the catechol (3, 4-dihydroxyphenyl)-moiety. Adrenaline  and noradrenaline are both metabolized to the inactive  4-hydroxy-3-methoxymandelic acid (HMMA),  incorrectly called vanillyl mandelic acid (VMA),  by similar pathways in which metadrenaline and  normetadrenaline, respectively, are intermediates (Fig.  24.1). Adrenaline, noradrenaline and their metabolites  can be measured in urine.   Noradrenaline causes generalized vasoconstriction,  with hypertension and pallor. Adrenaline dilates blood  vessels in muscles, with variable effects on blood  pressure and pulse rate. Adrenaline may also cause  hyperglycaemia due to stimulation of glycogenolysis  and other anti-insulin effects.   Phaeochromocytomas present mainly in adults and  occur in chromaffin tissue. About 10 per cent are  outside the adrenal medulla, 10 per cent are bilateral  and 10 per cent are malignant. They are associated with  the multiple endocrine neoplasia (MEN) syndrome,  neurofibromatosis and Von Hippel-Lindau disease   although many are isolated tumours. The symptoms and  signs can be related to very high plasma concentrations  of catecholamines.   The hypertension associated with phaeochromocy-  toma is potentially curable by surgery (Fig. 24.2).  Its presence should be excluded, particularly if a  young adult presents with paroxysmal or persistent  hypertension for no apparent reason or a patient  presents with refractory hypertension or shows the  classic triad of sweating, headaches and tachycardia  associated with hypertension. Hyperglycaemia and,  if the plasma glucose concentration is high enough,  glycosuria may occur during the attack.   The biochemical diagnosis of these tumours can  be made by measuring the daily urinary excretion  of catecholamines (adrenaline, noradrenaline and  dopamine) or HMMA, the major catabolic product  of catecholamines. Extra-adrenal tumours may  only secrete dopamine. Metanephrines (which are  also metabolites of catecholamines and include  metadrenaline, normetadrenaline and methoxy-  tyramine) are preferable because they are more  sensitive than catecholamines and can also be measured  in urine and their concentration may be raised in some  phaeochromocytomas when urinary catecholamines  are normal. A slightly increased excretion of urinary  catecholamines can be found in cases of essential    Figure 24.1 Synthesis and metabolism of catecholamines. DOPA, dihydroxyphenylalanine; HMMA, 4-hydroxy-3-  methoxymandelic acid.   Figure 24.2 Showing large fluctuations in systolic blood  pressure during manipulation of a phaeochromocytoma  during its surgical removal. The blood pressure was  treated with nitroprusside infusion. Adapted with kind  permission from Kinirons M and Ellis H. French’s  Index of Differential Diagnosis, 15th edition. London:  Hodder Arnold, 2011.   hypertension. Various dietary components and drugs  affect some of the analytical methods, and therefore it  is important to consult your laboratory before starting a  urine collection. Labetalol, paracetamol, a-methyldopa  and Sinemet (combined levodopa and carbidopa) may  interfere with some of these assays.   In some cases, particularly if the tumours are small  or hypertension is paroxysmal, metabolite excretion in  a 24-h urine collection may be misleading. If there is a  high index of suspicion, it may be useful to repeat this  estimation on three consecutive days. Raised plasma  metanephrine concentrations have nearly 100 per cent  sensitivity for the diagnosis of phaeochromocytoma  and may be particularly helpful if the patient is  unable to give a reliable urine sample. However, the  concentration of plasma metanephrines may also be  raised in renal failure.   The clonidine or pentolinium suppression tests may  have a place in the investigation if urinary catecholamines  are positive or equivocal and imaging studies are  negative. In normal individuals, concentration of  urinary and plasma catecholamines should decrease  after the administration of the suppressing agent, unlike   the situation in a phaeochromocytoma, in which there  is autonomous catecholamine secretion.   Imaging studies include abdominal computerized  tomography, magnetic resonance imaging (MRI)  and metaiodobenzylguanidine (MIBG) uptake  scan. The MIBG is taken up specifically by the  phaeochromocytoma tissue. The estimation of plasma  catecholamine concentrations in samples obtained by  selective venous catheterization may help to localize the  tumour before surgery.   Neuroblastomas are very malignant tumours of  the sympathetic nervous tissue and usually occur  in children. About 40 per cent occur in the adrenal  medulla, and about 60 per cent are extra- adrenal.   The plasma catecholamine concentrations may  be as high as, or higher than, those in patients with  phaeochromocytomas. Some neuroblastomas secrete  dopamine and its metabolite homovanillic acid (HVA)  whose concentration is raised in the urine and may be  used as a tumour marker.   Normal metabolism of 5-hydroxytryptamine  Some cells are called argentaffin because they reduce,  and therefore stain with, silver salts. These are normally  found in tissues derived from the embryonic gut and   A 56-year-old man attended the gastroenterology  department because of profuse diarrhoea. He also  mentioned that he had felt increasingly breathless, and  he looked flushed. A number of investigations were  requested, but it was a 24-h urinary 5-hydroxyindole  acetic acid (5-HIAA) concentration of 544 pmol/day  (less than 25) that suggested the diagnosis.   The patient had considerably raised urinary 5-HIAA  concentration and symptoms suggestive of carcinoid  syndrome. Note the flushing and diarrhoea as well  as the breathlessness, probably due to bronchospasm  induced by vasoactive and bronchoactive amines.  The patient was subsequently found to have  carcinoid syndrome involving a tumour of the ileum  that had metastasized to the liver. Urine 5-HIAA can  be used to monitor disease assuming that a change in  management would improve clinical prognosis.   are most abundant in the ileum and appendix, but  they are also found in the pancreas, stomach and  rectum. They synthesize the biologically active amine  5-hydroxytryptamine (5-HT; serotonin) from the amine  precursor tryptophan, the intermediate product being  5-hydroxytryptophan (5-HTP). 5-Hydroxytryptophan  is inactivated by deamination and oxidation by  monoamine oxidases to 5-hydroxyindole acetic acid  (5-HIAA) (Fig. 24.3).   The oxidases and aromatic amino acid decarboxylases  are present in other tissues as well as in argentaffin cells;  5-HIAA is usually the main urinary excretion product  of argentaffin cells. Argentaffin cells may also excrete the  peptide substance P, an excess of which causes flushing,  tachycardia, increased bowel motility and hypotension.   Causes of the carcinoid syndrome  The carcinoid syndrome is usually associated with  abnormally high concentrations of plasma 5-HT. Ileal  and appendiceal tumours only produce the typical clinical  syndrome when they have metastasized, usually to the  liver. The products of intestinal tumours are inactivated in  the liver, but those from other sites, such as the bronchus,  are released directly into the systemic circulation in an  active form and may therefore cause symptoms.   Only one enzyme, tryptophan-5-hydroxylase, is  needed to convert tryptophan to 5-HTP (see Fig. 24.3),  and its derepression in malignant syndromes leads  to excessive synthesis of 5-HTP. The decarboxylase  and monoamine oxidases are present in normal non-  argentaffin tissues, so 5-HTP accumulates.   • diarrhoea, which may be severe enough to cause the  malabsorption syndrome,   • bronchospasm and right-sided fibrotic lesions  of the heart, such as tricuspid incompetence and  pulmonary stenosis; the heart lesions do not occur if  the primary tumour is in the bronchus.   The symptoms and signs are more likely to be due  to the presence of substance P than of 5-HT. Histamine   has also been suggested as a cause of the flushing. A  pellagra-type syndrome may occasionally develop,  because tryptophan is diverted from nicotinamide  to 5-HT synthesis. A carcinoid crisis occurs when the  tumour releases large amounts of vasoactive substances  into the circulation.   Diagnosis of the carcinoid syndrome  Urinary 5-HIAA secretion is usually very high in the  carcinoid syndrome. A daily excretion of more than  25 pmol is elevated, and greater than 100 pmol indicates  carcinoid syndrome if plums, pineapples, kiwi fruit,  avocados, tomatoes, walnuts and bananas (which are  high in hydroxyindoles) have been excluded from  the diet for about 24 h before the urinary collection  is started. Elevated urinary 5-HIAA has also been  reported in association with small bowel disease and  intestinal obstruction, for example coeliac disease and  sprue, but usually concentrations are not as high as in  carcinoid syndrome.   In very rare cases, usually of bronchial or gastric  tumours, the argentaffin cells lack aromatic amino  acid decarboxylase. In such cases, despite increased  secretion of 5-HTP, urinary 5-HIAA excretion may  not be increased to diagnostic levels. If there is a strong  clinical suspicion of the carcinoid syndrome, despite  the finding of normal 5-HIAA excretion, estimation  of total 5-hydroxyindole (5-HTP, 5-HT and 5-HIAA)  excretion maybe indicated. In some cases, plasma 5-HT  or its metabolites may be measured.   Carcinoid tumours may also secrete  adrenocorticotrophic hormone (ACTH), which can  result in Cushing’s syndrome as well as being associated  with MEN 1 syndrome. Octreotide, a somatostatin  analogue, can be used to antagonize some of the effects  of the released mediators in carcinoid syndrome.   In the rare syndromes of MEN (pluriglandular  syndrome), two or more endocrine glands secrete  inappropriately high amounts of hormones, usually  from adenomas. There are two main groups of  syndromes.   MEN 1 may involve two or more of the following  endocrine tissues; the glands involved are listed in order  of decreasing incidence of involvement:   Remember the three Ps: parathyroids, pancreas and  pituitary. MEN 2 includes:   MEN 2 can be subdivided into 2A (Sipple’s  syndrome) and 2B, the latter also being associated with  marfanoid habitus, mucosal neuromas and abnormal  corneal nerve fibres.   Both types of MEN are usually familial, inherited as  a Mendelian dominant trait with variable penetrance.  MEN 1 is due to a mutation in the MENIN gene, a  tumour-suppressor gene; MEN 2 is the result of a  proto-oncogene mutation known as RET. Genetic tests  can be used to screen families.   Calcitonin (see Chapter 6) is secreted by the thyroid  parafollicular ‘C’ cells. Plasma calcitonin concentration  can be raised in medullary carcinoma of the thyroid  associated with MEN 2 syndrome (rarely in other  tumours). It can thus be used as a tumour marker  for medullary thyroid carcinoma. Plasma calcitonin  concentration can be used for screening for familial  thyroid medullary carcinoma. In some cases, basal  plasma calcitonin levels are not significantly raised  but may become so after intravenous pentagastrin  stimulation.   All cells have the potential to produce any peptide or  protein coded for in the fertilized ovum. Malignant cells  may produce ectopic hormones or other peptides if they  partly revert to their early embryonic, pluripotential  state. This may cause the histological changes associated   with neoplasia, which are sometimes associated with  the synthesis of significant amounts of a peptide not  normally detectable in the fully differentiated tissue.  There are two possible hypotheses, both of which  include derepression:   • of part of the genome of the tissue cell that codes for  the peptides, which are then synthesized in excess,   • of specialized neuroendocrine cells, which are  scattered through many tissues.   Some substances are secreted more often by one  type of tumour than by others, whereas some types of  tumour secrete more than one ‘foreign’ substance.   Hormones produced at ectopic sites, such as those  secreted by pathological overactivity of endocrine  glands, are not under normal feedback control, and  secretion continues under conditions in which it  should be suppressed; such secretion is therefore  inappropriate. Ectopic production is most clinically  obvious if the peptide has hormone-like effects. Many  of the syndromes associated with such hormonal  syndromes are discussed in the relevant chapters of this  book.   The following may be due to hormone secretion by  the tumour.   Although ectopic antidiuretic hormone (ADH)  production is commonly associated with the relatively  rare small cell carcinoma of the bronchus, the  syndrome of inappropriate ADH secretion has been  reported in a wide variety of tumours. This syndrome,  which is not confined to malignant disease, is discussed  in Chapter 2.   Ectopic ACTH secretion stimulates the secretion of  all adrenocortical hormones except aldosterone; the  clinical presentation often resembles that of primary  hyperaldosteronism (glucocorticoid hormones have  some mineralocorticoid action). The condition is  usually associated with a small cell carcinoma of  the bronchus but has occasionally been described in  association with a variety of other malignant lesions,  especially pulmonary carcinoid tumours. The patient  may present with severe hypokalaemic alkalosis (see  Chapters 4 and 5).   Hypercalcaemia due to the secretion of parathyroid  hormone-related protein (PTHRP) is one of the most  common of these syndromes. It is associated with  many types of malignant disease, including squamous  cell carcinoma of the bronchus. Parathyroid hormone-  related protein is structurally similar to PTH but not  usually detected by PTH immunoassay and may have  important actions in mineral metabolism in the fetus.  The onset of hypercalcaemia can be very rapid, in  contrast with that of primary hyperparathyroidism (see  Chapter 6).   Polycythaemia due to erythropoietin secretion  Polycythaemia due to the secretion of erythropoietin  or an erythropoietin-like molecule is a well-recognized  complication of renal carcinoma; erythropoietin  stimulates bone marrow erythropoiesis. Because  erythropoietin is a normal product of the kidney, this is  not an example of ectopic hormone secretion; however,  the syndrome has been reported in association with  other tumours, especially hepatocellular carcinoma  (primary hepatoma).   Although rare, hypoglycaemia due to insulin or an  insulin-like growth factor (IGF) has been reported  in association with various tumours. Severe  hypoglycaemia, with appropriately low plasma  immunoreactive insulin and C-peptide concentrations  and raised IGF-2 concentration, has been found in  association with very large retroperitoneal or thoracic  mesenchymal tumours resembling fibrosarcomas, or  with hepatocellular carcinoma (see Chapter 12).   This has been reported in association with various  tumours, including carcinoma of the bronchus, breast  and liver, due to raised circulating concentrations  of chorionic gonadotrophin. In children with  hepatoblastoma this may also cause precocious puberty.   Galactorrhoea may also occur due to the ectopic  secretion of prolactin or prolactin-like molecules (see  Chapter 7).   Some tumours of trophoblastic cells, such as  choriocarcinoma, hydatidiform mole and testicular  teratoma, have been shown to secrete a thyroid-  stimulating hormone-like substance similar to  human chorionic gonadotrophin (hCG). Clinical  hyperthyroidism is unusual, even when plasma  hormone concentrations are high (see Chapter 11).   For the measurement of a tumour marker to be  clinically useful, the result should clearly separate those  patients with from those without a tumour. Therefore  (although in practice this is not the case), a tumour  marker should ideally be:   • 100 per cent sensitive: levels should be raised if the  tumour is present,   • 100 per cent specific: levels should not be raised if the  tumour is not present.   A 68-year-old, non-smoking woman was under  the care of the general surgeons because of  colonic carcinoma, for which she had had a left  hemicholectomy 10 months previously. Her plasma  carcinoembryonic antigen (CEA) was 43pg/L  (normally less than 2.5 in non-smokers) prior to the  operation. At the time she left hospital for surgery,  her plasma CEA had reduced to less than 4.0 pg/L.  Three months after her surgery, her plasma CEA  was less than 2.5 pg/L. The results from a recent out-  patient follow-up clinic showed a plasma CEA of  19 pg/L.   The post-operative results indicate that her  tumour had been largely resected, as the plasma  CEA concentrations were reduced to near ‘normal’  levels. Tumour remission seemed likely at 3 months  post operation, as the plasma CEA concentrations  remained low. Unfortunately, tumour recurrence  had occurred at the time of her recent out-patient  follow-up visit, as evidenced by the considerable  rise in plasma CEA concentration. Tumour markers  are usually less useful for diagnosing a malignancy  than for following the progression of a tumour by  observing the changes in their concentration.   • To screen for disease Very few markers are sufficiently  sensitive or specific to be used to screen for the  presence of a tumour (see Chapter 1).   • To diagnose a tumour If a patient presents with  clinical signs or symptoms, the measurement of a  marker in plasma or urine may very occasionally be  used to confirm a diagnosis.   • To determine the prognosis In some cases the  concentration of a specific marker is related to the  mass or spread of the tumour.   • To monitor the response to treatment If a tumour  marker is present, the rate of its decrease in  concentration may be used to assess the response  to treatment such as surgery, chemotherapy or  radiotherapy.   • To identify the recurrence of a tumour If the  concentration of the marker was previously raised,  intermittent measurement during remission  may sometimes be used to identify recurrence.  Occasionally, however, tumours may dedifferentiate  and fail to express the marker despite continued  growth and spread.   A 67-year-old man attended the urology out-patient  clinic because of urinary hesitancy, lower back  pain and nocturia. Rectal examination revealed a  large, firm and craggy prostate gland. Some of his  biochemistry results were as follows:   Prostate-specific antigen (PSA) 28pg/L (<4)  Albumin-adjusted calcium 2.98mmol/L (2.15-2.55)  Phosphate 0.92mmol/L (0.80-1.35)   The grossly elevated plasma PSA concentration is  suggestive of malignant prostate disease. A bone  scan showed osteosclerotic bony secondaries  in his lumbar spine. Further investigations,  including prostatic biopsy, confirmed metastatic  prostatic carcinoma. This would also be a likely  explanation for his hypercalcaemia. The raised  plasma ALP suggests a bone source due to the  bony secondaries.   Prostate-specific antigen (PSA) is a marker for prostatic  carcinoma, a common male tumour, and is a 33-kDa  protein and is homologous with the protease kallikrein  family; it has a plasma half-life of about 3 days. One  of its probable functions is to help liquidize semen. Its  level is raised in benign prostatic hyperplasia (BPH)  and prostatic carcinoma but also in prostate infection,  for example prostatitis, and after rectal examination.   Levels of PSA increase with age, which is mainly  due to the increase in the volume of the prostate that  occurs. Therefore age-adjusted reference ranges should  be used. There may also be a place for expressing  plasma PSA in terms of prostate volume as found on  ultrasound examination.   One diagnostic limitation is that the values of PSA  overlap in BPH and prostatic carcinoma. After a radical  prostatectomy, plasma PSA levels become undetectable  at 2-3 weeks. Finasteride, a 5-oc-reductase inhibitor  that is sometimes used to treat BPH, decreases plasma  PSA by up to 50 per cent.   The PSA is bound in the plasma to either oCj-  antichymotrypsin or a, -macroglobulin. The  concentration of bound or complexed PSA is higher  in prostate carcinoma, whereas that of free PSA is  higher in BPH. The ratio of free to total PSA is lower  in men with prostatic carcinoma. The PSA index  is expressed as the percentage of the total plasma  PSA that is free; an index above about 17 per cent is  suggestive of BPH and one of less than 17 per cent of  prostate carcinoma.   Plasma PSA concentrations greater than lOpg/L are  strongly suggestive of carcinoma, although carcinoma  may be present even if values fall within the reference  range. A PSA above 20 pg/L is suggestive of prostatic  carcinoma that has spread beyond the prostate gland.   Plasma PSA assays in conjunction with digital  rectal examination may be used as part of a screening  programme for prostatic carcinoma in at-risk males.  There is, however, no universally agreed screening  protocol for prostatic carcinoma in the general  population. Prostate cancer antigen 3 ( PCA3 ) and  specific PSA isoforms may also prove useful markers in  conjunction with PSA.   Prostate biopsy is usually necessary if the PSA  concentration is above lOpg/L; however, the decision  regarding biopsy is more difficult if PSA levels are  4-10 pg/L, although the PSA index may help.   Carcinoembryonic antigen may be produced by some  malignant tumours, especially colorectal carcinomas. If  the initial plasma concentration is raised, serial plasma  CEA estimations may sometimes help to monitor the  effectiveness of, or recurrence after, treatment (Fig.  24.4). Plasma concentrations correlate poorly with  tumour mass, but a very high concentration usually  indicates a bad prognosis. Plasma concentrations may  also rise in non-malignant disease of the gastrointestinal  tract and in smokers. Thus, the test is non-specific and  thus lacks value in diagnosis.   a.-Fetoprotcin (AFP) is an oncofetal protein, the  synthesis of which is suppressed as the fetus matures.  Concentrations may be very high in the plasma of  patients with certain tumours such as hepatocellular  carcinoma (primary hepatomas and hepatoblastomas)  and teratoma. Moderately raised concentrations may  be due to non-malignant liver disease.   Human chorionic gonadotrophin (hCG) is normally  produced by the placenta, but also by trophoblastic  cells of gonadal and extragonadal germ cell tumours.  Ectopic secretion has been observed in some bronchial  carcinomas. The measurement of hCG can be used  to screen for choriocarcinoma in women who have   Figure 24.4 The use of the tumour marker  carcinoembryonic antigen (CEA) in monitoring  disease progression. Patient A had an advanced  gastrointestinal tumour and after initial chemotherapy  died. Patient B had initial remission as a result of  surgery but died after tumour relapse. Patient C had  successful surgery with CEA essentially normalizing.  Reproduced with kind permission from Candlish  JK and Crook M. Notes on Clinical Biochemistry.  Singapore: World Scientific Publishing, 1993.   had a hydatidiform mole (see Chapter 10). Plasma  concentrations may be raised in patients with  malignancy of the gonads such as seminomas, and hCG  may be used to monitor the response to treatment and  tumour recurrence.   Carbohydrate antigens (CAs) are a group of tumour  markers, raised plasma concentrations of which may  be used to monitor the response to treatment and the  recurrence of certain tumours.   • CA-125 concentration may be raised in the plasma  of patients with ovarian carcinoma. It can also be  raised in pregnancy, fibroids, liver and pancreatic  disease, endometriosis and pelvic inflammatory  disease. Additionally, it can also be raised in other  malignant diseases such as lung, breast or colon  carcinoma. If plasma CA-125 is 35kU/F or more,  an ultrasound scan of the abdomen and pelvis has  been proposed as a means of screening for ovarian  carcinoma.   • CA-15-3 concentration may be raised in the plasma  of some patients with advanced breast carcinoma,  although it can also be raised in cirrhosis, and with  ovarian cysts.   • CA-19-9 concentration may be raised in the plasma  of patients with pancreatic or colorectal carcinoma  and those with obstructive liver disease.   None of the CA tumour markers fulfils the criteria  of an ideal marker, as none is sufficiently sensitive  or specific to be used to screen for early disease.  Furthermore, some advanced tumours dedifferentiate  and fail to produce a marker despite continued growth  and spread. These tumour markers may, however, be  useful in the monitoring of patients with tumours and  in the assessment of therapy. High levels are associated  with tumour spread and relapse, and low levels are  suggestive of tumour remission.   • Serum paraprotein and urinary Bence Jones protein  (discussed in Chapter 19 for multiple myeloma).   • Plasma lactate dehydrogenase (LDH): the activity can  be raised in certain haematological tumours such as  lymphomas.   • Placental alkaline phosphatase: true placental alkaline  phosphatase and placental-like isoenzyme levels are  raised in seminoma and dysgerminoma. bevels are  not usually raised in teratomata. In conjunction   with AFP and hCG, it is useful in the diagnosis and  monitoring of extragonadal and gonadal germ cell  tumours. However, plasma levels are also elevated in  smokers (see Chapter 18).   • Tiiyroglobulin : this high-molecular-weight protein  is produced in the follicular cells of the thyroid.  Its concentration is raised in follicular or papillary  carcinoma of the thyroid. Spuriously low levels may  be found in the presence of thyroglobulin antibodies,  which interfere with the assay.   • Neuronal-specific enolase: plasma levels maybe raised  in small cell lung carcinoma and neuroblastoma; it is  derived from neurodectal tissue.   • Inhibin: this is secreted by the granulosa cells of  the ovary and by the Sertoli cells of the testis. It  can be used as a plasma tumour marker of ovarian  granulosa cell tumours and testicular Sertoli cell  tumours.   • Squamous cell carcinoma antigen: this is a plasma  tumour marker of potential use in squamous cell  carcinoma of the cervix.   • Chromogranin A is released from neuroendocrine  cells such as in phaeochromocytoma and carcinoid  tumours.   • Protein SIOOB is a calcium-binding protein. It is  expressed in brain astrocytes and glial cells and also  in melanocytes and may be useful in monitoring  therapy in malignant melanoma.   • Human epididymis protein (HE4) is being used as a  tumour marker for ovarian carcinoma.   New tumour markers are likely to be revealed in  the future and this chapter is not all-inclusive. Genetic  tests are also being developed that may be useful to  predict those individuals at risk of developing various  carcinomas, for example BRCA gene mutations for breast  carcinoma. Tumour-modified deoxyribonucleic acid  (DNA), ribonucleic acid (RNA) and nucleic acids also  circulate in the blood and may be useful in the diagnosis  of certain cancers.   • Laboratory biochemical tests can be useful in the  investigation and management of certain malignant  disorders.   • Tumours alter metabolism and thus may produce  clinical effects, some of which are hormonal  syndromes. In addition, tumours may release  compounds that, although biologically inactive,  may be analytically detectable in body fluids. These  are sometimes used as tumour markers.   • In the rare syndromes of MEN (pluriglandular  syndrome), two or more endocrine glands secrete  inappropriately high amounts of hormones, usually  from adenomas.   • The diagnosis of phaeochromocytomas may be  facilitated by the assay of urinary catecholamines  or metanephrines.   Similarly, raised urinary 5-HIAA concentration may  be useful in the diagnosis of carcinoid syndrome.  For the measurement of a tumour marker to be  clinically useful, the result should clearly separate  those patients with a tumour from those without.  Therefore a tumour marker should ideally be  sensitive (concentrations should be raised if the  tumour is present) and specific (levels should not  be raised if the tumour is not present).   Tumour markers are generally best used for  monitoring the progression of tumours with  therapy (remission or relapse). Many are of little  value in diagnosis or screening of disease.   Drug overdose and the consequences of drug side effects  are common causes of hospital medical admissions.  This chapter also looks at therapeutic monitoring of  particular drugs and how the clinical biochemistry  laboratory can be involved in the management of  various drug overdoses.   The blood (usually plasma or serum) concentrations  of many drugs can be measured or, more rarely, those  of other body fluids, although in only a few situations is  this of proven benefit.   Pharmacokinetics is the study of the fate of drugs after  administration and is concerned with their absorption,  distribution in body compartments, metabolism and  excretion. Absorption depends on whether the drug is  taken orally, by intravenous or intramuscular injection,  sublingually or rectally.   Possible indications for measuring drug  concentrations in various body fluids are to:   • check that the patient is taking the drug as prescribed  (compliance),   • ensure that the dose is sufficient to produce the  required effect but not so high as to be likely to cause  toxic effects,   • determine the type of drug or drugs taken in cases  of suspected overdose and to assess the need for  treatment.   One can assess whether the dose of a drug is optimal  either clinically or by laboratory assays. An example  of the former would be monitoring the action of  antihypertensive drugs by measuring the blood pressure.  Examples of laboratory biochemical effects, which are  discussed in the relevant chapters, include the following:   • thyroid- stimulating hormone concentrations while  on thyroxine therapy (Chapter 11),   • plasma calcium concentration and alkaline  phosphatase activity during vitamin D treatment for  hypocalcaemia or osteomalacia (Chapter 6),   • plasma cholesterol after hydroxy-malonyl-glutaryl  coenzyme A reductase inhibitor (statin) therapy  (Chapter 13),   • clotting status as judged by prothrombin time  and international normalized ratio (INR) for  determining warfarin dosage.   The drug or metabolite concentrations can also be  measured in biological fluids, although these may not  parallel cellular effects. However, the measurement  of plasma concentrations may be indicated in the  following situations:   • If the desired result cannot be measured precisely:  for example, the incidence of epileptic fits is a poor  indicator of the optimal dosage of anticonvulsants.   • If the range of plasma levels that is most effective  in producing the desired result without toxic side  effects (the therapeutic range) has been defined:  this is particularly true if there is a narrow margin  between therapeutic and toxic drug concentrations,  such as in the case of digoxin or lithium. This is  of particular value when the relation between  dose and plasma concentrations of a drug is  unpredictable.   • If the prescribed drug is the main active compound  and is not metabolized significantly to an active  metabolite: otherwise the active metabolite may be  measured, for example phenobarbital in primidone  treatment.   The total amount of drug in the extracellular fluid (ECF )  depends on the balance between that entering and that  leaving the compartment; the plasma concentration  depends on the volume of fluid through which the  retained drug is distributed.   Blood samples must be taken at a standard time after  ingestion of the drug, the exact time varying with the  known differences in the rate of absorption, metabolism  and excretion of different drugs (Table 25.1).   The following factors may affect the blood  concentration of some drugs.   It has been shown that not all patients take a drug  exactly as prescribed. The patient may not take the drug   at all, take more than the prescribed dose, take the drug  intermittently or become confused about the timing  and dose, particularly if taking more than one drug.   Clinicians should realize that poor compliance might  cause a poor clinical response or toxicity. A regular  review of therapy and careful explanation to the patient  are important. Assay of plasma concentrations is only  a crude method of assessing compliance and only tests  the situation at the time when blood was taken.   Entry of the drug into, and distribution  through, the extracellular fluid  Absorption   Lipid-soluble drugs can pass through cell membranes  more readily, and are therefore absorbed more rapidly,  than water-soluble ones; they reach the highest plasma  concentrations at between 30 and 60 min after ingestion.  The rate of absorption in an individual patient may be  affected by:    Risk of agranulocytosis  Risk of weight gain  Risk of diabetes mellitus  Possible cardiac toxicity   “Therapeutic ranges are less reliable for patients taking a number of drugs. Sampling times are only a general guide. In some cases, samples  taken at other times may be appropriate.   • the rate of gastric emptying: this must be allowed for,  for example during treatment with drugs affecting  gut motility or after gastric surgery,   • the administration of other compounds, such as the  bile acid sequestrants which may also bind certain  drugs within the intestinal lumen.   The final plasma concentration of a drug reached after  a standard amount has been absorbed depends on the  volume through which it has been distributed. For  example, it may be difficult to predict the appropriate  dose in oedematous or obese patients who, because  they have a larger than normal volume of distribution,  may have unexpectedly low plasma concentrations.  By contrast, in small children, with a low volume of  distribution, there is a danger of overdosage. The  patient’s weight or surface area may need to be allowed  for when the dose is calculated.   Many drugs, like many endogenous substances, are  partly inactivated by protein binding, usually albumin.  Most drug assays estimate the total concentration of the  free plus the protein-bound drug. Biological feedback  mechanisms do not control free drug concentrations as  they do those of plasma calcium and hormones; therefore,  the method of interpretation to allow for altered protein  binding is different. Measured plasma concentrations  fall little due to a reduction in protein binding; a larger  proportion of the measured drug will be in the unbound,  free, active form, so that metabolism and excretion will  increase. Unless this is realized, dangerously high plasma  free concentrations may be interpreted as being within,  or even below, the therapeutic range if the plasma  albumin concentration is very low.   The bound proportion varies with differing plasma  albumin concentrations, and there is no valid correction  factor that allows for protein abnormalities. About  90 per cent of phenytoin, 70 per cent of salicylic acid,  50 per cent of phenobarbital and 20 per cent of digoxin  is protein bound. However, binding may be affected by  the following:   • Abnormalities in plasma albumin concentration Blood  should be taken without stasis to minimize a possible  rise in plasma albumin, and albumin-bound drug,  concentrations. Low concentrations, such as those often   found in hepatic cirrhosis or the nephrotic syndrome,  may reduce the proportion of protein-bound drugs.   • Competition for binding sites on protein Many drugs,  unconjugated bilirubin fatty acids and hydrogen  ions compete with each other for binding sites.   Metabolism and excretion of drugs  The blood concentrations of a drug depend on normal  hepatic and renal function as well as on acid-base and  electrolyte balance. The time taken for the plasma drug  concentration to fall to half its original concentration  is called its effective half-life. To maintain a reasonably  steady plasma concentration, drugs with a short half-  life should be taken more frequently than those with  a long one. After starting treatment, a steady state is  usually reached after about five times its half-life has  elapsed; the first specimen of blood for monitoring  should not be taken earlier than this.   The rate at which the plasma drug concentration  falls after it has reached peak concentration depends on  the rate of distribution through the ECF (see above),  the rate of entry into cells and the rate at which it is  metabolized and excreted, whether in urine or bile.   The rate of elimination of most drugs depends on  their plasma concentration. A small increase in the dose  of some, such as phenytoin, may exceed the capacity  of the metabolic or excretory pathways and so cause a  disproportionate increase in plasma levels; the plasma  concentrations of these drugs should be monitored  carefully. The rate at which this occurs is termed  saturation kinetics.   Metabolic conversion to active or inactive metabolites  Some drugs are active only after metabolic conversion;  others are inactivated, usually by conjugation in the  liver. Ideally, a drug assay should measure all the active  forms, whether the parent compound or its active  metabolite, and none of the inactive forms.   If a number of different drugs are being prescribed,  one may affect the plasma concentration of another  by altering its binding to plasma proteins, rate of  metabolism or excretion. For example, sodium  valproate displaces phenytoin from its protein-binding  sites and reduces its rate of metabolism.   Some drugs (for example phenytoin) may induce  the synthesis of enzymes that inactivate them, and    consequently higher doses than usual may be needed  to produce the desired effect. In other cases, reduced  receptor sensitivity may require higher doses than  normal in order to increase the plasma concentration  and to achieve the desired effect.   The rate of metabolism of some drugs depends on the  age of the patient.   • Premature infants, with immature detoxifying  mechanisms, metabolize and excrete some drugs  more slowly than adults.   • Children, who have a higher metabolic rate than  adults, may eliminate some drugs more rapidly than  adults.   • The elderly may metabolize drugs slowly; they are  particularly sensitive to digoxin.   The rate of metabolism of some drugs maybe affected  by genetic or racial factors (see Pharmacogenetics  below). There are also metabolic factors causing  pharmacodynamic effects, for example increased  sensitivity to digoxin evoked by hypokalaemia or  hypercalcaemia.   Only a few drug assays are of proven clinical value. The  clinical picture must be taken into account and all the  above factors must be allowed for when interpreting the  results. The following are some of the drug measurements  that have been claimed to be useful for therapeutic  monitoring. The reader is also referred to Table 25.1.   This drug is used in congestive cardiac failure and  atrial fibrillation. Estimation of plasma digoxin  concentrations maybe useful in the following situations:   • To assess compliance: the elderly, for whom digoxin  is frequently prescribed, are often taking several  different drugs and are liable to be confused about  which tablets to take and at what time.   • In patients, such as the elderly, who are likely to  have a low threshold for toxicity, and in premature  infants, who have an increased elimination half-life.   • If it is difficult to calculate the appropriate dose  because of an abnormal volume of distribution due  to obesity or oedema, or in infants.   A 69-year-old man was on once-daily digoxin  250 pg, once-daily bendroflumethiazide 2.5 mg, and  once-daily warfarin 4 mg for atrial fibrillation and  hypertension. He attended his anticoagulant clinic to  have his blood warfarin levels checked. The following  results were returned:   International normalized ratio (INR) 2.7 (2-3)  Digoxin 3.3 pg/L (0. 8-2.2)   The attending doctor was worried about digoxin  toxicity given the raised plasma concentrations.  However, after discussion with the laboratory and  phlebotomy, it transpired that the blood samples  had been taken within 2 h of the patient taking his  digoxin tablet. Repeat testing 7h after taking the  digoxin showed a plasma concentration of 1.2 pg/L,  within the therapeutic range. This case illustrates the  importance of the correct timing of blood sampling  for therapeutic drug monitoring (in the case of  digoxin, this is optimally about 6-8 h post dose).   • If the patient is taking another drug, such as  amiodarone, which possibly reduces its rate of  excretion.   Plasma digoxin concentrations, even within the  therapeutic range, are very difficult to interpret in  the presence of conditions that may alter receptor  sensitivity, such as:   Blood should be taken at least 6 h after the last dose,  when absorption and distribution are usually complete  (see Table 25.1). It is also important to check plasma  potassium levels and renal function, as digoxin is renally  excreted. The assay of digoxin can be problematic, as  the antibodies used in certain immunoassays may   cross-react with endogenous compounds - so-called  digoxin-like immunoreactive substances. The latter  can occur in pregnancy. Sometimes digoxin overdose/  toxicity can be treated by the administration of  inactivating digoxin antibodies (Digibind). It should  be also remembered that Digibind interferes with some  immunoassays used to measure digoxin.   This is used to treat various cardiac arrhythmias,  including ventricular tachycardias and atrial  fibrillation. The drug is highly protein bound and has  a long half-life, up to 10-40 days after a single dose and  up to 100 days on chronic therapy. It is not excreted  in the urine, but is predominantly metabolized in  the liver. The metabolite desethylamiodarone is also  active. Amiodarone is potentially very toxic, sometimes  causing photosensitivity reactions, pulmonary fibrosis  and corneal microdeposits. Additionally, it blocks free  thyroxine to free tri-iodothyronine conversion and can  evoke hypothyroidism. Conversely, as it contains iodine,  it can also cause thyrotoxicosis in some circumstances  (see Chapter 11).   This is another antiarrhythmic drug for which  therapeutic drug monitoring may be beneficial.   Once the dose that gives stable plasma concentrations  within the therapeutic range has been determined,  monitoring is probably necessary only in the following  cases:   • if the frequency of fits increases in a previously well-  controlled patient,   • if another drug is prescribed that may affect the  plasma concentrations.   Plasma concentrations in children and in pregnant  women should be monitored more frequently, as these  groups are more likely to develop toxicity.   Phenytoin is used in the treatment of partial and  generalized tonic-clonic seizures by inhibiting voltage-  gated sodium channels. Measurement of plasma  phenytoin may be useful, because it exhibits saturation  kinetics. Thus a small increase in dose of phenytoin   may result in a large increase in plasma concentration  and be associated with toxicity. Sodium valproate  displaces phenytoin from its binding sites and reduces  its rate of metabolism; free plasma phenytoin may  then cause toxicity despite apparently appropriate total  plasma concentrations. Phenytoin is a potent enzyme-  inducing drug (as, indeed, are phenobarbital and  carbamazepine) and may enhance the metabolism of  other drugs as well as causing an elevation in plasma  y-glutamyl transferase, which does not necessarily  mean abnormal hepatic toxicity (see Chapter 17).   Like phenytoin, carbamazepine can be used in the  treatment of partial and generalized tonic-clonic  seizures by inhibiting voltage-gated sodium channels.  Additionally, it can be used in the treatment of  trigeminal neuralgia and diabetic neuropathy and also  for prophylaxis of bipolar disorders. It has an active  metabolite formed in the liver called carbamazepine  10,11-epoxide. Carbamazepine induces its own  metabolism and thus plasma concentrations may fall at  the end of the first month of therapy, which results in  the need for a dose increase.   This is used to treat myoclonic seizures, generalized  absence seizures and partial and generalized tonic-  clonic seizures. It is also used in the treatment of bipolar  affective disorders.   Plasma concentrations of sodium valproate correlate  poorly with the dose prescribed, the clinical response  and toxicity, probably because of the presence of active  metabolites. Consequently, the main justification  for measuring plasma concentrations is to assess  compliance. However, valproate is hepatotoxic and  it is therefore important to check liver function tests.  Phenobarbital, primidone and ethosuximide are less  used today in the treatment of epilepsy, although  therapeutic drug monitoring may be useful.   There are several new antiepileptic drugs now  available, for which the need to monitor plasma  concentrations is under evaluation. Vigabatrin, an  inhibitor of y-aminobutyric acid metabolism, has  a long action because it binds avidly to receptors;  therefore, plasma concentrations are unlikely to  predict therapeutic response or toxicity. Other newer  antiepileptic drugs include lamotrigine, which  inhibits excitatory neurotransmitter release possibly  by action upon voltage-sensitive sodium channels,   and gabapentin, but it remains to be seen how useful  therapeutic drug monitoring will be in patient  management. Lamotrigine also has found use in the  treatment of bipolar disorders as well as partial and  tonic-clonic seizures.   The margin between therapeutic and toxic  concentrations of lithium at high dosage is narrow, and  therefore plasma concentrations should be monitored  regularly. Lithium may be given to psychiatric patients  for bipolar affective disorders. It is not protein  bound, and interpretation of the results of assays is  not complicated by protein abnormalities. Plasma  concentrations should be measured on a specimen  taken 12 h after the evening dose.   Lithium usage is associated with hypothyroidism  (see Chapter 11), and plasma concentrations above  about 1.4mmol/L can be nephrotoxic. Oliguria and  acute kidney injury, which can be precipitated by   A 40-year-old woman was admitted to casualty  because of confusion. She was known to be on  lithium therapy for a bipolar disorder. The following  blood results were obtained:   The elevated plasma lithium concentration indicates  a considerable risk of toxicity. The level should  be checked to ensure correct sample timing, as  incorrect timing, i.e. not trough levels, is a common  cause of elevated drug levels. The hypernatraemia  can be explained by the polyuria and nephrogenic  diabetes insipidus secondary to lithium toxicity  upon the kidney. Primary hypothyroidism can  also be secondary to lithium toxicity. Note also  the abnormal renal function, which is essential to  monitor in patients on lithium.   reduced salt intake or diuretic usage, can reduce  lithium clearance. It is important, therefore, to monitor  thyroid and renal function tests in such patients.  Plasma lithium levels above 2.5mmol/L are associated  with significant mortality and may necessitate dialysis.  Rarely hypercalcaemia may result possibly mediated by  hyperparathyroidism.   Theophylline assays may be useful, especially in acute  asthmatic attacks that are not responding clinically; the  results may help to ensure that the poor response is not  due to underdosage and, if it is not, that increasing the  dosage will not lead to toxic plasma levels.   Theophylline and its active metabolite caffeine are  used in the management of recurrent apnoea in the  newborn infant; at this age, their clearance is slow and  theophylline is partially metabolized to caffeine. Both  plasma theophylline and caffeine concentrations may  need to be assayed if theophylline has been given, in  order to assess therapeutic or toxic effects.   Samples for theophylline assays should be taken at  between 2 and 4h after the last dose. The half-life of  theophylline is shortened in smokers.   Aminoglycosides, such as gentamicin, are given  intramuscularly or intravenously and, in contrast  to many other antibiotics, the margin between the  therapeutic range and toxic levels is narrow. Toxic levels  may cause ototoxicity and nephrotoxicity. Measurement  is particularly indicated if there is serious infection or  the treatment is so prolonged that the risk of toxicity is  increased or renal function is impaired.   Aminoglycoside concentrations are often measured  twice. The first specimen should be taken immediately  before injection, when the ‘trough level’ is expected, to  ensure that concentrations are not already high and that  further administration is not likely to cause toxicity,  or that they are not so low as to be ineffective. The  second should be taken about an hour after injection,  at the time of the anticipated ‘peak level’, to ensure  that an adequate concentration has been achieved  for antibacterial action. Two glycopeptide antibiotics,  namely vancomycin and teicoplanin, are used to  treat aerobic and anaerobic gram-positive bacteria.  Like gentamicin, they may cause nephrotoxicity  and ototoxicty, a risk increased by concomitant  aminoglycoside usage. Therapeutic drug monitoring  also has a role in the use of these drugs.   Methotrexate is used as a cytotoxic agent and, to achieve  this, plasma concentrations should exceed 1 pmol/L  for at least 36 h. It is a folate antagonist, which inhibits  deoxyribonucleic acid (DNA) synthesis at lower  concentrations. It is also used as an immunosuppressant  in psoriasis and rheumatoid arthritis.   When used as chemotherapy, plasma levels above  5-10 pmol/L at 24h, 0. 5-1.0 pmol/L at 48h and  0.1 pmol/L at 72 h post dose are associated with an  increased risk of marrow suppression. Therapeutic drug  monitoringis less useful when lower immunosuppressant  doses are used unless toxicity is suspected.   Methotrexate excretion is predominantly renal and  is facilitated by a good urine flow and an alkaline pH  greater than 6.5. After high-dose methotrexate, ‘rescue  therapy’ with folinic acid is given to reduce toxicity by  providing cells with folate co-factors for DNA synthesis.  For liver monitoring and methotrexate use, see Chapter  17.   This is an immunosuppressant that inhibits  T-lymphocyte activation by binding to calcineurin  phosphatase and is used to prevent the rejection of  transplanted organs, for example kidney. Ciclosporin is  a cyclic peptide derived from the fungus Tolypocladium  inflatum (Gams). It is also used (usually at lower  dose) in the treatment of inflammatory bowel disease,  rheumatoid arthritis and psoriasis.   The dosage may be difficult to assess because there  is considerable variation among individuals in the rate  of absorption and clearance; there is also a relatively  narrow margin between therapeutic and toxic drug  concentrations. The main toxic action is on the kidney,  although hepatic dysfunction can also occur. During the  first 6 months, when the risks of rejection are highest,  plasma concentrations should be measured regularly.   Peak ciclosporin concentrations occur 1-4 h after oral  dosage and require adequate bile flow. Over half of the  drug binds to red blood cells and then redistributes in  plasma, where a majority is foundboundto lipoproteins,  as ciclosporin is strongly lipophilic. Ciclosporin levels  should be determined on trough ethylenediamine  tetra-acetic acid (EDTA) anticoagulated samples, that  is, 12 h after the previous dose on a twice-daily regimen.  Daily monitoring is usual after transplantation; in the  first few months, levels of 100-400 pg/L are usual,  decreasing to about 100-200 pg/L afterwards.   This is a macrolide lactone and, like ciclosporin,  inhibits calcineurin phosphatase, thereby inhibiting  T-lymphocyte activation.   Although useful as an immunosuppressant, it is  nephrotoxic and cardiomyopathy may occur. It has an  advantage over ciclosporin in having a greater potency,  and impaired biliary function is less likely to alter its  absorption. Thereapeutic drug monitoring seems to  be of value, and levels over 25 pg/L are associated with  increased toxicity.   Atypical antipsychotic drugs  These drugs, such as clozapine, may benefit from  therapeutic drug monitoring to monitor compliance  and help detect toxicity. There is a risk of agranulocytosis  with some agents and full blood counts should be  monitored. Some of these agents may cause weight gain  and risk of diabetes mellitus, as well as cardiac toxicity.   Various enzymes are responsible for the metabolism  of drugs. For example, the inactivation of isoniazid by  acetylation depends on whether the patient is genetically  capable of carrying out this process at a normal rate;  toxicity is likely at lower plasma concentrations in ‘slow  acetylators’ than in ‘fast acetylators’.   Thiopurine methyltransferase (TPMT) is involved  in the metabolism of azathioprine, which is used as a  cytotoxic and also as an immunosuppressant agent. The  enzyme TPMT shows pharmocogenetic variation and  about 90 per cent of the UK population have high enzyme  levels. However, about 10 per cent are heterozygotic for  TPMT deficiency and about 0.3 per cent are homozygotic  for no functional activity. The latter two groups are more  susceptible to azathioprine toxicity. Determination of red  cell TPMT activity prior to azathioprine administration  may give a guide as to in whom the drug should be  avoided or given at a lower dose.   Similarly, debrisoquine hydroxylase activity can  be used as a guide to cytochrome P450 phenotype.  The cytochrome family is involved in the metabolism  of various drugs. Debrisoquine hydroxylase activity  reflects cytochrome (CY) 2D6 status, which is involved  in the metabolism of a number of drugs, including  certain antiarrhythmics and antidepressants.   Some drugs have harmful side effects. For example,  the plasma urea, creatinine, sodium and potassium   concentrations should be checked for patients on diuretic  therapy or taking angiotensin-converting enzyme (ACE)  inhibitors. The assessment of liver damage (for example  plasma transaminase activities) or of renal function  may be indicated during treatment with potentially  hepatotoxic (for example valproate) or nephrotoxic (for  example ciclosporin) drugs, respectively. The statins may  sometimes cause muscle damage or rhabdomyolysis,  which can be detected by measuring the plasma  concentration of creatine kinase (CK).   Drug overdosage must always be excluded as a cause  of coma of unknown origin. Unexplained clinical or  laboratory findings may suggest effects due to drug  or alcohol ingestion despite denial by the patient.  Hypokalaemia, for example, may be due to purgative  abuse, and hypoglycaemia may be a presenting finding  associated with alcohol ingestion. Raised plasma  transaminase activities, may be due to alcoholic liver  disease,. A raised plasma CK concentration may be  indicative of ‘Ecstasy’ or cocaine abuse. Measurement  of blood gases is useful to determine whether there is an  acid-base disturbance, for example a metabolic acidosis  may be seen in paracetamol overdose. As discussed  above, some drugs, such as digoxin and lithium, may  cause renal impairment.   About half the patients attempting suicide take  several drugs, sometimes with alcohol. Qualitative  screening of plasma, urine or gastric aspirate may be  needed to help identify the drugs. The measurement  of plasma drug concentrations (and also sometimes  of other biological fluids such as vomit or gastric  contents) may be supplemented by screening the  urine for drugs or their metabolites. Screening is rarely  advised and it is best to discuss cases with a toxicologist  to decide whether this is necessary. The careful labelling  and identification of samples that may have associated  medicolegal issues is essential.   It is often unnecessary to measure the plasma  concentrations of drugs that the patient is known to  have taken in overdose. The need for gastric lavage  and measures to increase the urinary excretion of  the drug and to maintain adequate respiration and  circulation are not usually affected by knowledge of  drug concentrations. The treatment of many drug  overdoses is often supportive, with close monitoring  of the patient and clinical intervention when necessary.  However, in some cases specific antidotes are indicated.   Drugs or poisons for which there is a  specific antidote  Paracetamol (acetaminophen)   It is essential to measure plasma drug concentrations  in suspected paracetamol poisoning, for the following  reasons:   • A metabolite of paracetamol is hepatotoxic:  its metabolism is saturable and is detoxified  by conjugation with glutathione. Depletion of  glutathione is implicated in the liver damage. The  patient may die of liver failure despite recovery from  the immediate effects.   • The antidote is only useful if given within a defined  period of time and if defined plasma concentrations  are reached.   • The likelihood of hep ato toxicity cannot be predicted  from the clinical picture at presentation (Fig. 25.1).   A 23 -year-old woman took a paracetamol overdose  after an argument with her boyfriend. She had a  history of anorexia nervosa. The following results  were obtained 2 days after treatment with intravenous  iV-acetylcysteine.   Alanine aminotransferase (ALT) 881 U/L (<42)  Alkaline phosphatase 127 U/L (<250)   The results show severely abnormal liver function  tests secondary to paracetamol overdose. The raised  ALT activity suggests hepatocyte damage evoked by  the paracetamol. Paracetamol overdose can induce  a number of biochemical disturbances, including a  metabolic acidosis, raised plasma anion and osmolar  gap. The raised INR is concerning and suggests severe  paracetamol toxicity and the risk of life-threatening  hepatic damage, in some cases necessitating liver  transplant. It should be remembered that those  who are undernourished or have alcoholism or are  on certain medications, such as enzyme-inducing  drugs, may be more at risk of paracetamol toxicity;  see Fig. 25.1   Figure 25.1 Patients whose plasma paracetamol concentrations are above the normal treatment line should  be treated with acetylcysteine by intravenous infusion (or, if acetylcysteine cannot be used, with methionine  by mouth provided the overdose has been taken within 10-12 h and the patient is not vomiting). Patients on  enzyme-inducing drugs (e.g. carbamazepine, phenobarbital, phenytoin, primidone, rifampicin, alcohol and  St John’s wort) or who are undernourished (e.g. in anorexia nervosa, in alcoholism, or those who are human  immunodeficiency virus-positive) should be treated if their plasma paracetamol concentrations are above the  high-risk treatment line. The prognostic accuracy after 15 h is uncertain, but a plasma paracetamol concentration  above the relevant treatment line should be regarded as carrying a serious risk of liver damage. Reproduced  courtesy of University of Wales College of Medicine Therapeutics and Toxicology Centre.   N - acetylcysteine and methionine are both relatively  effective antidotes, because they enable paracetamol to  be converted to non-toxic metabolites. Methionine is  given orally but may cause vomiting. N - acetylcysteine  is most often used and is given intravenously. It is only  effective in clearly defined circumstances and should  only be given after the following factors have been  taken into account.   Plasma concentrations of paracetamol cannot  be interpreted and should not be measured until  absorption and distribution are nearly complete, at  about 4 h after ingestion. If treatment is to be effective,  it should be started as soon as possible following   ingestion. Concentrations should be measured on  specimens taken as early as possible, ideally between 4  and 20 h after ingestion (see Fig. 25.1).   Patients taking enzyme-inducing agents such as  phenytoin, rifampicin, carbamazepine or alcohol or  those who are undernourished, for example because of  anorexia or infection with human immunodeficiency  virus (HIV), are more at risk of paracetamol toxicity.  Monitor plasma transaminase activities (which may  rise to 10000U/L or more), renal function (as renal  failure can develop) and prothrombin time or INR.  Prothrombin time or INR is probably the best marker  of the severity of liver failure.   This is more likely in children, who may mistake the  tablets for sweets. Desferrioxamine chelates iron and the  chelate is excreted in the urine. Concentrations above  90 |jmol/L in young children and above 1 50 |jmol/L in  adults are said to be indications for treatment.   The following are some of the other drug and chemical  poisonings for which there are specific antidotes.   Respiratory depression, leading to a respiratory  acidosis, can occur and measurement of blood gases  may therefore be useful. Flumazenil may reverse some  of their actions.   A specific assay for this is unlikely to be available in  the emergency situation, although a raised plasma  anion gap may be an indirect clue as to the presence  of ethylene glycol (see Chapter 4). This may cause a  metabolic acidosis, renal and hepatic dysfunction and  also hypocalcaemia (due to oxalate conversion and  calcium chelation as calcium oxalate). Therapeutic  ethanol administration may reduce the conversion of  this to toxic metabolites by competition with alcohol  dehydrogenase.   This can be rapidly fatal, although measurement of  arterial blood gases may be useful as a lactic acidosis  occurs. Antidotes may include dicobalt edetate, sodium  nitrite and sodium thiosulphate, which may protect the  blocking of cyanide on the electron transport system in  the mitochondria.   Renal failure, hypoglycaemia, metabolic acidosis  and rhabdomyolysis may occur, as can raised plasma  amylase concentration. Ethanol infusion inhibits  alcohol dehydrogenase, which converts methanol to  toxic formic acid.   These can cause respiratory depression leading to a  respiratory acidosis. Naloxone may reverse some of the  effects of opiates such as morphine or heroin.   Carbon monoxide can cause hypoxia without cyanosis.  Carboxyhaemoglobin above 60 per cent can cause   respiratory failure and death. Arterial blood gases  are important, and a metabolic acidosis may ensue.  Oxygen therapy, sometimes hyperbaric, may increase  the washout of carboxyhaemoglobin as oxygen  displaces haemoglobin-bound carbon monoxide.   Antidotes include cholinesterase inhibitors such as  atropine or pralidoxime, with the treatment being  monitored using acetylcholinesterase activity.   There are no specific antidotes for the majority of drug  overdoses.   Salicylate overdose is worth considering in further  detail, as it is probably one of the most common  overdoses encountered. Forced diuresis may increase  the excretion of salicylate in severe overdose (induction   A 24-year-old woman being treated for depression  took an aspirin overdose of about forty 300 mg tablets  that she had purchased from the local chemist. The  following biochemical results were obtained in the  casualty department 8 h after ingestion.   The patient displays a respiratory alkalosis due to the  aspirin (salicylate) overdose as this can stimulate the  respiratory centre. Aspirin can, however, also result  in a metabolic acidosis, particularly in children,  which can present with an increased plasma anion  gap. Other biochemical changes that may be  seen with aspirin overdose are hypoglycaemia or  hyperglycaemia and disturbances in liver function  tests and electrolytes.   of alkalosis often further increases the excretion rate of  acidic drugs), but neither forced diuresis nor induction  of alkalosis is without risk. If an alkaline diuresis is  induced, plasma potassium concentrations, as well as  blood pH, must be monitored because of the danger of  acute hypokalaemia.   In adults, a respiratory alkalosis occurs initially,  followed by a metabolic acidosis (possibly due to   uncoupling of oxidative phosphorylation). Children  seem less likely to develop the respiratory alkalosis.  Hypokalaemia and either hyperglycaemia or  hypoglycaemia may also occur.   Where there is no specific drug antidote, treatment is  supportive, although biochemical tests for electrolytes,  acid-base balance, renal and hepatic function and  blood gases are often useful.   The biochemical laboratory plays an important role  in the monitoring of the concentrations of some  drugs, usually those for which there is a narrow  therapeutic window. The common drugs for which  this may be useful are the antiepileptic drugs and  some of the cardiac antiarrhythmic agents, as well as  certain antibiotics, lithium and chemotherapy drugs.  Therapeutic drug monitoring may be useful for  detecting patient drug compliance and for observing   changes in drug levels if used in combination  with other drugs to help avoid drug interaction.  Therapeutic drug monitoring can be used to ‘tailor’  drug dosage to patient needs.   Drug or substance overdoses are a common cause  of hospital admissions, and measuring drug  concentrations and biochemical monitoring, for  example of renal, liver and acid-base status, can be  useful.   This chapter looks at clinical biochemistry at the  extremes of age, that is, in neonates and the elderly.   Some preliminary definitions are useful. A premature  or preterm baby is one born before 37 completed weeks’  gestation. A neonate is a live baby within 1 month of  birth. A low-birthweight baby is one less than 2.5 kg  and a very low-birthweight baby is one less than 1.5 kg.   One of the most significant advances in recent medical  care has been the survival rate of very small preterm  infants, which has increased because of improved  specialized medical and nursing techniques for treating  the neonate. However, these infants can experience a  number of abnormal biochemical conditions, which  are briefly summarized in this chapter.   Diseases occurring during the neonatal period can  be divided into two main groups: those of infants born  before term, in whom immaturity contributes to the  severity of the disease, and those of full-term infants.  The most common disorders in both groups are  perinatal asphyxia, the respiratory distress syndrome  (RDS), infection and inborn errors.   Compared with about 5L in a 70-kg adult, the  blood volume of a premature infant weighing 1 kg is  only about 90 mL. Therefore only a small amount of  blood can be taken without causing volume depletion  or anaemia. Sometimes capillary samples are used as  an alternative to venous (or arterial) samples, although  the former may be more prone to contamination from  the interstitial and cellular fluids. Prior to requesting  tests on small samples, the clinician should contact the  laboratory to discuss the best strategy for obtaining  test samples and prioritizing of investigations. The  interpretation of test results may be influenced by the  different reference ranges at different ages, which can  be difficult to define.   The kidneys usually have a full complement of nephrons  by about the 36th week of gestation, but renal function   is not fully developed until the age of about 2 years.  Glomerular function develops more rapidly than that  of the tubules. Fetal urine is produced from about the  ninth week.   The glomerular filtration rate (GFR) doubles during  the first 2 weeks of life because of an increase in renal  blood flow. The GFR (which is corrected for surface  area) reaches adult values by about 6 months of age.   The plasma urea concentration is low in newborn  infants compared with that in adults, despite the  relatively low GFR; the high anabolic rate results in more  nitrogen being incorporated into protein rather than  into urea than in adults. Plasma urea concentrations  fluctuate markedly with varying nutritional states,  metabolic rates and states of hydration.   The plasma creatinine concentration (which is  inversely related to GFR) at birth is similar to the  mother’s. It decreases rapidly at first, averaging about  35 pmol/L, the fall being slower in preterm infants, and  then rises before reaching adult values (corrected for  the body surface area) by about 6 months.   Renal function in newborn infants can maintain  basic homeostasis but may not be able to respond  adequately to illness or other stresses. It is often  difficult to determine if there is renal impairment  because of the unsatisfactory nature of renal function  tests at this age. A plasma urea concentration above  about 8mmol/L suggests retention due to glomerular  impairment, whether parenchymal or pre-renal in  origin, especially if the urinary output can be shown  to be low. Management depends to a large extent on  clinical criteria. In the neonate, neither the plasma  urea nor the creatinine concentration is a very sensitive  indicator of renal function.   Renal dysfunction in neonates can be due to factors  such as hypotension, birth asphyxia, renal toxic drugs,  renal agenesis, septicaemia or dehydration or can occur  post surgery.   The daily excretion of urine is about 15-60mL in  full-term neonates, increasing to about 500-600 mL/  day at about 1 year (see Chapter 3).   About 80 per cent of the weight of a neonate consists  of water, compared with about 60 per cent of that of an  adult; the relative amount of water is related more to  the proportion of fat to lean tissue than to total body  weight. Proportionally, there is probably more water in  the extracellular than in the intracellular compartment  at birth. During the first week of life, the extracellular  fluid (ECF) compartment contracts; urinary loss may  account for the relatively high total amount of sodium  excreted by preterm infants and does not necessarily  indicate a disturbance in sodium homeostasis.   Renal concentrating capacity is lower in the newborn  infant than in the adult and increases during the first  2 years; the maximum urinary osmolality that can  be achieved in response to water deprivation, even if  stimulated by exogenous antidiuretic hormone (ADH),  is between 500 and 700 mmol/kg. The countercurrent  mechanism is relatively inefficient, partly because the  loops of Henle do not penetrate as deeply into the  renal medulla as in the adult and partly because of the  relatively low rate of urea production and therefore  excretion. Reabsorption of urea from the collecting ducts  contributes to the interstitial medullary osmolality, so  facilitating water reabsorption in response to ADH;  renal concentrating ability may rise if urea production  is increased when a high-protein diet is given.   ‘Insensible’ water loss is proportionately much higher  in infants than in adults because the ratio of surface  area to body volume is high, more fluid is lost through  the skin because the epidermis is not fully developed  before about the 28th week of gestation, there is very  little subcutaneous fat, and metabolic and respiratory  rates are high. Insensible transepidermal water loss  can increase by up to 50 per cent due to phototherapy  or overhead baby warmers - hence the importance  of maintaining high ambient humidity. Daily fluid  requirements are therefore up to five times higher per  kilogram of body weight than in adults. It is important  to pay close attention to this, as high insensible losses  can occur, leading to renal and circulatory failure and  haemoconcentration and hypernatraemia. The average  basal water loss in a neonate is about 20 mL/kg per day  (see Chapter 2).   The total body sodium content of a newborn infant  of 1kg is less than 100 mmol, compared with about  3000 mmol in the adult. A premature infant may  need up to 6 mmol/kg of sodium a day because of   the high losses, and a normal full-term infant about  2-4 mmol/kg, compared with about 1 mmol/kg in an  adult. In preterm infants, plasma renin, aldosterone  and angiotensin II concentrations are high.   Renal conservation of sodium is inefficient in  premature infants, despite relatively high plasma renin  activities and aldosterone concentrations compared  with adults, because of immature renal tubular  function. Excretion of a sodium load is impaired,  perhaps because of the low GFR. Sodium balance  should be carefully controlled, bearing in mind the very  low total body sodium content at this age.   The total body sodium and the plasma sodium  concentrations (and therefore osmolality) may fluctuate  because renal function is immature. Sodium excretion  declines after birth. Preterm babies may have a failure of  sodium tubular reabsorption leading to sodium deficit,  particularly if there is low sodium input. The newborn  infant, unlike fully conscious children and adults, is  unable to make its need for water clear and it may be  difficult to assess the requirements accurately. Rapid  changes in extracellular osmolality due to inappropriate  intake of water cause significant shifts of water between  the intracellular and extracellular compartments; signs  varying from listlessness to convulsions, or even coma,  may be caused by changes in cerebral hydration. The  plasma sodium concentration should be monitored to  ensure that the proportion of sodium to water is correct.  Hyponatraemia   • Prolonged maternal infusion of oxytocin or other  hypo-osmolal fluid, for example 5 per cent dextrose,  during labour. Oxytocin has an antidiuretic action  similar to that of ADH and may cause dilutional  hyponatraemia in both mother and infant.   • Inappropriate ADH secretion: following post-partum  intracranial haemorrhage or meningitis in severe  pulmonary disease, such as respiratory distress  syndrome (RDS; also called hyaline membrane  disease) or pneumonia.   • Infusion of water in excess of sodium to the baby, either  as 5 per cent dextrose or as hypo-osmolal sodium  solutions.   • Acute renal disease, including acute tubular necrosis,  especially if hypo-osmolal fluid is given to a child  with glomerular dysfunction.   Congenital adrenal hyperplasia, adrenal insufficiency,  hypothyroidism and cystic fibrosis should always be   Clinical signs of hyponatraemia may be associated  with hypotension, drowsiness and convulsions.   Hypernatraemia develops if water loss exceeds that of  sodium, or if an excess of sodium relative to water is  infused or fed.   If replacement is inadequate, the high ‘insensible’  water loss, aggravated by impaired urinary water  retention, may cause rapid ECF depletion with  hypotension and, if water depletion is predominant,  hypernatraemia. This is particularly likely if ‘insensible’  loss is increased through the:   • skin - for example due to sweating caused by pyrexia  or overhead heaters or to phototherapy for jaundice,   • lungs - if the infant is hyperventilating, for example  because of pneumonia,   Iatrogenic causes in infants include giving sodium  bicarbonate or excess salt. Neonates are more  susceptible than adults to developing hypernatraemia  because the addition of a given amount of extra sodium  increases the very low extracellular sodium content  proportionally more than if it were diluted in a larger  pool, as it is in adults; this tendency is aggravated  by impaired renal excretion. Nephrogenic diabetes  insipidus is a rare cause of neonatal hypernatraemia.   The total body potassium of a newborn infant of 1 kg is  less than 100 mmol, compared with about 3000 mmol  in an adult. A normal full-term infant requires about  2-4 mmol/kg of potassium a day (compared with about  1 mmol/kg in an adult) to replace losses.   As in the adult, artefactual causes of abnormal  plasma potassium concentrations must be excluded.  Pseudohyp erkalaemia is especially likely if capillary  samples are used, because tissue cells may be damaged  if the skin is squeezed as in a heel prick. It may also be  due to in vitro haemolysis, or to withdrawal of blood  from a cannula through which a potassium solution is  being infused. Conversely, pseudohypokalaemia may  result if the infused fluid is potassium free.   Hypokalaemia may be caused by increased  gastrointestinal loss due to diarrhoea or an alkalosis,   for example pyloric stenosis. In this age group also  consider renal tubular acidosis (type I or II). Iatrogenic  causes include diuretic use.   Iatrogenic causes include excess potassium treatment.  Other common causes include acute kidney injury,  exchange transfusion and tissue damage resulting  from hypoxia or birth trauma, for example severe  bruising or haematoma. Consider also congenital  adrenal hyperplasia or adrenal insufficiency (see  Chapter 5).   Renal complications and disturbances of electrolyte  balance are more likely to develop in infants with  perinatal asphyxia. Cerebral oedema or haemorrhage  may stimulate ADH secretion, causing oliguria and  a dilutional hyponatraemia with hypo-osmolality,  accompanied by a high urinary sodium concentration  due to plasma volume expansion.   The hypotension occurring during asphyxia may  reduce renal blood flow enough to cause acute oliguric  renal failure (acute kidney injury). In addition to the  oliguria and hyponatraemia, there maybe uraemia and  hyperkalaemia, with proteinuria. Hypoglycaemia and  hypocalcaemia may also occur.   Fetal pH can be measured during delivery, usually  from a capillary sample from the baby’s scalp. This  may be indicated if changes in cardiotocograph or fetal  heart rate indicate fetal distress. A pH value below 7.2  indicates the need for urgent delivery.   Fetal lung fluid production ceases at birth, probably as  a result of increases in adrenaline concentration during  delivery. Fabour squeezes liquid from the mouth, and  absorption takes place into the pulmonary lymphatics  and capillaries. Type 2 pneumocytes produce surfactant,  which serves to reduce lung surface tension, thus  facilitating lung expansion. Deficiency of surfactant as  in prematurity can cause RDS.   Normal term neonatal arterial Po 2 breathing air is  about 8-llkPa. Oxygen therapy can cause toxicity,  including bronchopulmonary dysplasia and stiff lungs,  and should be closely monitored, for example by  transcutaneous gas analysis. Retinopathy of prematurity  (previously known as retrolental fibroplasia) may lead to  blindness.   The plasma bicarbonate concentration is normally  about 3mmol/L lower in newborn infants than in  adults. This is due partly to renal immaturity and  partly to the low concentration of urinary buffers;  both these factors impair bicarbonate ‘reabsorption’  and regeneration. However, the plasma concentrations  must be interpreted with caution, as they are likely to be  artefactually low in very small samples. The neonate is  more vulnerable to acid-base disturbances (for further  discussion see Chapter 4). The specific conditions that  affect neonates are discussed here.   - tissue hypoxia resulting from poor tissue  perfusion caused by hypotension and the low  Po 2 accompanying asphyxia or sepsis,   • inborn errors of amino acid or organic acid  metabolism (see Chapter 27).   This can result from pyloric stenosis, with which  projectile vomiting can occur. This is associated with  hypokalaemic alkalosis.   Respiratory distress may be caused by pulmonary  disorders such as RDS, pneumonia and meconium  aspiration during birth. The most common cause in the  preterm infant is RDS, the incidence of which is inversely  related to the gestational age of the infant at birth.  Respiratory distress syndrome is due to immaturity of  the enzymes responsible for the intrauterine synthesis  of pulmonary surfactant, which maintains the patency  of the alveoli. Surfactant synthesis begins at about the  20th week of gestation and increases rapidly after about  the 34th week following maturation of the alveolar cells.  Surfactant deficiency is more common in male infants,  those of diabetic mothers and those with asphyxia and  hypothermia.   The condition presents with pulmonary collapse  (atelectasis), with secondary lung infections being  common. The blood Pco 2 is high, causing respiratory  acidosis. The low blood Po 2 and reduced blood flow  due to hypotension cause tissue hypoxia with lactic  acidosis; renal dysfunction may aggravate the metabolic  acidosis. The combination of respiratory and metabolic  components may cause severe acidosis.   An infant with RDS may benefit from the  administration of surfactant, given through an  endotracheal tube, and from positive-pressure  ventilation, which help to expand the lungs and  correct blood gas abnormalities. If these treatments are  successful, the improved general condition increases  tissue perfusion, correcting the lactic acidosis and  improving renal function.   The Po 2 and Pco 2 of cutaneous capillary blood can  be monitored continuously using electrodes placed on  the skin. The electrodes bring the capillary Po 2 and Pay  to near arterial levels by heating the skin to about 44°C,  thus increasing cutaneous blood flow. The electrodes  must be repositioned every 4h to prevent local burns  and need to be recalibrated frequently. Transcutaneous  blood gas monitoring supplements, but does not replace,  arterial blood gas analysis (see Chapter 4).   The conditions associated with a respiratory alkalosis  that occur in neonates include septicaemia, meningitis,  hyperammonaemia and hepatic failure.   The fetal gastrointestinal tract has limited functional  development before 26 weeks. This helps to explain  why premature babies may have poor tolerance of  enteral feeding. Lactose may be poorly absorbed within  the first week of life.   The metabolism of the neonatal liver is initially slower  than in adults. As will now be seen, this is important with  regard to neonatal bilirubin metabolism, particularly  that of conjugation.   Plasma transaminase activities are up to twice the  upper limit of the adult reference range during the first  3 months of life and fall to adult levels by the age of about  1 year. The plasma total alkaline phosphatase activity  is higher in infancy and during childhood because of  the contribution from actively growing bone; it falls to  adult levels after the pubertal growth spurt.   A full-term infant was jaundiced and his plasma  bilirubin concentration was 182pmol/L 2 days  after delivery. Further testing showed that this was  predominantly unconjugated bilirubin (176 pmol/L).  The baby was otherwise well, and within 5 days  the plasma bilirubin concentration decreased to  36 pmol/L (< 20).   The jaundice was attributed to physiological  jaundice of the newborn, which occurs in some  infants. This is the most likely explanation, given the  only moderate elevation of bilirubin concentration  that presented 2 days after delivery. The bilirubin was  predominantly unconjugated due to ‘immaturity’  of the glucuronyltransferase system, which is  responsible for conjugation of bilirubin.   Neonatal abnormalities of bilirubin metabolism are  discussed here (see Chapter 17 for further details).   Proportionally more unconjugated bilirubin reaches  the liver in the newborn infant than in the adult, for the  following reasons:   • Delayed clamping of the umbilical cord may  significantly increase red cell mass.   • Bruises may occur during birth, and resorption  of haemoglobin breakdown products from these  increases the plasma bilirubin concentration.  A cephalohaematoma can release large amounts of  bilirubin.   • The red cell half-life is shorter; the blood haemoglobin  concentration falls rapidly during the first week of  life, even in normal infants.   The mature liver can conjugate very large amounts  of bilirubin and, due to increased bilirubin production,  jaundice is rarely severe in adults. In the newborn  infant, even at term, the conjugating process is not fully  developed.   Physiological jaundice (unconjugated hyperbilirubi-  naemia) is defined as mild jaundice which is not  present at birth but which develops during the first  few days and continues during the first 10 days of  life, and for which there is no obvious pathological   reason. Such jaundice is very common in normal  newborn infants (about 50 per cent of normal babies  develop this after 48 h), the incidence being inversely  related to the gestational age at birth. The plasma  total bilirubin rarely exceeds 200 pmol/L, with the  conjugated bilirubin unlikely to be greater than  40 pmol/L. Physiological jaundice can be aggravated  by prematurity, infections, dehydration, hypoxia and  poor nutrition, to name but a few.   Plasma unconjugated bilirubin concentrations may  be very high in premature infants because of hepatic  immaturity. If the bilirubin concentration exceeds the  albumin-binding capacity, the unbound, fat-soluble  unconjugated bilirubin may cross cell membranes and  be deposited in the brain, causing kernicterus. This is a  serious complication, which may result in permanent  brain damage or death.   • if the plasma unconjugated bilirubin concentration  is rising rapidly, perhaps due to haemolysis,   - displacement of bilirubin from albumin by  hydrogen ions in acidosis due to hypoxia or  other serious illness.   Jaundice during the first 24 h of life is more likely to  be pathological than physiological. It may have the  following causes:   • Maternofetal rhesus or ABO blood group  incompatibility: this is particularly likely in infants  born to multiparous mothers, who may have  developed antibodies during previous pregnancies.   • Inherited erythrocyte abnormalities associated  with haemolysis, such as glucose-6-phosphate  dehydrogenase deficiency, pyruvate kinase  deficiency or hereditary spherocytosis. The first of  these is X-linked and more common in people of  Mediterranean or African origin.   • Intrauterine infections that affect the liver, such as  syphilis, rubella or toxoplasmosis.   Bilirubin should be measured in blood taken from  the umbilical cord of all infants known to be at risk  for one of the above reasons; blood group typing and  Coombs’ testing for red cell antibodies can also be  performed on cord blood.   If the plasma bilirubin concentration is rising rapidly  or exceeds about 340 pmol/L in full-term infants,  exchange transfusion may be needed. This ‘action  level’ may be significantly lower in the preterm  infant. Biochemical complications of exchange  transfusions are usually due to the anticoagulant used  in the infused blood, and are transitory. They include  hyperkalaemia, hypocalcaemia, metabolic acidosis  and hypoglycaemia.   Bilirubin is destroyed by intense visible light and  lesser degrees of unconjugated hyperbilirubinaemia  may be treated by phototherapy. Water loss from the  skin may be high and fluid balance must be carefully  monitored. There are action charts available that help  with treatment decisions based on hours after birth and  the plasma bilirubin concentration.   Prolonged unconjugated hyperbilirubinaemia, that  is, persisting beyond 10 days in a term baby, not in itself  requiring treatment, may accompany chronic infections,  such as with cytomegalovirus, or hypothyroidism. It is  more common in breast-fed infants than in those given  formula, possibly because a factor(s) in maternal milk  inhibits uridine-diphosphate-glucuronyltransferase  activity (see Chapter 17).   Impaired excretion of bilirubin that has been conjugated  in hepatocytes may be due to the following:   • Congenital biliary atresia (Byler’s disease): it is  important to make this diagnosis early because some  forms may be amenable to surgical treatment. The  stools are usually pale and there is increased urinary  bilirubin concentration with abnormal liver function  tests. Biliary tree ultrasound and radioisotope scans  together with liver biopsy can aid diagnosis. Early  surgery by the Kasai hepatoportoenterostomy  procedure may be life saving.   • Biliary obstruction caused by pressure on the bile  ducts by, for example, extrabiliary tumours, although  this is a rare cause in children. Alagille’s syndrome  is a very rare condition associated with decreased  intrahepatic bile ducts, congenital cardiac defects  and facial dysmorphism.   Neonatal hepatitis rarely presents clinically  until after the first week of life. Its causes include  intrauterine infection, such as syphilis, toxoplasmosis,  cytomegalovirus infection, hepatitis A or B and  rubella.   - it is severe, for example plasma bilirubin greater  than 300 pmol/L,   • Plasma bilirubin concentration should be quantified  and separated into unconjugated and conjugated  fractions. Liver function tests, urinary bilirubin and  stool colour should be checked.   • Consideration should also be given to any  medications that might be implicated in the jaundice  or whether the neonate is on parenteral nutrition.   • Neonatal infection and congenital hypothyroidism  should be excluded as causes of the jaundice.   • If unconjugated bilirubin predominates, the blood  film and reticulocytes should be checked and a  Coombs’ test performed; these are useful to exclude  haemolysis.   • Remember also that unconjugated hyperbilirubi-  naemia can be seen in breast-milk jaundice.   • In cases of severe unconjugated bilirubinaemia,  Crigler-Najjar syndrome should be considered.   • If conjugated bilirubin predominates, it is important  to exclude liver disease, including hepatitis.   • Depending on the clinical findings, if liver function  is found to be abnormal, various biochemical tests  may be indicated in an inborn error of metabolism   such as plasma a - antitrypsin deficiency, plasma  and urinary amino acids. Exclude galactosaemia (see  Chapter 27). Liver ultrasound may also be useful.   • It is important that biliary atresia is excluded, for  example with imaging techniques.   • In the presence of conjugated hyperbilirubinaemia  and normal liver function tests, Dubin-Johnson or  Rotor’s syndrome should be considered.   Causes of hypoglycaemia in the newborn period  (and, for completeness, also in childhood) are  shown in Box 26.1. Hepatic glycogen stores increase  about three-fold and adipose tissue (another source  of energy) is laid down during the last 10 weeks of   Disorders of fat oxidation, e.g. medium-chain acyl  coenzyme A dehydrogenase deficiency  Disorders of gluconeogenesis, e.g. pyruvate  carboxylase deficiency   pregnancy. Very premature infants therefore have little  liver glycogen and adipose tissue and are especially  prone to hypoglycaemia. Full-term infants may  become hypoglycaemic if initially adequate stores  are drawn on more rapidly than normal, for example  during perinatal asphyxia. Infants at greater risk of  hypoglycaemia include those with infections, asphyxia,  rhesus haemolytic disease and exchange transfusion,  those who are small for dates and those born to  diabetic mothers.   Plasma glucose concentrations as low as 1.7 mmol/L  during the first 72 h of life in the preterm infant, or  2.0 mmol/L during the later neonatal period, may not  be associated with any clinical signs. When clinical  features do occur they include tremors, apnoeic attacks  and convulsions. However, impaired neurological  development has been reported in full-term infants  in whom the plasma glucose concentration repeatedly  fell to below 2.0 mmol/L despite the absence of clinical  signs. It has been recommended that the plasma glucose  concentration be maintained above 2.0 mmol/L in at-  risk infants.   If a baby has hypoglycaemia and is asymptomatic,  milk feeds are usually given until blood glucose levels  are above 2.0 mmol/L. However, in the presence of  symptoms of hypoglycaemia, intravenous 10 per cent  dextrose (glucose) mayneedtobegiven.Nesidioblastosis  is due to overgrowth of insulin-producing (3-cells in the  pancreas and presents with severe hypoglycaemia and  hyperinsulinaemia. Beckwith-Wiedemann syndrome  is due to a short-arm deletion of chromosome 11  and is associated with hypoglycaemia, visceromegaly,  exomphalos and intellectual disabilities.   Hypoglycaemia determined by near-patient glucose  tests must be confirmed by proper laboratory testing in  a fluoride sample. Lack of ketonuria in the presence of  hypoglycaemia implies hyperinsulinaemia or a defect  of fat oxidation.   The babies of diabetic mothers (including gestational  diabetes) tend to be large, jaundiced, hypocalcaemic,  hypomagnesaemic, polycythaemic and to have lung  immaturity and congenital defects. These pregnancies are  associated with greater need for instrumental deliveries  and caesarean section. Neonatal hypoglycaemia is related  to the degree of maternal glycaemic control.   Soon after birth, or the introduction of milk to the  diet, hypoglycaemia may be due to one of the following   A deficiency of one ofthe enzymesinvolvedin glycogenesis  or glycogenolysis results in the accumulation of normal  or abnormal glycogen with hepatomegaly. In von  Gierke’s disease, the least rare glycogen storage disorder,  there is a deficiency of glucose-6-phosphatase. Fasting  hypoglycaemia occurs because the enzyme is essential  for the conversion of glucose-6-phosphate (G6P) to  glucose. There may also be ketosis and endogenous  hypertriglyceridaemia due to excessive lipolysis caused  by low insulin activity, lactic acidosis due to excessive  anaerobic glycolysis, and hyperuricaemia.   The diagnosis is made either directly, by  demonstrating the absence of the enzyme in a liver  biopsy specimen, or indirectly, by demonstrating  failure of plasma glucose concentrations to rise after  giving glucagon to stimulate glycogenolysis. Infusion  of galactose or fructose, which are normally converted  to glucose via G6P, also fails to increase plasma glucose  concentrations because G6P cannot be converted to  glucose. Treatment involves giving frequent meals to  maintain normal plasma glucose levels and so prevent  cerebral damage.   This is a rare cause of hypoglycaemia. It is due to  deficiency of fructose- 1 -phosphate aldolase. The  accumulation of fructose- 1 -phosphate in several tissues  causes many of the clinical features; hypoglycaemia  may be due to the inhibition of glycogenolysis and  gluconeogenesis. Symptoms only present after sucrose-  containing (fructose and glucose) and fructose-  containing fruit or fruit drinks have been introduced  into the diet. Hypoglycaemia, with nausea, vomiting  and abdominal pain, and fructosuria follow about  30 min after fructose ingestion or intravenous infusion;  this can be used as a diagnostic test. The infant fails  to thrive. Liver accumulation of fructose- 1 -phosphate  causes hepatomegaly with jaundice; this may progress  to cirrhosis with ascites.   The diagnosis of this condition is made by excluding  other causes of hypoglycaemia. Symptoms usually  develop after fasting or a febrile illness. Brain damage  is a risk. In some cases there is excessive insulin  secretion and it may not be possible to differentiate  this condition from an insulinoma. Islet cell  hyperplasia (nesidioblastosis or endogenous persistent   hyperinsulinaemic hypoglycaemia of infancy) is a  difficult diagnosis to make and is an uncommon cause  of hypoglycaemia occurring before the age of 3 years.   During the second year of life, hypoglycaemia  associated with ketosis may also develop after fasting or  a febrile illness (ketotic hypoglycaemia). These children  were usually ‘small for dates’ at birth. Adult causes of  hypoglycaemia, including insulinoma, must always be  considered in the differential diagnosis.   There is often a familial incidence of leucine sensitivity.  During the first 6 months of life, casein (present in  milk) may cause severe hypoglycaemia due to its high  leucine content. Leucine sensitivity is probably due to  the stimulation of insulin secretion by the amino acid.  The condition appears to be self-limiting and does not  usually persist beyond the age of 6 years. The diagnosis  is confirmed by demonstrating hypoglycaemia within  30 min of an oral dose of leucine or casein. Normal  subjects do not respond to leucine with a significant  fall in plasma glucose concentration, but a number  of patients with insulinoma are leucine sensitive.  Treatment involves giving a diet low in leucine-  containing food.   Medium-chain acyl coenzyme A dehydrogenase  deficiency (MCADD) can also present with  hypoglycaemia (see Chapter 27). Other causes of  hypoglycaemia in childhood are shown in Box 26.1 and  discussed in Chapter 12.   Calcium and phosphate are actively transported  across the placenta; total and free ionized calcium  concentrations are higher in fetal than in maternal  plasma. 25-Hydroxycholecalciferol, but not its active  metabolite 1,25-dihydroxycholecalciferol or parathyroid  hormone (PTH), can pass placental membranes.  Calcium and phosphate accumulate rapidly only  between the 30th and 36th week of fetal life; therefore,  a very premature infant may become calcium and  phosphate deficient when feeds are not supplemented.   During intrauterine life, the relatively high  maternally derived fetal plasma free ionized calcium  concentration suppresses PTH release from the  fetus’s own parathyroid glands. Fetal plasma calcium  concentration is usually higher than in the mother due  to active calcium transport across the placenta. The   A preterm baby was born at 28 weeks weighing only  900 g. The following biochemical results were obtained:   The results show hypocalcaemia and hypoglycaemia,  which are sometimes seen in severe prematurity,  although other causes may need to be excluded.  Preterm neonates may also show renal, liver  and acid-base abnormalities. The baby here was  also jaundiced (note the raised plasma bilirubin  concentration). Note the higher reference range for  plasma phosphate in neonates than that in adults.   glands may take time to recover after birth and there  may be transient hypoparathyroidism. Plasma total  and free ionized calcium concentrations fall by up to  30 per cent immediately after birth, but, in the normal  infant, spontaneously regain adult concentrations  within about 3 days; this fall rarely needs to be treated.   The reference range for plasma total calcium  concentration in the newborn period is wider than  that for adults. Plasma phosphate concentrations are  higher in actively growing infants and in children than  in adults, as is the concentration of plasma alkaline  phosphatase.   Hypocalcaemia during the first 2 weeks of life can be  divided into two groups (Box 26.2).   Hypocalcaemia of early onset occurs during the first 3  days of life and is most common:   It may also occur if the mother was hypercalcaemic  during pregnancy, for example due to primary  hyperparathyroidism; the maternal hypercalcaemia  is reflected in the fetus, and the suppressed fetal  parathyroid glands may take some time to recover. This  condition is a more severe form of the ‘physiological’  condition described above.   Hypocalcaemia of late onset is less common. It usually  followed the use of high-phosphate-containing feeds,  such as unmodified cow’s milk formulas, which are less  used nowadays.   Primary hypoparathyroidism in neonates is rare but  can be inherited, including an X-linked form. Maternal  hyperparathyroidism may suppress neonatal PTH,  evoking hypocalcaemia. There is also a rare transient  form of hypoparathyroidism presenting up to a year  of age. Di George’s syndrome is a disorder in which  hypoparathyroidism is associated with cardiac defects,  thymic aplasia and immunodeficiency.   Bone demineralization (osteopenia) is common in  small preterm infants and often resolves spontaneously  before obvious clinical features of rickets develop. The  plasma alkaline phosphatase activity may rise to more  than six times the upper adult reference limit; the  plasma calcium concentrations are usually low-normal  and those of phosphate are low.   Rickets of prematurity usually becomes clinically  evident between the 4th and 12th weeks of life, and  occurs more commonly in very premature, low-  birthweight infants than in infants of older gestational  age at birth. Longitudinal growth slows, and the  decalcification of the bones predisposes to pathological  fractures; in severe cases, respiration is impaired by the  soft ribs.   Radiograph changes may be minimal or absent  in early cases. In more advanced disease, the classic  radiological changes appear at the ends of long bones.  Rickets of prematurity may be due to the following:   • Calcium and phosphate depletion : if an infant is born  prematurely, unsupplemented breast milk may not  contain enough of these minerals to replace that  which should have accumulated in utero during the  last 10 weeks of gestation.   Phosphate depletion is probably the most  common cause and is indicated by a very low  plasma phosphate concentration, compared with  the appropriate reference range, and low urinary  phosphate excretion.   • Maternal vitamin D deficiency during pregnancy or in  the infant after birth: in very premature infants, such  deficiency may be due to low activity of the renal  1 a -hydroxylase neededto convert 25-hydroxyvitamin  D to the active 1,25-dihydroxyvitamin D.   • Renal tubular disorders of phosphate reabsorption,  which may cause phosphate depletion: in such cases  the plasma calcium concentration is usually normal,  but may even be high because the calcium cannot be  deposited in bone without phosphate.   Vitamin D and calcium and phosphate supplementation  may be monitored by measuring serial plasma alkaline  phosphatase activities. Despite successful treatment,  these may continue to rise for several weeks, when bone  is being actively laid down, before falling once the bone  is adequately calcified.   Hypercalcaemia in the newborn period  Hypercalcaemia is less common than hypocalcaemia at  this age. It may be associated with phosphate depletion  and hypophosphataemia because calcium cannot be  deposited in bone without phosphate, and because  hypophosphataemia enhances la-hydroxylase activity  and therefore the formation of the active vitamin D   metabolite. After treatment with phosphorus, the  plasma calcium concentration may fall rapidly enough  to cause clinical symptoms.   Idiopathic infantile hypercalcaemia may occur in  full-term infants receiving inappropriately high-dose  vitamin D prophylaxis who have abnormalities of the  CYP24A1 gene, which encodes for a major enzyme  (25-hydroxyvitamin D 24-hydroxylase) that metabolizes  vitamin D. Other conditions include vitamin D excess,  familial hypocalciuric hypercalcaemia and Williams’  syndrome. The last mentioned is associated with  intellectual disabilities, ‘elfin’ facies and cardiac defects.   Hypomagnesaemia often accompanies hypocalcaemia  and may be caused by dietary deficiency or by increased  intestinal or urinary loss. Low plasma magnesium  concentrations may impair the release and action of PTH  and so delay correction of plasma calcium concentrations.  Hypomagnesaemia should be considered if the infant  has convulsions despite normocalcaemia.   At birth, the relative concentrations of individual  plasma proteins differ from those found in adults. The  concentration of total protein is about 12g/L lower  than in adults, but that of albumin is only slightly  lower; as always, the latter may fall during illness. The  acute-phase proteins (reflected in the a-globulins and  (3-globulins in the electrophoretic pattern) reach adult  concentrations by about 6 months, but are affected by  illness, again as they are in adults.   The immunoglobulin pattern differs significantly  from that of adults. During normal pregnancy, placental  transfer of maternal immunoglobulin G (IgG) leads to  a gradual increase in fetal plasma concentrations of this  immunoglobulin. After birth, maternally derived IgG is  degraded and endogenous immunoglobulin synthesis  starts. Adult concentrations of plasma IgM are reached  by about 9 months, of IgG by between 3 and 5 years,  and of IgA by the age of 15.   Measurement of plasma immunoglobulin  concentrations may be indicated if an immune-  deficiency state, whetherprim ary, secondary or transient,  is suspected. It may also help to detect infection,  whether it occurred during the intrauterine period or  has developed after birth. Results must be compared  with age-matched reference ranges, allowance being  made for both the time since conception (gestational  age) and age since birth (post-natal age).   A high plasma IgM concentration in blood obtained  from the umbilical cord, or within the first 4 weeks of life,  may indicate intrauterine or neonatal infections such  as syphilis, rubella, toxoplasmosis or cytomegalovirus.  Allowance must be made for the normal rise in plasma  IgM that starts after about 6 weeks of post-natal age.  Specific inborn errors, such as 0 ^- antitrypsin deficiency,  maybe diagnosed by measuring the appropriate plasma  protein.   Plasma ammonia should be less than lOOpmol/L  in full-term neonates and infants, and less than  200pmol/L in preterm neonates. Clinical features of  hyperammonaemia include seizures, coma, lethargy  and respiratory alkalosis. Plasma ammonia should be  assayed in fresh samples collected in heparin.   Sepsis (including urinary tract infections),  cardiac failure and certain tumours can result in  hyperammonaemia, as can valproate treatment.  Acute hepatic failure or chronic liver disease can also  evoke hyperammonaemia, as the liver is an important  deaminating organ; Reye’s syndrome is also associated  with hyperammonaemia. This is thought to be due  to a mitochondrial disorder leading to fatty liver  degeneration and acute encephalopathy.   The urea cycle disorders are an important cause  of severe hyperammonaemia, and if urinary organic  acid concentrations are raised, this is suggestive of an  organic aciduria (see Chapter 27). The plasma amino  acid pattern may give clues as to the amino acid disorder  causing the hyperammonaemia, such as arginaemia,  citrullinaemia, 3H syndrome and arginosuccinic  aciduria. Inborn errors of metabolism are covered in  more detail in Chapter 27.   Immediately after birth, plasma thyroid- stimulating  hormone (TSH) concentrations rise rapidly, probably  in response to the stress of birth, to about 15 times the  upper adult reference limit. They reach a peak within  the first hour, before falling, rapidly at first, during  the next week. Plasma total thyroxine concentrations  peak within the first 24-48 h and then fall gradually  (Fig. 26.1).   Most laboratories in the UK screen with TSH alone,  using a heel prick sample, although this may miss  the even rarer cases of secondary hypothyroidism  (Fig. 26.2). Screening tests for neonatal congenital  hypothyroidism should be delayed for about a week   Figure 26.1 Thyroid function in the newborn infant.  TSH, thyroid-stimulating hormone.   after birth to allow the plasma TSH concentration to  stabilize. In premature babies TSH may not increase  after birth and thus give false-negative results; therefore,  testing should be repeated when they are equivalent to  40 weeks.   About 1 in 3500 neonates have congenital  hypothyroidism, with most cases being due to thyroid  dysgenesis and about 10 per cent resulting from  dyshormonogenesis. Thyroid function tests should be  repeated in infants found to have a positive screening  test at a week after birth; if the diagnosis is confirmed,  thyroid replacement should be started immediately.  Thyroid function should be reassessed, after the  withdrawal of treatment, at the age of 1 year because  neonatal hypothyroidism is sometimes transient.   Hypothyroidism may be suspected clinically, for  example because of failure to thrive or persistent   jaundice. (See also Chapter 11 for discussion of thyroid  disease.) Neonatal screening is discussed further in  Chapter 27.   Within the next 50 years or so, about one-quarter of  the world’s population will be over the age of 65 years.  Just as in the neonate, it is important to understand the  biochemical changes that occur with age when it comes  to interpreting chemical pathology results in the elderly.   First, it is pertinent to realize that multiple  pathologies are sometimes present in the elderly. In   An 83 -year- old woman was seen by her general  practitioner because of increasing confusion. She was  known to have congestive cardiac failure, hypertension,  diabetes mellitus, osteoporosis and diverticular  disease. She was taking 1 0 different medications. Some  of her biochemical results were as follows:   Estimated glomerular filtration rate (eGFR)  34mL/min per 1.73 m 2  Albumin 36g/L (35-45)   Glycated haemoglobin (HbA lc ) 8.0% (64 mmol/mol)  Thyroid- stimulating hormone 11.9mU/L (0. 2-5.0)  Lree thyroxine 8.5pmol/L (12-25)   The patient was subsequently shown to have had  a cerebrovascular accident. Her biochemical tests  suggest hypothyroidism, hypocalcaemia (found to  be due to osteomalacia) and impaired renal function.  The elderly may present with multiple pathologies.  Renal function declines with age. Type 2 diabetes  mellitus is also relatively common in the elderly.  Another clinical problem is ‘poly-pharmacy’, with  some elderly patients being treated with multiple  drugs for multiple conditions and therefore running  the risk of dangerous drug interactions.   addition, they may well be on numerous medications,  and therefore drug interactions are more likely. Ageing  may be associated with changes in the reference  range of certain analytes; for example plasma alkaline  phosphatase may increase, probably of bone origin.   Some diseases are more common in the elderly,  including cardiovascular disease, type 2 diabetes  mellitus, Paget’s disease, osteoporosis, renal  impairment, thyroid disease and certain tumours.  Multiple myeloma is far more prevalent as age increases  as is other malignant disease.   The increased morbidity and mortality of the elderly  are not helped by the fact that nutritional disorders are  also more common. Some elderly people, particularly  if on low incomes, may have poor diets, and deficiency  states have been described, including osteomalacia and  scurvy.   Ischaemic heart disease and cerebrovascular disease  constitute the major causes of death in the elderly,  and their risk increases with age. Plasma cholesterol  concentration also increases with age, although there  may be a decline in those over the age of 75 years.   Renal function may deteriorate with age over the age  of 70 years, Remember that plasma creatinine may  be within the adult reference range but estimated  glomerular filtration rate (eGFR) abnormally low.  This partly relates to the decrease in muscle ‘bulk’ that  sometimes occurs with ageing, and a corresponding  decrease in plasma creatinine. This is important to  remember when it comes to prescribing medications  that are dependent on renal excretion (see Chapter 3).   This is more common in the elderly, in whom it is  usually type 2. Generally, glucose intolerance increases  with age (see Chapter 12).   Abnormalities of thyroid function also increase with  age; both hyperthyroidism and hypothyroidism are  more common in the elderly. The former is more likely  to be associated with atrial fibrillation and the latter  with hypothermia. Because of the increased likelihood  of disease in the elderly, sick euthyroidism is also  more common (see Chapter 11). The pituitary gland  diminishes in size in the elderly. Microadenomas are  more likely, as is growth hormone deficiency.   The female menopause usually occurs around the  age of 51 years and is associated with decreased  plasma oestrogen and raised luteinizing hormone  and follicle-stimulating hormone concentrations. The  use of hormone replacement therapy is controversial.  Gonadal decline is more variable in the male, as fertility  may continue into the seventies and beyond, when  plasma testosterone concentration may be reduced.   The most common bone disorder is osteoporosis (see  Chapter 6). The reason is multifactorial, but includes  decreasing endocrine function, including reduced  oestrogen and testosterone, as well as nutritional  factors. Paget’s disease is also more prevalent and partly   accounts for the higher plasma alkaline phosphatase  activities found in this age group. Additionally,  osteomalacia is increased due to vitamin D deficiency.   About 10 per cent of people over the age of 65 years  have dementia. This can be defined as a decline in  global cognition in clear consciousness. It may be due  to many factors, although the most common causes  are vascular and Alzheimer’s disease. It is essential  to exclude treatable causes such as hypothyroidism,  infection and nutritional deficiencies. Potentially useful  laboratory tests include full blood count, plasma folate  and vitamin B 12 , plasma glucose, thyroid function tests,  liver and renal function tests, plasma calcium, syphilis  screen and paraprotein screen.   • Neonates show a number of biochemical features  that are different from those found in adults.   • Unconjugated hyperbilirubinaemia can be  physiological in neonates, but severely raised plasma  bilirubin concentration (more than 350pmol/L  approximately) can result in kernicterus associated  with neurological damage.   • Premature neonates may present with hypocalcaemia,  jaundice, hypoglycaemia and renal, hepatic and acid-  base disorders.   The elderly may present with multiple pathologies.  Renal function declines with age. Type 2 diabetes  mellitus is also relatively common in the elderly.  Another clinical problem is ‘poly-pharmacy’, with  many elderly people being treated with multiple  drugs for multiple conditions, which runs the risk  of drug interaction.   Reference ranges for certain analytes may be  different in the very young and the elderly.   Principles of treatment of inborn errors of metabolism 373  Diseases due to inborn errors of metabolism 373   The inherited characteristics of an individual are  determined by about 50000 gene pairs, arranged on 23  pairs of chromosomes, one of each pair coming from  the father and one from the mother. Genotype diversity  is introduced by random selection and recombination  during meiosis, as well as by occasional mutation. These  genetic variants may at one extreme be incompatible  with life, or at the other produce biochemical  differences detectable only by special techniques, if at  all. Between the two extremes there are many variations  that produce functional abnormalities or inborn  errors of metabolism (IEM). Incidences of IEM range  from about 1 in 100 to 1 in 200 000, depending on the  disorder and the population involved.   Inherited inborn disorders may involve any peptide  or protein, and are usually most obvious if there is an  enzyme abnormality. Deficiency of a single enzyme in  a metabolic pathway may produce its effects in several  ways.   Suppose that substance A is acted on by enzyme X  to produce substance B, and that substance C is on an  alternative pathway (Fig. 27.1). A deficiency of X may  cause:   • deficiency of the product (B) of the enzyme reaction,  for example cortisol deficiency in congenital adrenal  hyperplasia,   • accumulation of the substance (A) acted on  by the enzyme, for example phenylalanine in  phenylketonuria (PKU),   • diversion through an alternative pathway: some  product(s) of the latter (C) may accumulate and  produce effects, for example congenital adrenal  hyperplasia when accumulation of androgens causes  virilization.   The effects of the last two types of abnormality  will be aggravated if the whole metabolic pathway is  controlled by negative feedback from the final product.  For example, in congenital adrenal hyperplasia,  cortisol deficiency reduces negative feedback, thereby  increasing the rate of steroid synthesis and therefore the  accumulation of androgens, causing virilization in the  female (see Chapter 8).   The clinical effects of some IEM may be modified by,  or depend entirely on, physiological or environmental  factors. For example, iron loss occurs during  menstruation and pregnancy; women with hereditary  haemochromatosis accumulate iron less rapidly than  men with the same condition and they rarely present  with clinical features before the menopause (see  Chapter 21). Patients with cholinesterase variants  develop symptoms only if the muscle relaxant  suxamethonium is given (see Chapter 18).   Some inborn errors are probably harmless. However,  they are important because they produce effects that  may lead to misdiagnosis, for example renal glycosuria  and Gilbert’s syndrome. In others, it is important to  make a diagnosis, even though no effective treatment is  yet available.   There is a group of diseases for which recognition in  early infancy is of great importance because treatment  may prevent irreversible clinical consequences or  death. Some of the more important of these are PKU,  galactosaemia and maple syrup urine disease.   Many countries have instituted programmes for  screening all newborn infants for certain inherited  metabolic disorders or congenital defects. The criteria  should depend on the following characteristics of the  disorder or of the test:   • The disease should not be clinically apparent at the  time of screening and should have a relatively high  incidence in the population screened.   • The disease should be treatable or early treatment  should improve outcome.   • It must be possible to obtain the result of the  screening test before irreversible damage is likely to  have occurred.   • The screening test should be simple and reliable  and the cost of the programme should, ideally, be  at least partly offset by the cost savings resulting  from early treatment. For example, such treatment  may sometimes eliminate the need for prolonged  institutional care.   Not all these criteria are necessarily fulfilled in all  screening programmes.   In the UK, at between 5 and 8 days, babies are screened  for certain conditions by taking a small capillary blood  sample from a heel prick (see Chapter 26, Fig. 26.2).  Blood spots are placed on a paper card, which can be  posted to the regional laboratory for assay.   In the UK, screening is generally carried out for  neonatal hypothyroidism (see Chapters 11 and 26)  and PKU. Other conditions that may be screened for  in certain regions include cystic fibrosis, sickle cell  disease or thalassaemia, medium-chain acyl coenzyme  A dehydrogenase deficiency (MCADD), glucose-6-  phosphatase deficiency, galactosaemia and congenital  adrenal hyperplasia. (These conditions are discussed  elsewhere in this book.) The use of deoxyribonucleic  acid (DNA) technology and tandem mass spectroscopy  in antenatal screening can be expected to increase in the  future.   Prenatal screening, of high-risk groups only, may  be performed for some disorders in order to plan   the appropriate place and method of delivery for the  well-being of the infant or to offer termination, if the  diagnosis is made early enough and if it is acceptable.   Prenatal screening for inherited metabolic  disorders most commonly involves demonstrating the  metabolic defect in cultured fetal fibroblasts obtained  by amniocentesis early in the second trimester, or by  chorionic villus sampling during the first trimester.  Examples of those groups in whom such screening  may be indicated include women with a previously  affected infant and ethnic groups thought to have a  relatively high incidence of the carrier state, such as  of Tay-Sachs disease in Ashkenazi Jews. In these high-  risk populations, screening is often performed before  conception, enabling genetic advice and prenatal  diagnosis to be offered to couples who are carriers.   If, as in cystic fibrosis, the gene defect of the parent  of an affected infant is known, there may be a case for  selective screening of subsequent pregnancies using  molecular biological techniques. Prenatal screening for  congenital disorders, for example neural tube defects,  or chromosomal abnormalities may also be performed.   The possibility of an inherited metabolic defect should  be considered if there are unusual, unexplained clinical  features (Box 27.1) or abnormal laboratory findings in   A peculiar smell, or staining, of the nappies  Death of child in family and positive family history  Cataracts or retinitis pigmentosa   infancy or early childhood, especially if more than one  infant in the family has been affected or there has been  a consanguineous marriage.   Screening tests should be interpreted with caution  and a suspected diagnosis confirmed by more specific  techniques in a laboratory specializing in such disorders.   Inborn errors presenting acutely are usually due to  an enzyme abnormality. This may be demonstrated  indirectly, by detecting a high concentration of the  substance normally metabolized by the enzyme, or  a low concentration of the product; or directly, by  demonstrating a low enzyme activity in the appropriate  tissue or blood cells; these assays may only be available  at special centres. If possible, all cases should be  confirmed in this way.   • estimation of plasma ammonia concentration to test  for disorders of the urea cycle or organic acidurias,  in which it accumulates,   • chromatography of plasma and urine for amino  acids for the detection of disorders of amino acid  metabolism,   • detection of organic acids in urine in disorders of  branched-chain amino acid metabolism and organic  acidurias.   If the clinical signs and symptoms are  strongly suggestive of a particular disorder,  specific measurements, such as those of urinary  glycosaminoglycan excretion and white cell enzymes  characteristic of mucopolysaccharidoses (MPSs), may  be used. The technique of tandem mass spectroscopy is  proving useful in the investigation of various IEM.   Genetic tests are being used more frequently, and  their use is likely to increase still further. Box 27.2  gives a suggested investigation plan.   • limiting the dietary intake of precursors in the  affected metabolic pathway, such as phenylalanine in  PKU or lactose in galactosaemia,   • supplying the missing metabolic product, such as  cortisol in congenital adrenal hyperplasia,   • removing or reducing the accumulated product,  such as ammonia in urea cycle disorders.   Experimental treatments may be tried for some  disorders with particularly poor prognoses. One   Serum cholesterol and triglyceride  Plasma lactate  Plasma uric acid  Thyroid function tests  Porphyrins   of these is enzyme replacement by bone marrow  transplantation, but the results have sometimes  been disappointing and it is not without its own  complications. Insertion of the missing or defective  gene is being attempted for disorders such as  adenosine deaminase deficiency. However, for many  disorders there is, at the time of writing, still no  treatment unless they respond to one of the measures  listed above.   Only a few of the known IEM are discussed here. Some  of these conditions are mentioned briefly in the relevant  chapters in this book.   For the sake of convenience, nine general categories  of IEM are arbitrarily defined:   4. disorders of carbohydrate metabolism, for example  glycogen storage disorders, gluconeogenesis and  carbohydrate intolerance defects,   A 3-month-old boy was seen in the paediatric out-  patient department because of failure to thrive and  hypotonia. The family had previously lost a male  child, who had died at the age of 9 months. Some of  the presenting child’s abnormal biochemistry results  were as follows:   Plasma amino acid analysis revealed elevated alanine,  glutamine and orotic acid concentrations.   The diagnosis was established as ornithine  transcarbamylase (OTC) deficiency, one of the urea  cycle defects, which is X-linked. Note that the child  shows hyperammonaemia and low plasma urea  concentration and that he is male, in keeping with a  sex-linked condition. There are relatively few causes  of a low plasma urea concentration in neonates,  and this combined with severe hyperammonaemia  supports a diagnosis of a urea cycle disorder. An  inborn error of metabolism should be suspected  on the death of a baby or when a child presents  with failure to thrive, particularly if the parents are  consanguineous, for example cousins.   urinary orotic acid concentration, an intermediate  metabolite of pyrimidine synthesis derived from  carbamyl phosphate. The urea cycle defects can present  not only with severe hyperammonaemia, but also with a  respiratory alkalosis and low plasma urea concentration  (Fig. 27.2). Carbamyl phosphate synthetase (CPS)  deficiency is a urea cycle disorder in which, unlike other  defects in this pathway, urinary orotic acid is not raised.  Ornithine transcarbamylase deficiency is probably the  most common urea cycle defect and is sex linked.   2 Disorders of amino acid metabolism  Many disorders of amino acid metabolism are  characterized by raised plasma concentrations of one  or more amino acids, with overflow amino aciduria.   The main metabolic pathway for aromatic amino  acids is outlined in Figure 27.3, which also indicates the  known enzyme defects. Tyrosine, normally produced  from phenylalanine, is the precursor of several  important substances, inherited disorders of which are  considered briefly.   Phenylketonuria is an autosomal recessive disorder  caused by an abnormality of the phenylalanine  hydroxylase system. In the UK the incidence is about  1 in 10 000. This is the enzyme most commonly affected,  but in about 3 per cent of cases the enzymes responsible    Figure 27.2 Summary of the urea cycle. OTC, ornithine  transcarbamylase. Reproduced with kind permission  from Candlish JK and Crook M. Notes on Clinical  Biochemistry. Singapore: World Scientific Publishing,  1993.   Figure 27.3 Diagram showing the metabolism of tyrosine and some inborn errors of the aromatic amino acid  pathway. Substances highlighted may be present in abnormal amounts in certain inborn errors of metabolism. (1)  Phenylalanine hydroxylase - phenylketonuria (PKU); (2) homogentisic acid oxidase - alkaptonuria; (3) tyrosinase  - albinism; (4) thyroid enzymes - thyroid dyshormonogenesis. TCA, tricarboxylic acid.   Therefore several different inherited deficiencies  may have very similar biochemical and clinical  consequences. Phenylalanine cannot be converted to  tyrosine, and accumulates in plasma and is excreted in  the urine with its metabolites, such as phenylpyruvic  acid (a phenylketone).   • intellectual disabilities developing at between 4 and  6 months, with psychomotor irritability,   • a tendency to reduced melanin formation because of  reduced production of tyrosine - many patients are  pale skinned, fair haired and blue eyed,   • irritability, feeding problems, vomiting and fits  during the first few weeks of life,   Diagnosis may involve measuring the phenylalanine  concentration in blood taken from a heel prick.  The microbiological Guthrie test was used to assay  phenylalanine, but now many laboratories use  chromatography methods or tandem mass spectroscopy.  In the newborn, and especially in preterm infants, the  enzyme system may not be fully developed and false-  positive results are likely if the test is performed too  early. If a positive result is found, the test should be  repeated later, to allow time for development of the  enzyme. The phenylalanine concentrations may be  greater than 240 pmol/T.   Heterozygotes may be clinically normal, but can be  detected by biochemical tests. A variant - persistent  hyperphenylalaninaemia - without intellectual  disabilities has been described. Infants who are exposed   in utero to the high phenylalanine concentrations of  undiagnosed or poorly controlled phenylketonuric  mothers may have intellectual disabilities, although  they themselves do not have detectable PKU (maternal  phenylketonuric syndrome).   The aim of treatment is to lower plasma phenylalanine  concentrations by giving a low-phenylalanine diet. Such  treatment should be monitored carefully, especially if  the patient is planning to conceive or is pregnant. It is  now generally recommended that in proven cases dietary  restriction should be life long, with supervision by a  metabolic physician and expert dietitian. Remember  that the artificial sweetener aspartame is metabolized  to phenylalanine.   Tyrosinaemia presents with renal tubular dysfunction,  hypoglycaemia and severe liver disease with very  raised plasma alkaline phosphatase concentration.  The defect is due to abnormal fumarylacetoacetase  leading to raised tyrosine, succinylacetone and  hydroxyphenylpyruvate. Diagnosis is by showing  raised urinary succinylacetone concentration and  assay of fumarylacetoacetase in cultured leucocytes  or fibroblasts. Treatment can be dietary, by liver  transplantation or by nitro-trifluoromethylbenzoyl  cyclohexanedione, which is thought to reduce the  accumulation of some of the toxic metabolites.   Alkaptonuria is an autosomal recessive disorder  associated with a deficiency of homogentisic acid  oxidase. Homogentisic acid accumulates in tissues  and blood, and is passed in the urine. Oxidation   and polymerization of homogentisic acid produce  the pigment alkapton, in much the same way as  polymerization of dihydroxyphenylalanine results  in melanin. The deposition of alkapton in cartilages,  with consequent darkening, is called ochronosis and  results in visible darkening of the cartilages of the  ears and often arthritis in later life. The conversion  of homogentisic acid to alkapton is accelerated in  alkaline conditions, and sometimes the most obvious  abnormality in alkaptonuria is darkening of the urine  as it becomes more alkaline on standing. Homogentisic  acid, a reducing substance, reacts with Clinitest tablets  (Fig. 27.4).   A deficiency of tyrosinase in melanocytes causes one  form of albinism; it is inherited as an autosomal recessive  disorder. Pigmentation of the skin, hair and iris is reduced  and the eyes may appear pink. Reduced pigmentation  of the iris causes photosensitivity, and decreased skin  pigmentation is associated with an increased incidence  of certain skin cancers. The tyrosinase involved in  catecholamine synthesis is a different isoenzyme,  controlled by a different gene; consequently, adrenaline  (epinephrine) metabolism is normal.   involve sulphur-containing amino acids. Patients  may show progressive central nervous system (CNS)  dysfunction, thrombotic disease, eye disease, including  cataracts, and cardiovascular problems. The diagnosis  of homocystinuria is based on the presence of raised  urinary and plasma homocysteine with low plasma  methionine concentrations. The defective enzyme can  be assayed in cultured skin fibroblasts.   In maple syrup urine disease, which is inherited as  an autosomal recessive condition, there is deficient  decarboxylation of the oxoacids resulting from  deamination of the three branched- chain amino  acids, leucine, isoleucine and valine. These amino  acids accumulate in the plasma and are excreted in the  urine with their corresponding oxoacids. The sweet  smell of the urine is like that of maple syrup, hence the  condition’s name (Fig. 27.5).   The disease presents during the first week of life  and, if not treated, severe neurological lesions develop  which cause death within a few weeks or months.  If a diet low in branched-chain amino acids is given,  normal development is possible. The diagnosis of   Homocystinuria is an autosomal recessive disorder due  to deficiency of cystathionine synthase. These pathways    Figure 27.4 Alkaptonuric urine. The flask on the right  contains fresh urine darkened somewhat; the flask on  the left, to which sodium hydroxide has been added,  contains a black suspension. Reproduced with kind  permission from Nyhan WL and Barshop BA. Atlas  of Inherited Metabolic Diseases, 3rd edition. Hodder  Arnold, 2012.    Figure 27.5 Frozen urine from a patient with untreated  maple syrup urine disease. The odour of maple syrup  is concentrated in an oil at the top. Reproduced with  kind permission from Nyhan WL and Barshop BA.  Atlas of Inherited Metabolic Diseases, 3rd edition.  Hodder Arnold, 2012.   maple syrup urine disease is made by demonstrating  raised concentrations of branched-chain amino  acids in plasma and urine and low plasma alanine  concentration. It may be confirmed by demonstrating  the enzyme defect in leucocytes.   Histidinaemia is associated with deficiency of  histidinase, an enzyme needed for normal histidine  metabolism, and is probably inherited as an autosomal  recessive trait. Some individuals may have intellectual  disabilities and speech defects, but others may be  normal.   The diagnosis is made by demonstrating raised  plasma levels of histidine, and by finding histidine and  the metabolite imidazole pyruvic acid in the urine.   Groups of chemically similar substances are often  transported by shared or inter-related pathways. Such  group -specific mechanisms usually affect transport  across all cell membranes, and defects often involve  both the renal tubules and intestinal mucosa. Inborn  errors of the following amino acid group pathways have  been identified:   • the dibasic amino acids (with two amino groups)  cystine, ornithine, arginine and lysine (cystinuria) -  COAL is a useful mnemonic,   • many neutral amino acids (with one amino and one  carboxyl group) (Hartnup’s disease),   • the imino acids proline and hydroxyproline, which  probably share a pathway with glycine (familial  iminoglycinuria) .   Amino acids are usually filtered by the glomeruli, reach  the proximal tubules at concentrations equal to those  in plasma and are almost completely reabsorbed as they  pass through this part of the nephron. Amino aciduria  may therefore be of two types.   • Overflow amino aciduria In which, because of  raised plasma concentrations, amino acids reach the  proximal tubules at concentrations higher than the  reabsorptive capacity of the cells.   • Renal amino aciduria In which plasma  concentrations are low because of urinary loss due  to defective tubular reabsorption.   Amino aciduria may also be subdivided according to  the pattern of excreted amino acids.   • Specific amino aciduria is due to increased excretion  of either a single amino acid or a group of chemically  related amino acids. It may be overflow or renal in  type.   • Non-specific amino aciduria, in which there is  increased excretion of a number of unrelated amino  acids, is almost always due to an acquired disorder. It  may be overflow in type, as in severe hepatic disease  when impaired deamination of amino acids causes  raised plasma concentrations; more commonly,  renal amino aciduria results from non-specific  proximal tubular damage, and other substances that  are usually almost completely reabsorbed by the  proximal tubule are also lost (phosphoglucoamino  aciduria; Fanconi’s syndrome). If it occurs due to  an inborn error of metabolism, it is rarely a direct  result of the genetic defect, but more commonly  secondary to tubular damage caused by deposition  of the substance not metabolized normally, such as  copper in Wilson’s disease (Fig. 27.6).   Cystinuria is the result of an autosomal recessive  inherited abnormality of tubular reabsorption, with  excessive urinary excretion, of the dibasic amino  acids cystine, ornithine, arginine and lysine. A similar  transport defect has been demonstrated in the intestinal  mucosa, but, although dibasic amino acid absorption is  reduced, deficiencies do not occur because they can be  synthesized in the body. Cystine is relatively insoluble  and, because of the high urinary concentrations in  homozygotes, may precipitate and form calculi in the  renal tract. In heterozygotes, increased excretion can  be demonstrated, but concentrations are rarely high  enough to cause precipitation.   The diagnosis of cystinuria is made by demonstrating  excessive urinary excretion of the characteristic amino  acids. All these amino acids must be identified to  distinguish this from cystinuria occurring as part of a  generalized amino aciduria.   The management of cystinuria aims to prevent calculi  formation by reducing urinary concentration. The  patient should drink plenty of fluid. Alkalinizing the  urine increases the solubility of cystine. If these measures  prove inadequate, D-penicillamine may be given; this  forms a chelate, which is more soluble than cystine alone  (see Chapter 3 for a discussion of renal calculi).    Figure 27.6 Chromatographic pattern of amino aciduria. Reproduced with kind permission from Nyhan WL and  Barshop BA. Atlas of Inherited Metabolic Diseases, 3rd edition. Hodder Arnold, 2012.   This is a very rare but serious disorder of cystine  metabolism, characterized by intracellular accumulation  and storage of cystine in many tissues. It must be  distinguished from cystinuria, a relatively harmless  condition. Renal tubular damage by cystine causes  Fanconi’s syndrome. Amino aciduria is non-specific and  of renal origin. Affected individuals may die young.   Hartnup’s disease is a rare autosomal recessive disorder  in which there are renal and intestinal transport defects  involving neutral amino acids. Many of the clinical  manifestations can be ascribed to reduced intestinal  absorption and increased urinary loss of tryptophan.  This amino acid is normally partly converted to  nicotinamide, the conversion being especially important  if the dietary intake of nicotinamide is low.   The clinical features of Hartnup’s disease are  intermittent and resemble those of pellagra, namely  a red, scaly rash on exposed areas of skin, reversible  cerebellar ataxia and mental confusion of variable  degree. Despite the generalized defect of amino   acid absorption, there is no evidence of protein  undernutrition; this may be because intact peptides can  be absorbed by a different pathway.   Excessive amounts of indole compounds, originating  from bacterial action on unabsorbed tryptophan, are  absorbed from the gut and excreted in the urine. The  diagnosis of homozygotes is made by demonstrating  the characteristic amino acid pattern in the urine.   Increased urinary excretion of the imino acids proline  and hydroxyproline, and of glycine, despite normal  plasma concentrations, is due to a transport defect for  these three compounds. The condition is inherited as  an autosomal recessive trait. It is apparently harmless,  but must be differentiated from other more serious  causes of iminoglycinuria, such as the defect of proline  metabolism, hyperprolinaemia.   The MPSs are rare conditions caused by defects  of any of the several enzymes that hydrolyse   mucopolysaccharides (glycosaminoglycans), which  therefore accumulate in tissues such as the liver,  spleen, eyes, CNS, cartilage and bone.   Hurler’s syndrome (MPS IH) is the least rare,  and is inherited as an autosomal recessive disorder.  Patients present in infancy or early childhood with the  characteristic coarse features, short stature, intellectual  disabilities and clouding of the cornea.   They usually die young of cardiorespiratory disease.  Scheie’s syndrome (MPS IS) is difficult to distinguish  clinically from Hurler’s syndrome at the time of  diagnosis, but has a much better prognosis; there is  little intellectual disability.   Hunter’s syndrome (MPS II), in contrast to all the  other MPSs, is inherited as a sex-linked recessive trait.   There are other mucopolysaccharide conditions,  including Sanfilippo’s syndrome, which manifests severe  CNS abnormalities, and Morquio’s syndrome, which is  associated with short stature, barrel chest, genu valgum  and other skeletal abnormalities. The MPSs can initially  be diagnosed biochemically by demonstrating increased  urinary excretion of sulphated glycosaminoglycans,  such as dermatan, heparan and keratan sulphates,  the excretion pattern being characteristic of each  syndrome. The diagnosis should be confirmed by direct  enzyme assay and gene tests with family studies. There  is current research looking at enzyme therapy.   Here a deficiency of a lysosomal hydrolase is inherited,  resulting in the accumulation of sphingolipid. The  following are some examples:   • GM2 gangliosidosis such as Tay-Sachs disease, due  to hexosaminidase deficiency,   Clinical features of these conditions may include  organomegaly, skeletal abnormalities, pulmonary  infiltration and cherry-red macular spot on  ophthalmologic examination.   4 Carbohydrate disorders  A variety of inborn disorders involve carbohydrate  metabolism. Some are discussed in Chapter 26 in the  context of neonatal hypoglycaemia.   Galactosaemia is an autosomal recessive disorder due to  galactose- 1 -phosphate uridyl transferase (Gal- 1 -PUT)  deficiency (Fig. 27.7). Galactose is necessary for the  formation of cerebrosides, of some glycoproteins and,  during lactation, of milk. Excess is rapidly converted  into glucose. The symptoms of galactosaemia become  apparent only if the infant is taking milk; the plasma  galactose concentrations then rise. The main features  include:   • renal tubular damage due to the deposition of  galactose- 1 -phosphate in the tubular cells (Fanconi’s  syndrome).   Galactose is a reducing substance. The urine may  give a positive reaction with Clinitest tablets; this  feature maybe absent if the subject is not receiving milk  and therefore galactose. Tubular damage may cause a  generalized amino aciduria.   The diagnosis is made by identifying galactose by  thin-layer chromatography and by demonstrating  a deficiency of Gal- 1 -PUT activity in erythrocytes.  Urinary reducing substances are usually positive  provided the infant is on a lactose-containing milk diet.   Treatment involves eliminating galactose in milk  and milk products from the diet. Sufficient galactose  for the body’s requirements can be synthesized  endogenously as uridyl disphosphate galactose. This  will reverse the acute symptoms but not some of the  chronic long-term neurological complications.   This is known as von Gierke’s disease and is a deficiency of  glucose-6-phosphatase (see Fig. 27.8 and Chapters 12 and  26). Patients may display a lactic acidosis, hypoglycaemia,  hyperuricaemia and hypertriglyceridaemia.   Pompe’s disease or maltase deficiency (a- 1,4-  glucosidase) is a lyosomal defect. It is associated with  skeletal myopathy, including muscular hypotonia and  cardiomyopathy.   This is a defect of debranching enzyme and is known  as Forbes-Cori disease. Abnormal glycogen with short   Figure 27.8 Summary of glycogen metabolism in relation to glycogen storage disease types I and III. Glycogen  is converted to limit dextran with four-unit stubs, after which a transferase removes a trisaccharide and attaches  it to a free end, leaving a dextran with single 1,6-linked glucosyl units. If amylo-l,6-glucosidase is lacking, as  in glycogen storage disease type III, the process stops at that point and hypoglycaemia results. In type I, where  the glucose-6-phosphatase is lacking, formation of glucose for the maintenance of euglycaemia is blocked, and  the enhanced alternative pathways tend to produce lactic acidosis and hyperuricaemia. PRPP, 5-phosphoribosyl  pyrophosphate; o, glucose units in the dextrans. Reproduced with kind permission from Candlish JK and Crook  M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993.   A 4-year-old boy was seen in the paediatric out-  patient department because of hepatomegaly,  metabolic acidosis and growth retardation. Some of  his abnormal fasting blood results were as follows:   The child has hyperlactataemia, hypoglycaemia,  hyperuricaemia and hyperlipidaemia. He was  later found to have von Gierke’s disease (or type  I glycogen storage disease) due to glucose-6-  phosphatase deficiency. This enzyme deficiency leads  to abnormalities of glycolysis and gluconeogenesis,  resulting in the hypoglycaemia and lactic acidosis.  The raised plasma lactic acid concentration may  interfere with uric acid renal excretion, leading to  hyperuricaemia.   external branches accumulates in skeletal muscle,  heart and liver. This can result in growth retardation,  muscular weakness and cardiomyopathy.   This is a defect of glycogen branching enzyme and is also  called Andersen’s disease. There is hepatosplenomegaly  and also cardiac and skeletal muscle defects.   McArdle’s disease is a deficiency of muscle  phosphorylase. Muscle cramps and fatigue occur on  heavy exertion. The urine may be burgundy-red in  colour due to myoglobin from muscle breakdown.   Hers’ disease is due to hepatic phosphorylase deficiency.  Symptoms may be mild, although growth retardation  may occur.   Tarui’s disease is due to phosphofructokinase deficiency.  The symptoms are similar to those of type V.   5 Lipid disorders and organic acidurias  Some inborn errors involving lipid disorders are  discussed in Chapter 13.   Those organic acids derived from the metabolism  of amino acids, carbohydrates and lipids are often  detectable in the urine; others accumulate if there is  an enzyme deficiency in a specific metabolic pathway.  Examples include methylmalonic acidaemia, glutaric  acidaemia, isovaleric acidaemia and proprionic  acidaemia. These disorders, known as organic acidurias,  are individually rare, but collectively have an incidence  of about 1 in 12 000 births, similar to that of PKU. They  may present in the neonatal period with life-threatening  metabolic acidosis, vomiting and hypotonia, or in early  infancy with failure to thrive, a Reye-like syndrome and  convulsions associated with profound hypoglycaemia.  They may also be a cause of sudden infant death.   The diagnosis is suggested by the clinical findings  and supported by initial tests demonstrating a  metabolic acidosis and sometimes hyperammonaemia,  with or without ketosis. It is confirmed by measuring  urinary organic acid excretion and subsequent enzyme  analysis, which should be performed only in specialized  laboratories.   A 5-month-old girl had been referred to the teaching  hospital paediatric department because of failure  to thrive, lethargy and convulsions. Some of her  biochemistry results were as follows:   Plasma amino acid analysis showed increased glycine  concentration, and the urine showed increased  concentrations of ketones and methylmalonic acid.   The findings support the diagnosis of methylmalonic  acidaemia, one of the organic acidurias. Note the  severe hyperammonaemia and raised methylmalonic  acid concentration, which confirm the diagnosis.  The child had a metabolic acidosis. Remember to  consider inborn errors of metabolism in children  who fail to thrive.   Medium-chain acyl coenzyme A dehydrogenase  deficiency is autosomal recessive and is one of the most  common fatty acid oxidation defects (about 1 in 10000  live births). This potentially fatal condition may present  with hypoketotic hypoglycaemia, encephalopathy,  seizures and hepatomegaly following diarrhoea and  vomiting (reduced food intake). Urinary dicarboxylic  aciduria with glycine conjugates may occur, along with  increased plasma octanoylcarnitine (an acylcarnitine).   6 Mitochondrial disorders  Mitochondrial DNA (mtDNA) is derived from the  mother. This differs from nuclear DNA in that there are  no introns and replication of mtDNA lacks proofreading,  and the mutation rate is thus about 10-100 times  greater than that of nuclear DNA. Mitochondria lack  an adequate DNA repair mechanism. A number of  clinical features may be present, including neuropathy,  intellectual disabilities, lactic acidosis, myopathy, ocular  defects, diabetes mellitus, anaemia and hearing loss  (Fig. 27.9).   There are a number of mitochondrial disorders,  including Leigh’s syndrome, MELAS syndrome  (mitochondrial encephalomyopathy, lactic acidosis,  stroke), Kearns-Sayre syndrome, NARP syndrome  (neuropathy, ataxia, retinitis pigmentosa) and LHON  syndrome (Leber’s hereditary optic neuropathy).    Figure 27.9 The circular deoxyribonucleic acid (DNA)  of the human mitochondrial genome. Reproduced  with kind permission from Nyhan WL and Barshop BA.  Atlas of Inherited Metabolic Diseases, 3rd edition.  Hodder Arnold, 2012.   The arterial or venous lactate to pyruvate ratio may  be high (more than 50:1), which suggests a metabolic  block in the respiratory chain system. There is often  a high plasma lactate at rest. Plasma creatine kinase  activity may be raised and rhabdomyolysis can occur  with myoglobinuria. Specialized muscle histology may  be useful and also genetic tests and family studies.   In this group of disorders there is either a deficiency  of a peroxisomal enzyme or a defect in forming intact  peroxisomes. There are probably about 20 of these  disorders affecting about 1 in 30 000 individuals.  Peroxisomes are involved in a number of metabolic  processes. The following are some of the defects that  have been described: defects of phytanic acid oxidation  (Refsum’s disease), dihydroxyacetone phosphate  acyltransferase abnormality (Zellweger’s syndrome),  catalase defects (neonatal adrenoleucodystrophy),  and abnormal plasmalogen biosynthesis (rhizomelic  chondrodysplasia punctata).   These conditions may present with a variety of  features, including dysmorphia, cataracts, liver disease,  retinitis pigmentosa, adrenal insufficiency, peripheral  neuropathy, deafness and ataxia.   8 Drugs and inherited metabolic disorders  The variation in individual response to drugs may be  partly due to genetic variation. There are a number of  well-defined inherited disorders that are aggravated by,  or which become apparent only after, the administration  of certain drugs. These disorders may be classified into  two groups.   The muscle relaxant suxamethonium (succinyl choline,  or scoline) normally has a very brief action because  it is rapidly broken down by plasma cholinesterase.  In suxamethonium sensitivity (see Chapter 18), a  cholinesterase variant of low biological activity impairs  the breakdown of the drug, and prolonged post-  operative respiratory paralysis may result (‘scoline  apnoea’).   Two other inherited disorders are characterized  by defective metabolism of the drugs isoniazid and  phenytoin. In both, toxic effects occur more frequently,  and at lower dosages, than in normal individuals. The  genetic differences in the metabolism of azathioprine  are discussed in Chapter 25.   Deficiency of glucose-6-phosphate dehydrogenase  (G6PD) may cause haemolytic anaemia, and is relatively  common in ethnic groups of Mediterranean origin. It  is X-linked. This enzyme catalyses the first step in the  hexose monophosphate pathway and is needed for  the formation of nicotinamide adenine dinucleotide  phosphate, which is important for the maintenance  of intact red cell membranes. Numerous variants of  G6PD deficiency have been described. Haemolysis may  be precipitated by certain antimalarial drugs, such as  primaquine, and by sulphonamides. In the inherited  hepatic porphyrias (see Chapter 21), acute attacks may  be precipitated by various drugs, such as barbiturates.  So many drugs have been implicated that it is probably   Some people react to general anaesthetics (most  commonly halothane with suxamethonium) with  a rapidly rising temperature, muscular rigidity and  acidosis (malignant hyperpyrexia), which is associated  with high mortality. Many, but not all, susceptible  subjects in affected families have a high plasma creatine  kinase activity.   9 Miscellaneous disorders  There are many other IEMs in addition to those  mentioned above, including congenital adrenal  hyperplasia (see Chapter 8), adenosine deaminase  deficiency, electron transport chain defects and sulphite  oxidase deficiency.   • The incidence of IEMs ranges from about 1 in 100  to 1 in 200000, depending on the disorder and the  population involved.   • The presence of an inborn error should be  considered in children with family histories of IEM,  failure to thrive, convulsions and other metabolic  abnormalities.   • Neonatal screening programmes based on the  analysis of neonate blood spots are used to  screen for certain metabolic disorders, including  hypothyroidism and PKU.   Neonatal screening laboratories use highly skilled  biochemical and molecular biology techniques.  Some inborn errors can be treated by:   - limiting the dietary intake of precursors  in the affected metabolic pathway, such  as phenylalanine in PKU or lactose in  galactosaemia,   - supplying the missing metabolic product, such  as cortisol in congenital adrenal hyperplasia,   - removing or reducing the accumulated product,  such as ammonia in urea cycle disorders.   • Chromosomal disorders due to the absence, or  abnormal arrangement, of chromosomes affecting  many genes and therefore many gene products.  Examples include Down’s syndrome (trisomy for  chromosome 21), Turner’s syndrome (45, XO) and  Klinefelter’s syndrome (47,XXY).   • Monogenic disorders due to an abnormality of a  single gene, which is the primary determinant of  the disorder and which is inherited in a predictable  pattern, such as phenylketonuria.   • Multifactorial or polygenic disorders due to the  interaction of multiple genes with environmental or  other exogenous factors, such as diabetes mellitus.   Genes, located on chromosomes, comprise a sequence  of bases on deoxyribonucleic acid (DNA) and code for  the synthesis of proteins by ribonucleic acid (RNA)  in its ‘messenger’ form (mRNA). All nucleated cells  contain an identical complement of genes, but only  about 1 per cent is expressed.   A genotype is the actual genes present in an  individual; phenotype is the physical expression of  that genotype, usually via production of a polypeptide  or protein. Genes differ in length, sometimes  containing many thousands of bases. However, only  10 per cent of the genome incorporates the protein-  coding sequences (exons) of genes. Interspersed  within many genes are intron sequences, which have  no coding function.   The rest of the genome consists of other non-coding  regions (such as control sequences and intergenic  regions). Humans can synthesize about 100 000 varying  polypeptides or proteins, which are usually composed  of about 20 different kinds of amino acids. Genes  contain specific sequences of three DNA bases (codons)  instructing the cell’s protein-synthesizing apparatus to  add specific amino acids.   In disorders of single genes (monogenic), the  abnormalities may be of:   • a structural gene, with production of an abnormal  protein: in this case all the biochemical abnormalities  can be explained by defective synthesis of a single  peptide;   • a controlling (enhancing) gene, which, by altering the  rate at which one or more structural genes function,  affects the amounts of one or more structurally  normal peptides.   The affected protein may be an enzyme, but in  other conditions it may, for example, be a receptor, a  transport or a structural protein, a peptide hormone,  an immunoglobulin or a coagulation factor. With the  Human Genome Project, more and more genes and  their functions are being discovered.   Every inherited characteristic is governed by a pair of  genes on homologous chromosomes, one gene being  received from each parent. Different genes governing  the same characteristic are called alleles. An individual  with two identical alleles is homozygous for that gene  or inherited characteristic; an individual with two  different alleles is heterozygous. Genes may be carried  on the autosomes (similar in both sexes) or on the sex  chromosomes (X and Y); the patterns of inheritance  differ.   If one parent (Parent 1 in the example in Fig. 28.1a) is  heterozygous for an abnormal gene (A) and the other  parent is homozygous for the normal gene (N), the  possible combinations in the offspring are shown in the  square in this figure.   On a statistical basis, half the offspring will be  heterozygous (AN) for gene A, like Parent 1. None will  be homozygous for the abnormal gene (AA).   If both parents are heterozygous (AN; Fig. 28.1b), a  quarter of the offspring will be homozygous (AA) and  half will be heterozygous (AN).   If one parent is homozygous (AA) and the other  normal (NN), all the offspring will be heterozygous.   The metabolic consequences of an abnormal gene  depend on the effectiveness of that gene compared with  the normal one.   Dominant abnormal genes affect both heterozygotes  (AN) and homozygotes (AA), although homozygotes  may be more severely affected. In the first example,  Parent 1 (AN) and half the offspring will be affected,  and in the second example both parents and three out  of four of the offspring (AA and AN) will be affected.  Characteristically, if there is autosomal dominant  inheritance:   • clinically normal offspring are not carriers of the  abnormal gene,   • statistically, three in four children are affected if  both parents are heterozygous - if one parent is  heterozygous, there is a one in two chance of an  offspring being the same.   Autosomal recessive inheritance  Recessive abnormal genes only affect homozygous  offspring (AA). In the first example, neither parent nor  the offspring will be affected, and in the second example  both parents will appear normal, but statistically one  in four of the offspring will be affected. Therefore, in  autosomal recessive inheritance:   • clinically normal children may be heterozygous and  therefore may be carriers of the abnormal gene,   • on average, one in four children of heterozygous  parents is clinically affected.   Disorders inherited as a recessive trait have a lower  expression frequency in affected families than dominant  disorders, but tend to be more severe. An example is  cystic fibrosis (Chapter 16).   Some abnormal genes are carried only on the sex  (almost always the X) chromosomes.   Women have two X chromosomes and men have  one X and one Y chromosome. In X-linked recessive   inheritance, an abnormal X chromosome (Xa) is latent  when combined with a normal X chromosome, but  active when combined with a Y chromosome. If the  mother carries Xa, she will appear to be normal, but  statistically half her sons will be affected (XaY). Halfher  daughters will be carriers (XaX), but all her daughters  will be clinically unaffected (Fig. 28.1c).   If the father is affected and the mother carries two  normal genes, none of the sons will be affected, but all  the daughters will be carriers (Fig. 28. Id).   Inherited disease manifesting in male offspring and  carried by females is typical of X-linked inheritance.  Females are only clinically affected in the extremely  rare circumstance when they are homozygous for  the abnormal gene. This will occur only if the female  inherited the abnormal genes from an affected father  and a carrier mother. Haemophilia is the classic example  of an X-linked recessive disorder.   In this type of very rare inheritance, both XaX women  and XaY men are affected. An example of this very rare  type of disorder is familial hypophosphataemia.   Occasionally there may be several alleles governing  the same characteristic. In such cases, different pair  combinations may produce different disease patterns,  for example some of the haemoglobinopathies, or the  variant may be detectable only by biochemical testing,  such as, for example, some of the plasma protein  variants.   In rare cases, spontaneous new mutations may occur  and may produce dominant disorders in unaffected  families. The terms ‘dominant’ and ‘recessive’ are  relative. A dominant gene may fail to manifest itself  (incomplete penetrance) and may therefore appear  to skip a generation. A gene may vary in its degree of  expression, and therefore in the degree of abnormality  that it produces. A recessive gene, which produces  disease only in homozygotes (AA), may be detectable by  laboratory tests in clinically unaffected heterozygotes.   In recent years there has been a huge growth in DNA  technology and research, such that clinical diagnosis  can now be made in certain conditions using small  tissue samples. This chapter gives a brief outline of  such technology; for a more detailed exposition, the  reader is advised to refer to a specific molecular biology   textbook. However, in some disorders, especially those  with multiple polymorphisms or mutations or with  incomplete penetrance, a biochemical or phenotypic  diagnosis is still preferable. Epigenetics is the  inheritance of a characteristic not by a change in DNA  sequence, such as may occur by histone acetylation  or DNA methylation, which modify gene expression.  See Wenham PR. DNA-based techniques in clinical  biochemistry: a beginner’s guide to theory and practice.  Ann Clin Biochem 1992; 29: 598-624.   Basics principles of molecular biology  Chromosomes consist of both protein and DNA;  chromosomal DNA contains an average of 100-200  million bases. DNA is a nucleoside polymer arranged  in two strands as a double helix. There are four bases in  DNA, namely adenine (A), cytosine (C), guanine (G)  and thymine (T). These nucleotides consist of ester-  linked phosphate residues at the 5' -hydroxyl residue of  a deoxyribose molecule that itself is linked to a purine  or pyrimidine base by its C- hydroxyl group. Each of  the DNA strands has polarity, namely 5' and 3' ends  with the two opposite-running strands in opposite  directions (anti-parallel).   The two DNA strands are complementary, so that  C on one strand bonds by hydrogen bonds with G on  another strand and A pairs with T. These bases are always  numbered from the 5' to the 3', with letters indicating  the nucleotide bases. Genetic material can copy itself  (replication) during the S phase of the cell cycle. DNA  polymerase needs a pre-existing DNA template and  moves along the two strands and adds the appropriate  nucleotides, following the base pairing rules, to the  growing chain. Two new double strands of DNA are  formed, identical to the original double strand and  the strands are anti-parallel. DNA polymerase always  moves from 3' to 5' along the template strand, i.e. the  reverse of the newly forming strand. Variable number  tandem repeats (VNTR) are short and repetitive  repeats distributed throughout the genome and that are  grouped near telomeres.   Transcription is the process whereby the polypeptide  or protein-coding instructions from the genes are  transcribed indirectly through mRNA; the latter moves  from the nucleus to the cellular cytoplasm, serving as  the template for protein synthesis. Translation is the  process whereby the ribosomal system then translates  the codons into an amino acid chain that will constitute  the polypeptide or protein molecule for which it codes.  A gene is a unique sequence of nucleotides that codes for   a particular protein or peptide and can be represented  by one or more alleles. The genetic code is thus a series  of codons that instruct which amino acids are needed  to synthesize specific polypeptides or proteins.   Deoxyribonucleic acid technologies  Deoxyribonucleic acid can easily be prepared from  very small amounts of biological samples, often  blood. This essentially involves three steps: leucocyte  lysis, chloroform/phenol extraction to remove  contamination by proteins, followed by proteinase and  RNAase treatment to remove remaining protein and  RNA contamination.   Samples of DNA after digestion are separated by  electrophoresis due to its strong negative charge at  neutral pH and, by running the DNA through agarose  gels, it can be separated into discrete DNA molecules  of 100-10000 base pairs. Smaller DNA pieces can be  separated by polyacrylamide gels, which are a tighter  molecular sieve than agarose. DNA probes can be a  piece of DNA of variable length that can be inserted  into the DNA of a plasmid. These circular pieces of  double-stranded DNA can be artificially introduced  into bacteria and incorporated into their DNA by a  process called transformation. By adding divalent  cations the bacteria are made permeable to DNA and  a selectable marker such as antibiotic resistance is then  inserted, and only those bacteria containing this will  grow in media containing the antibiotic. The probes can  be labelled to locate them in DNA experiments, using  either radiolabelled phosphorus-32 or, for example,  non-radiolabelled horseradish peroxidase-enhanced  chemiluminescence or digoxigenin.   If DNA in solution is heated, the hydrogen bonds  between the complementary strands become  disorganized and dissociate (denaturing or melting).  Renaturation or annealing occurs on lowering the  temperature when the two strands are able to rejoin.  This facilitates a complementary sequence of bases (the  target) to be annealed to a short piece of DNA (the  probe). By adjusting the reaction conditions (increasing  stringency), such as temperature and ionic strength, it  is possible to ensure that the probe hybridizes only to a  perfectly matching genomic DNA sequence.   The probes can be either complementary to lengths of  the DNA within the gene under study (genomic probe) or  complementary to exon regions of the gene that give rise  to mRNA (cDNA probe). Allele-specific oligonucleotide   probes are short, single-stranded DNA probes that differ  in composition by one single nucleotide and are thus  able to detect single DNA point mutations.   Deoxyribonucleic acid polymorphisms  DNA can be cut into smaller fragments by restriction  endonucleases derived from bacteria. These are named  by abbreviating the names of the originating bacteria.  These endonucleases often work in a palindromic way,  that is, the sequence of bases on one DNA strand is  repeated in reverse on the other. A restriction enzyme/  restriction endonuclease is an enzyme that recognizes a  specific nucleotide sequence (restriction site) and cuts  (restricts) the nucleic acid at that particular site. An  example of a restriction endonuclease is EcoRI made  by Escherichia coli, which is specific for the following  sequence:   The base pairs on the DNA strands throughout the  genome differ at particular locations in individuals.  This is called polymorphism, and, when the differences  occur in introns (non-coding sequences of DNA),  no overall change occurs in the final coded protein  product. If the polymorphic location occurs at a  particular restriction endonuclease site, either the site  is not recognized by the enzyme or an alternative site  may be created elsewhere in the DNA strand. Different  sized DNA fragments are produced - restriction  fragment length polymorphisms (RFLPs). The RFLPs  can, thus, be used as markers of certain loci that may  be relevant in disease processes (Fig. 28.2).   Specific DNA fragments can be separated by Southern  blotting, which is based on the reassociation of DNA  strands after denaturation and then detecting the  fragments by a labelled probe. Alkaline conditions  denature and separate the DNA fragments, and the  DNA is transferred to a membrane by capillary action  (blotting). It is then fixed to the membrane usually  nitrocellulose or nylon by baking or ultraviolet  irradiation. A probe is prepared which is either a  DNA or an RNA molecule that is complementary  to the particular sequence under study. The probe is  labelled, with radiolabelled phosphorus-32, fluorescent  or immunological techniques, and then incubated  with the blotted membrane under conditions that  favour reassociation of the DNA strands. Under    W/////////A i i i i v/M/////M/////M/nm. t///////m i i i i i i i r//////////   Figure 28.2 Summary of deoxyribonucleic acid (DNA) fingerprinting techniques. RE, restriction endonuclease; RF,  restriction fragment; VNTR, variable number of tandem repeats. Reproduced with kind permission from Candlish  JK and Crook M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993.   optimal stringency, the probe binds only to areas of  complementary DNA; thus the probe can then be  located for homologous DNA sequences.   Ribonucleic acid molecules can similarly be separated  by electrophoresis and can be hybridized with an RNA  or DNA probe. The mRNA from a particular gene  can be identified and characterized. This is based on  the principle that the mRNA will be abnormal either  in amount or size if there is a corresponding gene  mutation. Northern blots can also be used to study the  tissue-specific expression or development of particular  genes, as well as identifying and characterizing mRNAs  derived from genes of interest.   These are sometimes called DNA chips and are used to  detect changes in gene expression in different cells. It is  possible to bind very small amounts of defined DNA  molecules on to a membrane, which is then mixed  with a sample to see if RNA molecules are present -  somewhat like a reverse Northern blot.   A DNA segment is amplified by two primers using  repeated cycles of denaturation, primer annealing  and extension by DNA polymerase. The reaction  mixture consists of the original template DNA, the  four nucleotides, and a large excess of oligonucleotide  primers. The temperature is initially programmed to  95°C to separate the DNA strands. It is then reduced  to 50°C so that the complementary primers bind and  anneal, and then raised to 72°C for about 1 min, which is  the optimal temperature for the DNA polymerase. The  polymerase can usually incorporate 50-100 nucleotides  per second, following which the temperature is again  raised to 95°C to complete the cycle.   There is thus a cycle of denaturation, primer  annealing and primer-directed extension.   Taq polymerase is a heat-stable DNA polymerase  derived from the organism Thermus aquaticus.  Polymerase chain reaction (PCR) can amplify  DNA up to a million-fold. It can be used in the  diagnosis of known mutations of genomic DNA  using an amplification refractory mutation system  (ARMS). DNA cannot undergo PCR amplification  if an oligonucleotide primer contains a 3' nucleotide  mismatch. In site-directed mutagenesis, a mutation  or polymorphism destroys or creates a restriction  endonuclease cutting site, which can be identified by  PCR followed by endonuclease digestion (Fig. 28.3).  The ARMS allows diagnosis of single nucleotide  mutations, where specific priming of PCR allows  amplification to take place only if the mutation is  present. Gene sequencing can also be used in some  cases to diagnose certain mutations.   Variable number tandem repeats have been discussed  above, and embrace microsatellite repeat and  trinucleotide repeat conditions that occur in certain  diseases, e.g. Huntington’s disease and Friedreich’s  ataxia. The degree of repeats may differ from generation  to generation.   Cystic fibrosis is often due to a 3 base-pair deletion in  codon 508 of the cystic fibrosis chloride channel gene,  although other mutations may also occur. The genetic  defect can be detected using PCR and this can be used   Figure 28.3 Principles of the polymerase chain  reaction. Basically, polymerization can be conducted  in one vessel because the deoxyribonucleic acid (DNA)  polymerase can survive cycles of heating and cooling.  Initially the genomic DNA double helix is separated into  single strands (shown here as serrated lines) by heating,  then cooled and allowed to anneal to DNA primers (the  small serrated segments) complementary to the portion  of the gene to be amplified. Then the enzyme and the  added nucleotides form double-stranded DNA in the  primer extension step. In the second cycle there is  again denaturation by heating, annealing to primers in  the cold, then primer extension. The first segments of  DNA of the desired length are obtained, but annealed  to strands of indeterminate length. It is only in the  third cycle that the final products are obtained, but  are thereafter multiplied exponentially. Note also that  the base sequence of the gene, or part of it, has to be  known for the design of the primers. Reproduced with  kind permission from Candlish JK and Crook M. Notes  on Clinical Biochemistry. Singapore: World Scientific  Publishing, 1993.   to screen for carriers or affected pregnancies using  chorionic villous sampling (see Chapter 16).   Both Duchenne’s and Becker’s muscular dystrophies are  due to deletions in the dystrophin gene on chromosome  21. The former is due to an absence of dystrophin,   Figure 28.4 A hypothetical case of the use of  deoxyribonucleic acid (DNA) probes to diagnose  genetic conditions. Conventionally, squares represent  males and circles females. The deceased are  marked with crosses and the known male case is  represented as a solid square. After electrophoresis  of the fragments derived from the genomic DNA of  the subjects, the 2.8-kb band in the affected male is  found to be present in the mother, although in only  one of the sisters, who is almost certainly a carrier  if the mother is a carrier. Reproduced with kind  permission from Candlish JK and Crook M. Notes on  Clinical Biochemistry. Singapore: World Scientific  Publishing, 1993.    whereas the latter is due to partially functioning  dystrophin. Polymerase chain reaction techniques are  being used to detect carriers and those affected (see Figs  28.3 and 28.4).   oq-Antitrypsin deficiency can result in severe  emphysema or cirrhosis. The gene is polymorphic,  with more than 50 genetic variants. At one time the  abnormalities could be detected by protein isoelectric  focusing, but this has now been superseded by the  use of PCR to detect clinically important alleles (see  Chapter 19).   Phenylketonuria can be detected by measuring  phenylalanine on neonatal blood spots. However, new  PCR technology may allow affected pregnancies to be  tested by chorionic sampling (see Chapter 27).   Familial hypercholesterolaemia is an autosomal  dominant defect of the low-density lipoprotein (LDL)  receptor of chromosome 19. A variant is familial  defective apolipoprotein B-100 (apoB-100), caused  by a mutation of the apoB gene at the 3500 position.  These conditions may be screened for by PCR  technology. Apolipoprotein E shows polymorphism,  with the apoE2 homozygote being associated with  type III dyslipoproteinaemia and the apoE4 allele  associated with Alzheimer’s disease. Previously, apoE  phenotyping was performed using isoelectric focusing,  but this can give discordant results due to post-  translational modifications of the apoE lipoprotein  during its sojourn in the circulation. This method has  now been superseded by PCR methods to characterize  the apoE genotype (see Chapter 13).   The diagnosis of haemochromatosis is now more  commonly being made with DNA techniques. Genetic   tests are now available, for example a majority of cases  of haemochromatosis are due to the C282Y or H63D  HFE mutation (see Chapter 21).   Sickle cell anaemia is due to a single A to T substitution  in the sixth codon of the |3-globin gene. This can be  detected during amniocentesis, for example by allele-  specific hybridization.   Thalassaemia is characterized by the absence or size  alteration of the globin mRNA. This can be detected by  using Northern blotting techniques.   Polymerase chain reaction can be used to verify the  presence of specific pathogens. DNA primers are chosen  that are specific to DNA sequences of the pathogen.  Such pathogens include human immunodeficiency  virus (HIV), hepatitis C and Helicobacter pylori.   Deoxyribonucleic acid technology is now used to  determine the genetic father in paternity disputes and  also in forensic science to identify body tissues and  fluids.   • Deoxyribonucleic acid (DNA) technology will  undoubtedly increase over the next few years  and its use can be expected to increase in clinical  biochemistry testing.   The examples given in this chapter are only a  selection of the DNA tests that may become  available in the future. However, diseases such as  cystic fibrosis and muscular dystrophy are already  being diagnosed by DNA technology.   The polymerase chain reaction is an important  diagnostic molecular biology technique.   Most clinical laboratories take stringent precautions to  control the analytical accuracy and precision of results.  Indeed, many laboratories undergo carefully regulated  accreditation and inspection procedures to ensure that  they are fit for practice. Generally, laboratory users  need only limited knowledge of the technical details of  the laboratory tests. However, they should understand  that the appropriate collection of patient specimens can  affect results (Table 29.1), and they should therefore  work with the laboratory in its attempt to produce  answers rapidly and accurately, with appropriate  interpretation when required and identifiable with the  relevant patient. To this end, one should understand  the importance of:   • the collection of specimens by the correct technique  at the appropriate time,   Remember that treatment based on technically  correct results from a wrongly labelled or collected  specimen may be as dangerous as a faulty surgical  procedure. ‘Unlikely’ results are checked in most  laboratories to make sure that they have not been  transposed with the results for another patient.   All patient samples are potentially infection  risks. Informed consent may be needed for acquired  immunodeficiency syndrome (AIDS) testing and also  certain genetic tests. All blood specimens should be  sent in leak-proof, sealed plastic bags, with the request  form in a different pocket in the bag. Failure to comply  with these guidelines may put many people, including  porters and laboratory staff, at unnecessary risk.    Keeping blood overnight before sending it to the laboratory or refrigerating blood sample    Taking blood from an arm with an infusion running into it    Putting blood into wrong vial or tipping it from one vial into another    Palpation of prostate by rectal examination, passage of catheter, enema in last few days   ACTH, adrenocorticotrophic hormone; AST, aspartate transaminase; ALP, alkaline phosphatase; EDTA, ethylenediamine tetra-acetic acid;  LDH, lactate dehydrogenase; PTH, parathyroid hormone; PSA, prostate-specific antigen.   Accurate and legibly written information about the  patient is essential, although electronic requesting  systems are now widespread in some areas. This  information includes the patient’s:   These would usually be considered the minimum  acceptable dataset for patient identification details.   Any of these may be recorded inaccurately on the  form and, unless there is complete agreement with  previous details, results may be entered into the wrong  patient’s record either on a computer or in the patient’s  case notes, causing confusion and possible danger to the  patient. The National Health Service (NHS) number is  being used as a unique individual identifier in the UK.   It is important also to include relevant clinical details  so as to facilitate correct interpretation of the results.   It should be obvious that, if the ward or department  is not stated, it may take time and effort to determine  where the results should be sent. The requesting doctor  must sign the form legibly, and also state how he or she  can be notified rapidly, for example by ‘bleep number’,  in case abnormal results requiring urgent action are  found or advice needs to be sought about treatment.  The doctor must check the completed request form  to be sure that the information given is correct; it is  also important to include his or her name and contact  details.   Request forms designed by pathology and other  departments ask only for information that is essential to  ensure the most efficient possible service to the clinician  and therefore to the patient. Now quite widespread  is the use of electronic test requesting, which should  improve patient identification and speed up the process  and may replace ‘paper’ requests.   If a clinically improbable result has been checked  analytically and the second result is in close agreement  with the first, a fresh specimen should be analysed.   Although contamination at some stage of blood  collection is an obvious possibility, this is relatively rare  and should not be accepted until other, more common,  causes have been excluded. Of course, it should also  be remembered that the sample could be incorrectly  labelled with wrong patient details.   • Some tests may require the patient to fast, for example  plasma glucose or triglyceride (see Chapters 12  and 13).   • Oral medication: some assays may be affected by oral  medication.   - Blood should be taken for drug assays at a  standard time after the dose; misleadingly high  plasma concentrations may occur at the time  of peak absorption (see Chapter 25).   A 22-year-old man had the following post-operative  biochemical results after an appendectomy:   The clinical biochemistry laboratory suggested an  immediate repeat blood sample, which gave the  following results:   The first sample seems to display profound  hypernatraemia, hyperglycaemia and hypokalaemia.  In addition, the plasma urea and creatinine  concentrations are both low. The repeat sample values  are completely different. It later transpired that the  first sample had been taken out of the patient’s drip  arm, which had a dextrose-saline infusion going into  it. This had resulted in dilution of the analytes and  elevated sodium and glucose concentrations.   - There may be significant hypokalaemia for  a few hours after taking potassium-losing  diuretics due to rapid clearance of potassium  from the extracellular fluid (ECF). The plasma  concentration returns to its ‘true’ level as  equilibration occurs between cells and ECF  (see Chapter 5).   - Interfering substances: previous administration  of a substance may affect a plasma analyte  concentration for some time; for example certain  antibiotics may interfere with chemical reactions  used in creatinine assays (see Chapter 3).   • Effect of posture: for example the concentrations of  plasma proteins and of substances bound to them  are lower when the patient is supine than when  standing up (see Chapter 19).   • Intravenous infusion: for example a spuriously  low plasma sodium concentration may result if  the sample is taken from a dextrose drip arm (see  Chapter 2).   • Clinical procedures: for example palpation of  the prostate by rectal examination may possibly  release large amounts of prostate-specific antigen  (PSA) into the circulation; the spuriously elevated  concentrations in blood may persist for several days  (see Chapter 24).   It is usual to apply a tourniquet proximal to the site of  venepuncture to make it easier to enter the vein with  the needle. If occlusion is maintained for more than a  short time, the combined effect of raised intracapillary  pressure and hypoxia of the vessel wall increases the  rate of passage of water and small molecules from the  lumen into the surrounding interstitial fluid. Large  molecules, such as proteins, cannot pass through  the capillary wall at the same rate; their plasma  concentrations therefore rise.   Many plasma constituents are partly bound to  protein. Prolonged venous stasis can raise the plasma  total calcium concentration, sometimes to equivocal  or slightly high levels. Therefore, ideally, blood samples  for plasma calcium estimation should be taken without  stasis, especially if high plasma concentrations have  previously been found (see Chapter 6).   Prolonged stasis may also cause local hypoxia;  consequent leakage of intracellular constituents, such  as potassium and phosphate, may cause falsely high   plasma concentrations. It is sometimes difficult to enter  ‘bad veins’ without applying stasis. A tourniquet may  be used and released as soon as the needle is in the vein;  a suitable specimen may be obtained after waiting at  least a further 15 s before withdrawing blood.   If the patient is receiving an intravenous infusion, the  administered fluid in the veins of the same limb, whether  proximal or distal to the infusion site, has not mixed  with the total plasma volume; local concentrations  will therefore be unrepresentative of those circulating  through the rest of the body. Blood taken from the  opposite arm will give valid results. Note that glucose  infusion may cause systemic hyperglycaemia and  glycosuria. Only if the hyperglycaemia persists after  infusion has stopped should the diagnosis of diabetes  mellitus be considered (see Chapter 12).   Most hospital laboratories issue a list of the types of  container suitable for different assays (Fig 29.1); this list  may vary from hospital to hospital. For example, most  laboratories specify that:   • Blood for glucose estimation should be put into  a tube containing an inhibitor of erythrocyte  glycolysis, such as fluoride.   • Potassium should ideally be estimated on plasma  from heparinized blood rather than serum, although  many laboratories accept serum tubes for potassium  on the pragmatic grounds that the differences are   Figure 29.1 Blood collection tubes: if in doubt about  what tubes to use be sure to contact the laboratory for  advice. Reproduced with kind permission of Greiner  Bio-One.   usually non-significant. Potassium is released from  cells, especially platelets, during clotting. Serum  potassium concentrations are usually higher than  those of plasma by a variable amount; this difference  can be clinically misleading. Marked differences may  be found in patients with leukaemia, in whom the  number of white blood cells is usually significantly  increased.   Laboratories should accept blood only in the correct  containers. Even with this precaution, serious errors can  arise if blood is decanted from one container to another,  although this is less likely to occur if a closed vacuum  system is used for obtaining blood. The anticoagulant  actions of oxalate and of sequestrene (ethylenediamine  tetra-acetic acid, EDTA) depend on the precipitation  or chelation of calcium, respectively, thus invalidating   A blood sample had been taken from a 44-year-old  woman on the medical ward and the results were as  follows:   Albumin-adjusted calcium <0.5 mmol/L (2. 1 5-2.55)  Phosphate 0.92 mmol/L (0.80-1.35)   Repeat blood sampling on the same day gave the  following results:   The first blood sample had been collected in a  potassium ethylenediamine tetra-acetic acid (EDTA)  tube (this is normally used for certain haematology  tests such as full blood count) by mistake and then the  blood had been decanted by the doctor into the correct  chemistry lithium heparin tube. Note in the first  sample the raised plasma potassium and low calcium  concentrations due to chelation by potassium EDTA.   the results of calcium estimation. Ethylenediamine  tetra-acetic acid is usually in the form of its potassium  salt; therefore, it renders the sample unsuitable for  potassium analysis.   The use of sodium (instead of lithium) heparin or  trisodium citrate may give a falsely high plasma sodium  result. This anticoagulant is often used in specimens  taken for ‘blood gases’; apparent plasma sodium  concentrations of 160-170mmol/L can result from  transferring an aliquot of sodium heparinized blood  into a lithium heparin vial. The use of lithium heparin  leads to a falsely high plasma lithium concentration and  it should therefore not be used as an anticoagulant for  lithium determination.   The concentration of many substances is very different  in erythrocytes from that in the surrounding plasma.  Haemolysis releases the cell contents into plasma and,  consequently, if this occurs in vitro, the concentrations of  some plasma constituents, such as potassium, phosphate  and aspartate transaminase, may be falsely increased.  The increase is variable and is not related to the intensity  of the red colour of plasma due to haemoglobin. It is  uncommon for haemolysis to occur in vivo because  these constituents are distributed throughout the total  extracellular, not just plasma, volume.   Haemoglobin may interfere with some chemical  reactions, falsely increasing the apparent plasma  bilirubin concentration and lowering alkaline  phosphatase activity. The chance of haemolysis is  minimized if the blood is treated gently and if a closed  vacuum system is used.   It is important to check the sample date. The differential  concentrations of some analytes across cell membranes  are maintained by energy, derived from glycolysis. In  vitro, erythrocytes soon use up the available glucose  and therefore the energy source; concentrations of  these analytes in plasma will then tend to equalize with  those in erythrocytes by passive diffusion across cell  membranes. If plasma is not separated from blood cells  within a few hours, the effect on plasma concentrations  will be similar to that resulting from haemolysis.  However, there are a few important differences:   A blood sample had been taken in the morning  from a 43 -year-old man and was then sent to the  laboratory from the local health centre. The sample  was analysed in the evening of the same day, as there  had been a transport delay, and the following results  were obtained:   The laboratory contacted the patient’s general  practitioner and an urgent repeat sample showed the  following results.   The first patient sample shows hyperkalaemia, but  repeat analysis on a ‘fresh’ sample shows a ‘normal’  potassium concentration. The spuriously raised  potassium result in the first sample was due to the delay  in sample assay and leakage of intracellular potassium  ions out of cells, resulting in pseudohyperkalaemia. It  is important to transport samples to the laboratory as  quickly as possible to avoid storage artefacts.   • The plasma potassium concentration rises as the  plasma glucose falls; initially, there may be a slight  fall in the plasma potassium concentration as it  moves into the erythrocytes.   Many plasma constituents, such as bilirubin,  deteriorate even if the plasma is correctly separated and  stored. If the plasma sample is haemolysed or separation  has been delayed, the plasma potassium concentration  may still be clinically significant. For example, if the  sample is visibly haemolysed and the plasma potassium  concentration is only 2.8mmol/L, this may indicate  profound hypokalaemia; the laboratory staff should  contact the requesting doctor directly to discuss the  significance of the comment ‘haemolysed specimen’ on  the report form.   The refrigeration of whole blood has the effect  of raising the plasma potassium concentration  probably by reducing the activity of the adenosine  triphosphatase pump. Hence, blood samples for urea  and electrolytes should not be refrigerated. Transport  of samples in cold weather may have a similar effect.  Freezing will result in haemolysis. Therefore blood  specimens must be centrifuged and the plasma  separated from the cells before storing, for example  overnight.   Urine estimations performed on timed collections are  expressed as units/time (for example mmol/24h); this  figure is calculated by multiplying the concentration  by the volume collected during the timed period. The  accuracy of the final result depends largely on that of  the urine collection. Sometimes, if the patient is a child  or incontinent, accurate urine collection is particularly  difficult.   For example, a 24-h specimen may be collected  between 09.00 h on Sunday and 09.00 h on Monday.  The volume excreted by the kidneys during this period  is the crucial one: urine already in the bladder at 09.00 h  on Sunday was excreted earlier and should not be  included. Therefore the procedure is as follows.   • 09.00h on Sunday: the bladder is emptied completely,  whether or not the patient feels the need, and the  specimen is discarded.   • Collect all urine passed until 09.00h on Monday  at which point the bladder is completely emptied,  whether or not the patient feels the need, and the  urine specimen is added to the total collection.   The shorter the period of collection, the greater the  error if this procedure is not followed.   Before collection, a urine container should be  obtained from the laboratory; this may contain  a preservative to inhibit bacterial growth (which  might destroy the substance being estimated) or  acid preservative to prevent precipitation or sample  degradation, but that does not interfere with the  relevant assay. The patient must be told not to discard  the preservative in the container and that it might be  toxic and harmful if spilt.   Rectal emptying is usually erratic and, unlike that of  the bladder, can rarely be performed to order. Results   of faecal estimations of, for example, fat may vary by  several hundred per cent in consecutive 24-h collections.  If the collection period lasted for weeks, the mean 24-h  output would be very close to the true daily loss from  the body into the intestinal tract.   To render collection more accurate, orally  administeredTnarkers’ are usedin certain circumstances.  Faecal collections and estimations are time consuming  and unpleasant for all concerned and are now rarely  required.   Labelling patient specimens  All specimens must be accurately labelled and the  information should correspond with that on the  accompanying request form in every detail, as error  may cause a medicolegal disaster. The date, and  preferably the time, of specimen collection should   be included, and should be written at the time of  collection.   Sending the specimen to the laboratory  Many estimations can be performed rapidly if the result  is needed urgently. Non-urgent late blood samples can  be separated from cells and stored overnight, although  some laboratories work 24/7. In cases of true clinical  emergency, the requesting clinician should notify the  laboratory, preferably before the specimen is taken,  indicating the reason for the urgency, so that the  laboratory can be ready to deal with the specimen as  soon as it arrives. It is the clinician’s responsibility to  indicate the degree of urgency. The misuse of emergency  services may delay truly urgent results, and some urgent  samples may need to be separated immediately and  kept on ice.   • It is essential to liaise closely with the laboratory • Particular attention should be paid to avoiding  when collecting patient samples to help ensure blood samples being collected from the same arm as  correct sampling times and collection conditions. an intravenous infusion, leading to ‘drip arm’ results.   • In vitro haemolysis can lead to a spurious increase • It is also essential to ensure correct sample labelling   of predominantly intracellular ions in the plasma, and patient identification to avoid potentially life-  such as potassium (pseudohyperkalaemia). threatening errors and medicolegal disasters.   Some major advantages of point-of-care testing 397 Some possible clinical settings for point-of-care   Recently, laboratories have tended to become more  specialized and centralized and may process millions  of samples per year. High throughput, cost-effective  automation (perhaps including the use of robotics),  stringent quality assurance processes, computerization  with data storage and retrieval systems, and highly  skilled, and trained, personnel are now common.  In contrast, ‘point-of-care testing’ (POCT) has now  developed, which enables clinicians or patients to  perform tests without necessarily using the laboratory  directly.   One of the main advantages of POCT over laboratory  testing is the relative immediacy of results. This  may enable prompt treatment, shortened patient  waiting time and a reduced number of out-patient  appointments and clinic visits for the patient.   Many POCT devices require minimal specimen  preparation or collection (in some cases using a finger  prick of blood). The machine is in the near-patient  setting, thus reducing the delays associated with the  transport of specimens and reports.   Technological advances and ease of use  The recent increase in POCT has been due partly  to technological changes in the design of analysers.  With the advent of microchips, computerization and  miniaturization, it has become easier to bring analysis  nearer to the patient and for it to be performed by  personnel with minimal training or by the patients  themselves. Some of the modern POCT devices  incorporate biosensors, electrodes and dry- and solid-  phase chemistry reagents. These allow for small sample  and reagent volumes, quick assay reaction times, ease  of use and disposal of used reagents, more than one  analyte to be measured simultaneously and probably  less technical skill.   Transcutaneous biosensors allow continuous  measurements to be made through the patient’s skin  without the need for blood collection. Near-infrared  spectroscopy may allow continuous monitoring of more  than one analyte as well as in vivo glucose monitoring  with implantable sensors in diabetic patients.   In choosing a POCT analyser system, remember that  duplication may occur within the hospital at separate  sites. Different analysers on the same site may result in  the use of different reference ranges and thus difficulties  in comparing patient results. Many of the new POCT  analysers are relatively easy to maintain, but maintenance  may need to be carried out by non-laboratory staff out  of the laboratory setting. It is also likely that results will  need to be interpreted and troubleshooting performed  by non-laboratory personnel.   The reduction in turnaround time may result in a  reduction in total costs if patient episodes are shorter  and transport costs are reduced, for example courier  costs. However, on a direct charge basis, including  capital costs, POCT may be more expensive than  central laboratory testing. This can be due in part to  duplication of tests overall and to economies of scale.  The costs of reagents and machines, quality control  materials, maintenance, storage of report forms and  results and training may all need to be taken into  consideration when embarking on POCT. Labour  costs are more difficult to assess in POCT, but may  incorporate nursing staff; however, set against this are  possible savings of on-call or out-of-hours costs for  laboratory staff.   Depending on the structure and organization of  the POCT setting, the overall costs could be less when  the merits of rapid therapeutic responses and shorter  hospital stays are taken into consideration (Box 30.1).  It is essential that there is proper user training and  also maintenance of a complete audit trail for patient  results.   Reference ranges and results may differ from those  of the laboratory, thus making comparison difficult  Care is needed in machine maintenance and repairs  Poor training may lead to inadequate quality control  There may be a lack of back-up support should a  device fail   A few examples of clinical situations in which POCT  may be useful are given below.   One of the potential advantages of POCT is the  possibility of fast result turnaround time, which is  particularly important in the accident and emergency  (A&E) department.   A significant number of A&E departments have  blood gas machines, blood glucose meters and even  small hand-held analysers for common biochemical  tests. One area in A&E where POCT is important is  in the biochemical diagnosis of acute myocardial  infarction using troponin T or I. In cases of drug   A 13-year-old girl with known type 1 diabetes  mellitus was admitted to a casualty department  because of drowsiness. A blood stick finger prick  test done in the casualty department showed blood  glucose of 20mmol/L. The on-call doctor was going  to give her some more insulin but decided, wisely, to  confirm the result by sending a blood sample to the  laboratory. The result was returned as 1.8mmol/L!   It later transpired that the girl’s fingers had been  contaminated with vomit after she had been  given some orange squash to drink, which led to  a spuriously high blood glucose concentration  result by POCT. The moral of this case is to ensure  proper sample collection and staff training. Check  unexpected abnormal results with the laboratory.   overdose, it may take a few minutes for the results  of POCT measurement of plasma paracetamol  concentrations to be obtained.   A number of devices are available which, using capillary  or venous blood, can measure prothrombin or activated  partial thrombin time. These can be used in coagulation  clinics or by patients on warfarin to monitor their own  treatment.   Measuring for drugs of abuse or ethanol is eliciting  considerable interest in drug and alcohol addiction  clinics. Various drugs can be assayed, including opiates,  cocaine, cannabis, benzodiazepines, amphetamines  and methadone. In the case of methadone, its assay can  help determine compliance with replacement therapy,  and opiate assay is helpful to exclude ongoing use. It  may also be possible for individuals to test their level  of ethanol, which would be particularly useful in the  context of drink-driving.   Many out-patient clinics and wards as well as general  practices use urine dipstick testing for screening  patients, and various urine-testing strips are available.  Some of these tests can be useful to screen for urinary  tract infections (Fig. 30.1).    Figure 30.1 Point-of-care testing urine analysis strips.  Image courtesy of Siemens, used with permission.   There are also machines to determine cholesterol,  triglyceride and high-density lipoprotein (HDL)  cholesterol concentrations. Some desktop analysers  allow other parameters to be assayed, for example  creatinine, glucose, bilirubin and haemoglobin.   One blood test for which POCT is very relevant in the  special care baby units is the determination of bilirubin  using POCT bilirubinometers. In addition, blood gas  machines and mini- analysers have been developed. One  advantage is the need for only small blood samples.   Pregnancy testing is one of the most commonly used  forms of POCT that patients can perform themselves.  The test of faecal occult blood as a diagnostic aid for  colorectal carcinoma is another possibility for POCT  by the patient.   Near-patient testing and diabetes mellitus  The patient management of diabetes mellitus is one area in  medicine in which POCT or self-monitoring is frequently  used. Broadly, this can be considered as involving urinary  glucose concentration determinations (urine glucose  now rarely used), ketone urinary or plasma tests, blood  glucose measurements, glycated haemoglobin assays and  urinary microalbumin tests. Box 30. 1 gives some of the  advantages and disadvantages of POCT.   • POCT allows prompt turnaround of patients’  results and thus the potential for speedy clinical  management.   • POCT plays a large role in the management of  diabetes mellitus, with some patients testing their   However, POCT also has potential problems, and  there should be close liaison with the hospital  laboratory to ensure optimal usage. Good quality  control is essential.   Results in clinical chemistry have been expressed in  a variety of units. For example, the concentrations of  electrolytes were formerly usually quoted in mEq/L,  the concentration of protein in g/lOOmL and that of  cholesterol in mg/lOOmL. The units used might vary  from laboratory to laboratory: calcium concentrations  might be expressed as mg/100 mL or mEq/L; in  Britain plasma urea concentrations were expressed  as mg/lOOmL of urea, whereas in the United States it  is usual to report mg/lOOmL of urea nitrogen. This  situation can be confusing and with patients moving,  not only from one hospital to another, but from one  country to another, dangerous misunderstandings have  arisen.   International standardization is obviously desirable, and  such standardization has long existed in many branches  of science and technology. The main recommendations  for clinical chemistry are as follows:   • If the molecular weight (MW) of the substance being  measured is known, the unit of quantity should be  the mole or a subunit of a mole:   In clinical chemistry, in the UK, millimoles  (mmol), micromoles (pmol) and nanomoles (nmol)  are the most common units.   • The unit of volume should be the litre. Units of  concentration are therefore mmol/L, pmol/L or nmol/L.   • In the case of univalent ions, such as sodium and  potassium, the units will be numerically the same.  A sodium concentration of 140 mEq/L becomes  140 mmol/L.   • For polyvalent ions, such as calcium and magnesium  (both divalent), the old units are divided by the  valency. For example, a magnesium concentration of  2.0 mEq/L becomes 1.0 mmol/L.   If results were previously expressed in mg/lOOmL  the method of conversion to mmol/L is to divide by  the molecular weight (to convert from mg to mmol)  and to multiply by 10 (to convert from 100 mL to  L). Thus effectively the previous units are divided  by a tenth of the molecular weight. For example, the  molecular weight of urea is 60, and of glucose 180.  A urea concentration of 60mg/100mL and a glucose  concentration of 180mg/100mL are both equivalent  to 10 mmol/L. The factor of 10 is, of course, only  used for concentrations. The total amount of urea  excreted in 24 h in mg is numerically 60 times that  in mmol.   • Units of pressure (for example mmHg) are expressed  as pascals [or kilopascals (kPa)]. One kPa equals  7.5 mmHg, so that a Po 2 of 75 mmHg is lOkPa.  Pascals are SI units.   • Proteins. Body fluids contain a complex mixture of  proteins of varying molecular weights. It is therefore  recommended that the gram (g) be retained, but  that the unit of volume be the litre (L). Thus a total  protein of 7.0g/100mL becomes 70g/L.   • Enzyme units are not yet changed. Note that the  definition of international units for enzymes does  not state the conditions of the reaction.   A conversion table for some of the commoner results is  listed in Table A. 1 . Note that:   5-HIAA, 5-hydroxyindole acetic acid; HMMA, 4-hydroxy-3-methoxymandelic acid; TIBC, total iron-binding capacity.   abdominal pain 250-1  abetalipoproteinaemia 212-13  absorption in GI tract 235, 236-40  defects see malabsorption  drug 348-9  reduced areas 242-3  accident and emergency, point-of-care  testing 398   acid-base balance disturbances (=H +  disturbances) 65-77  arterial blood estimation in 77, 78, 80,  80-1   acyl-CoA dehydrogenase deficiency,  medium chain 365, 372, 382  addiction clinics, point-of-care testing 398  Addison’s disease (hypoadrenocorticism;  chronic adrenocortical insufficiency)  18,92, 132, 136-40  abdominal pain 251  hyperkalaemia 92  investigation in suspected disease  138-40   Addisonian crisis 138, 139  adenine phosphoribosyl transferase  (APRT) 303  deficiency 56   hyperfunction 132-6, 143-5  hypofunction/insufficiency  (hypoadrenalism) 132  acute (Addisonian crisis) 138,139  chronic see Addison’s disease  investigation 138-40, 144  secondary (ACTH deficiency) 119,  125, 138   phaeochromocytoma 339  adrenaline 178,338,339  adrenocorticotrophic hormone (ACTH;  corticotrophin) 117, 129, 132, 135-6  deficiency 119,125,139  ectopic 86,133-4,241,341   arterial blood (gases) findings in 77  hypokalaemic see hypokalaemia  metabolic see metabolic alkalosis  potassium abnormalities in 84, 88-9  respiratory see respiratory alkalosis  severe, blood pH indicating 60  symptoms 65-6  alkaptonuria 198, 375-6  alleles   p-aminobutyric acid (PABA) test 240, 248  aminoglycoside monitoring 352  5-aminolaevulinic acid (ALA) 310  raised levels 320,321  5-aminolaevulinic acid (ALA) synthase  310,319   amylase 237, 240, 270, 275-6  high levels 240,241,250,275-6  isoenzymes 276  macroenzyme 276, 277  measurements 240, 270, 275-6  in diabetic ketoacidosis 190  pleural fluid 336  amyloid 297  anaemia 316   sideroblastic, porphyrinuria of 322  vitamin A deficiency 225  vitamin B 6 deficiency 229  vitamin B ]2 and folate deficiency 229,  230   Conn’s syndrome diagnosis 144  diabetic nephropathy 188  hyperkalaemia 91  hypertension 330-1  angiotensin-converting enzyme 8, 280  angiotensin-converting enzyme inhibitors  8   high (and metabolic acidosis) 66, 67  normal (and metabolic acidosis) 68  urinary 67   antibodies/immunoglobulins  (autoimmune)  in coeliac disease 243  in diabetes mellitus 184, 196  to insulin 184, 196, 197  in primary biliary cirrhosis 257  to thyroid hormones 168, 169, 170, 171,  173   ApoB 3500 mutation (=familial defective  apolipoprotein B-100) 210,390  metabolic defect 212-13  apoC 2 deficiency 208  apoE   therapeutic drug monitoring 350-1  arterial blood see blood  arthritis, rheumatoid 336  arylsulphatase deficiency 379  ascites 336-7  in cirrhosis 261  ascorbate see vitamin C  asialotransferrin (tau) 334, 335  aspartate aminotransferases (AST) 255,  256, 272-3  in liver disease 268   B-type natriuretic peptide see brain/B-type  natriuretic peptide  bacterial flora, intestinal, alterations/  overgrowth 245  investigation/diagnosis 246, 247  bacterial infection of CSF (and meningitis)  332,333,335   Beckwith-Wiedemann syndrome 364  Bence-Jones proteins 294, 298, 345  benzodiazepine overdose 356  beriberi 227  beryllium poisoning 102  (3-cell (beta-cell), pancreatic islet 177, 179,  180   in intravenous fluids 10  loss in kidney, impaired ability leading  to metabolic alkalosis 74  measurement in blood/plasma 80  in hyperkalaemia 93  reclamation/reabsorption in kidney 62,  62-3   obstructed flow see biliary tract  stasis see cholestasis  bile acids (and salts) 206, 238, 264-5  deficiency 265  in obstetric cholestasis 256  sequestrants 213  synthesis 238, 252, 253, 264-5  biliary atresia, congenital 363, 364  biliary cirrhosis, primary 257, 258,   in primary hyperparathyroidism 101  in primary hypoparathyroidism 107  in secondary hypoparathyroidism  107-8   bicarbonate buffering system 61  blood 60,61-2  buffer pairs 60, 63-4, 64, 65  urinary 63-4   C-peptide (insulin) 177  measurements 195, 196  C-reactive protein (CRP) 287-8, 330  cardiovascular disease risk and 287-8,  330   infections 287, 335  C-type natriuretic peptide 328, 329  Cl esterase inhibitor defect/deficiency 293  C3 component of complement 288-9  CA- 1 5-3/CA- 19-9/CA- 125 (carbohydrate  antigens) 345  cabergoline 121, 148, 149  cadmium toxicity 234  caeruloplasmin 232, 233, 263, 269  caffeine as theophylline metabolite 352  calciferol see vitamin D  calcitonin 97   biliary (gallstones) 250, 257, 265  renal (nephroliths) 54-7  in cystinuria 55, 57, 377  in hypercalcaemia 55, 99, 100, 101  in hyperuricaemia 55, 305  treatment 57  in xanthinuria 55, 309  calprotectin 249  cancer see malignant tumours  capillary membrane   carbon monoxide poisoning 311, 356  pulse oximetry in 79  carbonate dehydratase (CD; carbonic  anhydrase) 61,62,63,64,68,69  inhibitors 85, 90   hyperaldosteronism) 22-3, 86, 143-5  diagnosing cause 144  hypertension 143, 330  hypokalaemia 22, 23, 86  investigations 143-4  treatment 144   in hyperuricaemia 307  in nutritional assessment 218  see also albuminxreatinine ratio;  proteinxreatinine ratio  Crigler-Najjar syndrome 264, 363  critical (intensive) care, point-of-care  testing 399  Crohn’s disease 249   ectopic tumours 86, 133-4, 135, 136  pituitary 127, 135, 136  hypertension 133, 330  hypokalaemia in 86  investigations/diagnosis 127, 134-6  mimicking conditions 135  treatment 136   cystathionine synthase deficiency 329, 376  cystatin C 51-2  cystic fibrosis 244, 388-9  diagnosis 240, 388-9   prenatal testing 240, 244, 372  cystine stones and cystinuria 55, 57, 377  cystinosis 378   nicotinamide deficiency 228  deoxyribonucleic acid see DNA  depression vs Cushing’s syndrome 135  dermatological lesions see eczema; skin  l-desamino-8-D-arginine vasopressin  (DDAVP) test 33  desferrioxamine 234,317,356  desmopressin (DDAVP) test 33  detoxification in liver 253  dexamethasone suppression test  high-dose 136  low-dose overnight 135  dextrose saline see glucose  diabetes insipidus 20   type 2 (non-insulin-dependent DM)  184, 185, 186, 187, 188  types other than 1 and 2 183-4  diagnostic performance 4-5  diagnostic value of test abnormalities 3  dialysis 53-4  machine 58  diarrhoea   adrenocortical hypofunction 137  biochemical monitoring 353-4  diabetes 184  on glucose tolerance 194  hypercalcaemia 99, 102  hyperkalaemia 91,92  hypermagnesaemia 114  hyperprolactinaemia 148, 149  hyperthyroxinaemia 174  hyperuricaemia 306-7  hypocalcaemia in pregnancy and  neonates 367  hypokalaemia 87, 89, 90  hypothyroidism 170  infertility 163  on liver 259,263,351,354  see also toxicology   hypothalamic-pituitary-gonadal axis  effects of 163  interactions between 349  metabolic disorders (inherited) and  382-3   diabetes (antidiabetic) 187  diabetic nephropathy 188  gastric acid suppressants 236  hypercalcaemia 104, 105  hyperlipidaemia see lipid-lowering  drugs   dysbetalipoproteinaemia, familial 210-11  dysgammaglobulinaemia 291-2  dyslipidaemias (commonly called  hyperlipidaemias) 207-15  in diabetes 189,190  familial combined 210  Fredrickson classification 202, 203, 207,  209-10   ectopic hormone production 342-3  by tumours 86, 133-4, 135, 136,272,  342-3   electrocardiogram (ECG) abnormalities  hypercalcaemia 99-100  hyperkalaemia 92  hypokalaemia 88  myocardial infarction 325  electrolytes   DNA (for Southern blots) 387  RNA (for Northern blots) 388  plasma protein 268,283-4,301,302  alkaline phosphatase isoenzymes 278  in cirrhosis 268, 284  in other diseases 284-5  urinary proteins 283-5,301   diabetes due to 184  diagnostic principles 118  hypercalcaemia due to 99-100, 102  hypoglycaemia due to 195  kidney disease (chronic) due to 48  osteoporosis due to 110  kidney 37   plasma (in general), and their  measurement 270-81  diagnostic precision 271  factors affecting results 270-1  hepatic enzymes 255, 256, 268-9, 280  non-specific causes of raised levels  271   creatine kinase 3, 4, 270  ethylene diamine tetra-acetic acid see  EDTA   deficiency 229-30, 239  folinic acid rescue 353  follicle-stimulating hormone (FSH) 117,  119, 146   in disease 284, 285  see also agammaglobulinaemia;  dysgammaglobulinaemia;  hypergammaglobulinaemia;  hypogammaglobulinaemia;  immunoglobulin G   gamma-glutamyltransferase (GGT) 256,  258, 259, 262, 268, 269, 271, 279  gangliosidoses 379  gastrectomy, complications 236, 243  gastric... see stomach  gastrin 236, 237   reduced tubular function and 43  filtration rate (GFR), reduced 43  acute oliguria with 44-5  in hypothyroidism 168  normal tubular function and 42  related to stage of renal failure 49  function 36, 37-8  tests 49-52   parenteral nutrition 220  y-glutamyltransferase (GGT) 256, 258,  259,262,268, 269, 271,279  gluten-sensitive enteropathy (coeliac  disease) 218,242-4  glycaemia see glucose, blood/plasma  glycated haemoglobin 187-8  glycogen 176,216   biochemical investigations 155-6  see also androgens; hypogonadism;  hypothalamic-pituitary-gonadal  axis; ovaries; sex hormones; testes  gonadotroph(s) 117  gonadotrophin(s), human chorionic see  human chorionic gonadotrophin  gonadotrophin(s), pituitary 117, 146  deficiency 125   hypogonadism due to see  hypogonadism  treatment 128  excess, hypogonadism due to  (hypergonadotrophic  hypogonadism) 145, 151, 152,  162   see also follicle-stimulating hormone;  luteinizing hormone  gonadotrophin-releasing hormone  (GnRH) 146  analogues 146  stimulation test 155,162  use in infertility 163  Gordon’s syndrome 92, 138, 330  gout 305, 305-6  treatment 307   carbohydrate metabolism 1 19, 178  deficiency 119, 122-3  excess 119,119-22  diagnosis 120-1  treatment 121-2  secretion (normal) 117,119  growth hormone-releasing hormone  (GHRH) 119,121,122  measurement 120-1  gut see gastrointestinal tract  gynaecomastia 154,343   hyperaldosteronism in 20-1  in hypercalcaemia 99-100  in hyperkalaemia 92  in hypokalaemia 92  see also cardiovascular disease; coronary  heart disease   3-methylglutaryl coA reductase  homocysteine 229, 329-30  homocystinuria 329, 376  homogentisic acid, urinary 198, 375-6  homogentisic acid oxidase deficiency  375-6   see also endocrine function; endocrine  system and specific hormones  hormone replacement therapy 151  human chorionic gonadotrophin 157-8  assays in pregnancy 157-8  in pregnancy 157-8  stimulation test 155-6  males 162 
ClinicalBiochemistryAndMetabolicMedicine Clinical_Biochemistry_and_Metabolic_Medicine_djvu.txt