CHAPTER 12 Drugs and the kidney 874 Assessing kidney function prior to prescribing \200 The most important measure is GFR, no t serum creatinine. \200 A formal creatinine clearance is ideal but cumbersome (requires 24h urine collection) and 6 rarely performed. (cid:2) MDRD eGFR was never intended to guide drug dosing. In particular, it may lead to ov erdosing of small patients and underdosing of large ones. Cockcroft \200\223 Gault estimation of CrCl incorporates body weight and is better suited to this purpose. 2 GFR may be rapidly changing, particularly in AKI or a critical care setting. This makes d rug selection and dosing a key component of management that requires constant attention and review. \200 If in any doubt, discuss with a renal pharmacist \200\224 they are an invaluable resource on all specialist units. PRESCRIBING IN RENAL IMPAIRMENT 875 CHAPTER 12 Drugs and the kidney 876 Prescribing for dialysis patients Patients on renal replacement therapy (RRT) will normally have an insig- ni \201 cant \200\230 native \200\231 GFR, and the removal of drugs usua lly dependent on renal excretion will 6 be negligible. 2 It is vital to know whether such d rugs will be removed by dialysis. Drug clearance will vary, according to modality of dialysis. Clearance by haemodialysis and peritoneal dialysis is contingent on diffusion down a concentration gradient between plasm a and dialysate. Clearance during haemo \201 ltration is reliant on convection alone. Two important factors that may in \202 uence drug removal require consid- eration when prescribing for patients on RRT: \200 Characteristics of the drug. \200 Ch aracteristics of the type of RRT. Drug characteristics \200 Molecular weight (MW): Haemodialysis membranes have relatively small pore sizes and will only clear molecules <500Da. Haemo \201 lters have larger pore sizes and can clear much larger molecules <5,000Da. Protein binding: Heavily protein-bound drugs are not well removed (even if low MW). Volume of distribution: Large volumes of distribution result in low plasma drug concentrations and reduced availability for removal by dialysis. Water solubility: Water-soluble drugs pass through \201 lters more readily than lipid-soluble ones. For patients receiving RRT, GFR cannot be calculated using standard equations. Even though clearance may be higher during treatment, dosing at CrCl <10mL/min is common fo r haemodialysis, as the treatment is generally intermittent. Clearance is higher in continuous techniques, such as continuous veno-venous haemo \201 ltration (CVVHF) or continuous veno-venous haemodia \201 ltration (CVVHDF) where clearances can be in the range 30 \200\223 40mL/min. This allows larger dos es to be prescribed. Generally, the manufacturer of machines and dialysers/ \201 lters will provide an estimate of CrCl by their system. Drugs will have a sieving coef \201 cient that indicates the proportion of drug that will pass through a membrane during treatment. A drug that passes freely has a si eving coef \201 cient of 1, and one does not pass at all will have a coef \201 cient of 0 . It is the knowledge of the system clearance and the drug-sieving coef \201 cient that informs guidance for drug prescription. PRESCRIBING FOR DIALYSIS PATIENTS 877 There are several comprehensive resources, such as renal formularies, that could, and should, be frequently consulted. Clinical decision support alerts within electronic prescribing systems can help reduce errors. Specialist renal and critical care pharmacists are an invaluable source of information and essential members of the relevant clinical teams. RRT characteristics \200 Properties of the dialyser or \201 lter (pore size, surface ar ea). \200 Drug adsorption onto some membrane types. \200 Duration of therapy, e.g. haem odialysis provides ef \201 cient clearance but is only an intermittent treatment. Peritoneal dialysis clears drugs much less ef \201 ciently than either haemodialysis or haemo \201 ltration but is a continuous technique. Dialysate and blood \202 ow rates (determine concentration gradient). \200 For peritone al dialysis: Peritoneal membrane status, e.g. clearance increases during the in \202 ammation caused by peritonitis. \200 Volume and frequency of exchanges. Drugs that are cleared by haemodialysis are usually administered after a treatment session. CHAPTER 12 Drugs and the kidney 878 Sepsis and antimicrobials 3 Sepsis is a relatively common cause and complication of renal f ailure. Presentations may be atypical and non-speci \201 c. Samples for culture and sensit ivity (including blood, urine, sputum, wound swabs, drain \202 uids, and any other releva nt locations) should be obtained (preferably before antibiotic administration) wherever pos sible. See Table 12.1 on choice of antimicrobial treatment and Table 12.2 for antibacterial , antiviral, and antifungal dosing Antimicrobials Choice of antibiotic \200 History and examination may identify the most li kely source of infection and guide initial antibiotic selection. However, prompt empirical treatment to cover all likely pathogens may be necessary in deteriorating patients. 2 Local antibiotic policies, developed with microbiology and drugstore input, should be adh ered to in all, but exceptional, circumstances. \200 Prudent antibiotic prescribing can r educe hospital-related infections, such as MRSA and C. dif \201 cile , and help to prevent antibiotic resistance. Always record indication and intended antibiotic duration or review date. IV antibiotics should be switched to oral equivalents after 48h if T <38 * C for 48h, patie nt is clinically improving, a suitable oral agent is available, and there is no evidence of malabsorption, gastric stasis, or vomiting. There are exceptions (e.g. PD peritonitis, end ocarditis, meningitis) \200\224 your microbiology team will advise. Numerous antimicrobials depend on renal clearance. However, most have a wide therapeutic index, and dose reductions are only necessary once GFR is <20mL/min ( (cid:2) aminoglycosides and vancomycin are important exceptions). High doses of B -lactam antibiotics can accumulate and cause neurological sequelae, including confusion, agitation, and seizures in advanced CKD, includ ing dialysis patients. Cephalosporins and quinolones require dose reductions in more severe renal impairment (see Table 12.2). Vancomycin is extensively used for staphylococcal infections (including dialysis line sepsis and PD-related peritonitis). Monitoring of drug levels is essential ( b p. 882). Aminoglycosides can be used with care ( b p. 880). Haemodialysis patients Central vascular dialysis catheter infection or PTFE access graft sepsis should always be considered ( b p. 297). Even if a PTFE graft has pre- viously been surgically removed, residual material may remain in situ . 2 Always consider immediate removal of any foreign body (particularly dial ysis lines). Do not wait for culture results. Empirical therapy often involves vancomycin a nd gentamicin, which are dosed according to trough drug levels ( b p. 882). Unlike gentamic in ( b p. 881), vancomycin SEPSIS AND ANTIMICROBIALS 879 is not effectively removed by standard HD, so speed of clearance will largely depend on res idual renal function (if any). In practice, doses may be days apart. (cid:2) Beware the com placency this can induce \200\224 it is crucial to proactively measure and respond to drug levels if periods of inadequate antibiotic activity are to be avoided. In addition to aminoglycosides, penicillins (e.g. benzylpenicillin, amoxicil- lin, and co-a moxiclav), meropenem, cefotaxime, ceftazidime, cipro \202 oxa- cin, metronidazole, clarith romycin, and trimethoprim are removed by HD and should be given post-treatment. 3 Severe se psis is an exception \200\224 administer immediately. Peritoneal dialysis patients ( b p. 322) In suspected PD peritonitis, perform an exchange, and send \202 uid for M,C+S. Perit onitis in overnight APD patients may be less easily diagnosed, as the patient may not witne ss a cloudy bag. Also consider (and exam- ine for) exit site or tunnel infections related t o Tenckhoff catheters. (cid:2) Remember: other causes of peritonitis or an acute abdomen ca n, and do, occur in peritoneal dialysis patients. All renal units will have a detailed anti biotic policy for the treatment of PD-related peritonitis and exit site infections. Interna tional guidance can be found at M http://www.ispd.org . Post-transplantation ( b p. 426) Pa tients are susceptible to a wide variety of infectious pathogens at differ- ent stages post -transplantation. Atypical infections must always enter the differential. Initial broad-spe ctrum antimicrobial treatment is often neces- sary until investigations are completed and c ulture results available. Also review the degree of immunosuppression, and consider a reduc tion. If overwhelming sepsis, stop all immunosuppression, and replace with IV steroid (e.g. hydrocortisone IV 50mg qds) until condition allows phased reintroduction. 2 Discuss with t ransplant team ASAP. Antivirals Many antivirals, including aciclovir, ganciclovir, valganciclovir, famciclo- vir , and lamivudine, are dependent on renal clearance and will begin to accumulate, even in re latively mild renal insuf \201 ciency (GFR <50mL/min). Dose-related (and usually reversibl e) neurological toxicity is typical ( l dizziness, tremor, confusion, ataxia, hallucination s, drowsiness, encepha- lopathy, seizures). Bone marrow suppression is also possible. Ribav irin and its metabolites rely on renal clearance, and their accumulation can cause severe a nemia. The neuraminidase inhibitor oseltamivir under- goes hepatic breakdown to active meta bolites, requiring subsequent renal clearance. An increased dose interval is 6 recommended once GFR <30mL/min. Many antivirals are removed freely by dialysis. Antifungals Unlike many other azole antifungals (e.g. ketoconazole, itraconazole), \202 u - conazole is renally excreted, so maintenance dosing requires adjustment in moderate to se vere CKD. It should also be administered after dialysis. Fluconazole \200\231 s urinary ex cretion means that it is useful in the treatment of fungal infections of the urinary tract. CHAPTER 12 Drugs and the kidney 880 Aminoglycosides \200 (cid:2) Aminoglycosides must always be used with caution in renal im pairment, as they undergo renal clearance, have a narrow therapeutic window, and are nephro toxic. A vicious cycle of rising drug levels and worsening renal function can (and often do es) result. \200 Aminoglycosides are freely \201 ltered by the glomerulus (so dose is adjusted according to GFR) and then partially taken up by tubular cells. This can cause tub ular cell injury ( l ATN). 2 Several factors increase risk of both nephrotoxicity (and ototoxicity): prolonged treatment, dehydration, concomitant diuretic use, obstructive jaund ice, hypokalaemia, and hypomagnesaemia. Dose adjustments aim to achieve peak plasma levels that are bactericidal whilst permitting low trough levels that avoid toxicity. SEPSIS AND ANTIMICROBIALS 881 (cid:2) Gentamicin dosing \200 Gentamicin does not enter fat, so obese patients should be dosed, according to ideal body weight (IBW), calculated as follows: \200 Adult \231\202 : IBW ( in kg) = 50 + (2.3 x height in inches over 5 feet). \200 Adult \231\200 : IBW (in kg) = 45.5 + (2.3 x height in inches over 5 feet). 2 Protocols will vary, so consult local policies, and discuss with microbiology. GFR >20mL/min: usual \201 rst dose for empirical treatment is 5mg/kg (according to ideal body weight), with a maximum dose of 450mg. \200 GFR <20mL/min in those who are not on di alysis treatment: usual recommended dose is 3mg/kg, according to ideal body weight, with a maximum of 280mg. Patients on HD or PD: usual recommended \201 rst dose is 2mg/kg, with a maximum dose of 180mg. Seek advice regarding subsequent doses. \200 Serum levels are maintained in the therapeut ic range for longer in patients with d GFR, and the dosing interval may be every 2 \200\223 5 days. A trough serum level, taken 20h post-dose, should be <1mg/L (unless the patient is on HD) before repeat administration. Peak level measurement is unnec essary in once daily dosing regimens. \200 If the level is not <1mg/L, then repeat every 24h, and redose when <1mg/L (and if there remains a clear indication for the drug). \200 In HD patients, redos e when pre-dialysis levels are <2mg/L. Dialysis removes a signi \201 cant proportion of gentamicin \200\224 waiting for pre-dial ysis trough levels to fall to <1mg/L risks sub-therapeutic periods. Gentamicin is generally administered at the end of HD and by intraperitoneal administration in PD patients. Amikacin, netilmicin, and tobramycin have similar pharmacokinetics and require similar management. Consult local policies. In HD and PD, the effect of aminoglycosides on residual \200\230 native \200\231 renal function remains unpredictable, but the risk of sepsis outweighs the risk of a fall i n residual urine output. It is 6 the risk of ototoxicity and devloping an irreversible bala nce disorder that is the paramount concern in this situation. CHAPTER 12 Drugs and the kidney 882 Table 12.1 Antimicrobial treatment in common renal scenarios (local policies will vary) Condition First-line Penicillin allergy Comments Modify treatment, according to culture results. Cipro \202 oxacin PO for 3 days in \231\200 and 7 days in \231\202 . 7 days in renal tra nsplant patients. Dose reduction required in renal impairment. UTI Co-amoxiclav 375 \200\223 625mg tds PO for 3 days in \231\200 and 7 days in \231\202 . G ive 7 days in renal transplant patients. Catheter-related bacteriuria does not require trea tment if no clinical evidence of a UTI. Modify, according to culture results. Gentamicin IV stat, and discuss with microbiology. Dose reduction if renal impairment (see b p. 881). Co-amoxiclav IV for 10 \200\223 14 days (+ gentamicin IV stat if renal transplant or sever e sepsis). Pyelonephritis (native or transplant kidney) See b p. 322. Flucloxacillin 500mg qds PO for 5 days. PD peritonitis If MRSA-colonized, seek microbiological advice. Clarithromycin 250mg bd PO for 5 days. Haemodialysis catheter exit site infection If MRSA- colonized, give vancomycin IV stat, and seek microbiological advice. Clarithromycin 250mg bd PO/IV for 5 \200\223 10 days. Consider line removal. Flucloxacillin 500mg qds PO for 5 days or 1g qds IV for 5 \200\223 10 days if severe. Cons ider line removal. Haemodialysis catheter tunnel infection Modify, according to culture results. Remove line if possible. Vancomycin 1g (500mg if <50kg) IV stat (check trough level at 20h, and redose if <15mg/L) + gentamicin 2mg/kg (max 280mg) IV stat through dialysis catheter (check trough level at 20h , and redose if <2mg/L (see b p. 297). (cid:2) If rigors or unwell, take blood cultures, an d administer immediately; do not wait until the end of dialysis. Haemodialysis catheter (or PTFE graft)-related bloodstream infection Empirical treatment of severe sepsis Co-amoxiclav IV 1.2g + gentamicin IV 2 \200\223 5mg/kg, depending on renal function ( b p. 881). Vancomycin 1g IV stat (regimen dependent on renal function, b Table 12.2) + gentamicin 2 \200\223 5mg/kg IV stat, depending on renal function ( b p. 881). Seek urgent advice. Use penicillin-allergic regimen if known to be MRSA-colonized. ) d e u n i t n o C ( r u c c o y t i c x o t o r u e N s t n e m m o C s s e n i s w o r d i r o / n o i s u f n o c l l i t s f h c t a y a m W y t i l a d o m n m / L m 0 2 \200\223 0 1 h 6 h 8 i g 5 7 3 3 \200\223 5 2 2 r o h 6 \200\223 g m 0 0 6 e g n a h c o N e g n a h c o N y r e v e y r e v e R F G e s a H V V C . y r e v e e s o D s i s y l a i d o t g n d r o c c a t n e m g 4 2 g 5 4 : V n . . . I i g 2 1 \200\223 g m 0 0 6 / L m 0 1 < R F G e / L m 0 1 < R F G e . n m / L m 0 1 < n m / L m 0 1 < n n n n i i i i i i i h 6 s a s a s a s a D P A C R F G e R F G e y r e v e e s o D e s o D e s o D e s o D n m n m : i i V I g 2 1 \200\223 g m 0 0 6 / L m 0 1 < R F G e / L m 0 1 < R F G e / L m 0 1 < R F G e . t s u d a e s o D n m / L m 0 1 < n n n n i i i i i j h 6 s a s a s a s a R F G e y r e v e e s o D e s o D e s o D e s o D D H n m n m n m : i i i V I h 4 2 / g 4 h 2 1 y r e v e h 6 l a t o t n m L m 0 1 < \200\223 g m 0 0 6 i e g n a h c o N \200\223 5 7 3 y r e v e y r e v e h 8 / a o t f g 2 1 o e s o d g m 5 2 6 y r e v e : l a r O . g 2 1 g 5 4 h 2 1 p U : : V V . . I I g 4 2 \200\223 g m 0 0 6 n m L m 0 2 \200\223 0 1 h 2 1 y r e v e i . e g n a h c o N e g n a h c o N \200\223 5 7 3 / y r e v e h 6 h 8 g 2 1 g m 5 2 6 y r e v e y r e v e : l a r O . g 5 4 h 2 1 R F G e o t : : V V . I I g n i s o d n m L m 0 5 \200\223 0 2 i e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N / g n d r o c c a l a g n u f i t n a i t n e m g m 0 0 5 \200\223 0 5 2 g m 5 2 6 \200\223 5 7 3 h 8 d n a y r e v e g 2 \200\223 g m 0 5 2 h 8 t s u d a g 4 2 \200\223 2 1 , l a r i v i t n a y r e v e g 1 \200\223 g m 0 5 2 j . h 8 h 6 h 6 h 6 h 8 l a m r o N g 2 1 e s o D y r e v e y r e v e y r e v e y r e v e y r e v e : l a r O : l a r O . . g 5 4 : : : V V V . I , l a i r e t c a b i t n A I I n i l l i c n e p l y z n e B v a l c x o m a - o C n i l l i c a x o l c u F m a t c a b o z a t n n i i i l l i c x o m A i l l i c i r e p P i l a i b o r c i m i l 2 2 1 . s n i l l i c n e P e l b a T i t n A i s i s p e s n o i t c u d e r r e b m u n e r o f e b r u c c o y t i c x o t o r u e N s t n e m m o C s t n e m m o C s s e n i s w o r d n o i t a c d e m n o i t c a r e t n i y l l a i t n e t o P r o / n o i s u f n o c r e d i s n o C e r e v e s r e h t o l l i t s f h 8 4 \200\223 4 2 s u o i r e s h c t a a i e s o d h t i y a m W f w n o i i g m 0 0 4 \200\223 0 0 2 y t i l a d o m y t i l a d o m n m / L m 0 2 \200\223 0 1 n m / L m 0 2 \200\223 0 1 e s o d i i y r e v e y r e v e s a \200\223 0 0 5 h 2 1 h 2 1 o n R F G e e s o d : V H V V C H V V C e g n a h c g m 0 5 7 I / l a r O R F G e y r e v e y r e v e : l a r O : l a r O s i s y l a i d o t g n d r o c c a t n e m s i s y l a i d o t g n d r o c c a t n e m g 2 \200\223 1 g 2 \200\223 1 n h h 2 1 i : : 2 1 V V s a n I I i R F G e n y r e v e / L m 0 1 < R F G e / L m 0 1 < R F G e e s o d n g m 0 5 2 i n m / L m 0 1 < n m / L m 0 1 < R F G e s a n i i g m 0 0 2 h 2 1 i i i i o n e s o d s a : s a e s o D R F G e V D P A C D P A C e g n a h c I / l a r O y r e v e e s o D : l a r O : l a r O n n m n m h 2 1 i : : i i V V s a I I y r e v e / L m 0 1 < R F G e / L m 0 1 < R F G e e s o d n t s u d a e s o D t s u d a e s o D g m 0 5 2 i n m / L m 0 1 < n m / L m 0 1 < R F G e s a n n i i g m 0 0 2 h 2 1 i i j j o n e s o d s a s a : V e g n a h c I / l a r O y r e v e e s o D e s o D : l a r O : l a r O n D H D H n m n m h 2 1 i : : i i V V I y r e v e s a I g m 0 0 5 \200\223 0 5 2 g 5 1 \200\223 g m 0 5 7 h 4 2 \200\223 2 1 y r e v e h 2 1 n m L m 0 1 < n m L m 0 1 < g m 0 5 2 . i i \200\223 5 2 1 \200\223 0 5 2 g 1 \200\223 g m 0 0 5 g m 0 0 2 h 2 1 h 2 1 h 2 1 h 4 2 h 2 1 y r e v e / / g m 0 0 5 g m 0 0 5 y r e v e y r e v e y r e v e y r e v e y r e v e : l a r O : l a r O : l a r O h 2 1 : : : g 1 V V V I I I y r e v e h 4 2 g 5 1 \200\223 g m 0 5 7 n m L m 0 2 \200\223 0 1 n m L m 0 2 g m 0 0 5 \200\223 0 5 2 h 8 \200\223 6 i i . g m 0 5 2 h 2 1 \200\223 8 y r e v e \200\223 0 5 2 / / \200\223 5 2 1 g m 0 0 2 h 2 1 h 2 1 h 2 1 h 2 1 y r e v e g m 0 0 5 g m 0 0 5 y r e v e y r e v e y r e v e y r e v e y r e v e : l a r O : l a r O : l a r O g 2 \200\223 1 h 2 1 R F G e o t R F G e o t : : : 0 1 g 1 V V V I I I n m L m 0 5 \200\223 0 2 n m L m 0 5 g 5 1 \200\223 g m 0 5 7 i i . e g n a h c o N e g n a h c o N y r e v e / / \200\223 5 2 1 h 2 1 g n d r o c c a g n d r o c c a y r e v e h 8 y r e v e i i g m 0 0 5 y r e v e : l a r O h 8 \200\223 6 g 2 \200\223 1 h 2 1 0 2 : g 1 V I t n e m t n e m g m 0 5 7 \200\223 0 5 2 g m 0 0 5 \200\223 0 5 2 g m 0 0 5 \200\223 5 2 1 y r e v e y r e v e g 5 1 \200\223 g m 0 5 7 g m 0 0 4 \200\223 0 0 1 h 8 \200\223 6 t s u d a t s u d a . h 8 \200\223 6 g 2 \200\223 g m 0 0 5 g m 0 0 5 h 2 1 h 2 1 h 2 1 h 2 1 j j l a m r o N l a m r o N y r e v e h 2 1 \200\223 8 e s o D e s o D y r e v e y r e v e y r e v e y r e v e y r e v e : l a r O : l a r O : l a r O h 2 1 : : : g 1 V V V I I I ) d e u n i t n o C n c y m o r h t i r a l C i n c a x o \202 o r p C i e m x o r u f e C e m d i z a t f e C e m x a t o f e C i i i ( l a i b o r c i m l a i b o r c i m i 2 2 1 . p s o l a h p e C s e d e n o o n u Q i l e l b a T l o r c a M s n i r o i t n A i t n A i ) d e u n i t n o C ( e d i s o c y l g o n m a s e s a e r c n f f d n a 1 2 1 o y l r a l u c i t r a P o g n i r o t i n o m s t n e m m o C . r e f e r f d n a n i r u e n c l a c o s r o t i b h n g n d r a g e r l e b a T o t n i r a f r a w e v e l i g n i s o d e s a e P i s l e v e e t o n l i i i e h t l l i y t i l a d o m n o i t c n u f n o i t c n u f n m / L m 0 7 \200\223 0 3 e s o d e s o d i l a m r o n l a m r o n h 4 2 g k / g m 5 7 R F G e s a : : V V H V V C I / l a r O I / l a r O y r e v e e s o D R F G e l a n e r l a n e r s i s y l a i d o t g n d r o c c a t n e m n n i i s a s a n . i R F G e n o i t c n u f e s o d e s o d l a m r o n n m / L m 0 1 < n m / L m 0 1 < n m / L m 0 1 < R F G e n n i i i i i i s a s a : : V V D P A C I / l a r O I / l a r O e s o D R F G e e s o D l a n e r n n i i s a s a s a l a n e r e s o d r e t f a t s u d a e s o D n m / L m 0 1 < n m / L m 0 1 < R F G e n i i l a m r o n i j n o i t c n u f s a : : V V g k / g m 5 s i s y l a d I / l a r O I / l a r O e s o D e s o d n D H i s a i n i g n d r o c c a f / g 2 m u m x a m y r e v e i , o % 5 7 \200\223 0 5 y r e v e g k / g m 2 e s o d n m L m 0 1 < h 2 1 \200\223 8 d n a h 2 7 \200\223 8 4 i \200\223 0 0 2 \200\223 0 5 2 g m 0 0 5 h 8 h 6 y r e v e / s l e v e h 8 4 \200\223 4 2 g k / g m 2 l a m r o n g m 0 0 5 g m 0 0 5 t s u d a y r e v e y r e v e : l a r O : l a r O i y a d l j : : o t V V I I t s u d a e c n o g k / g m 3 \200\223 2 n m L m 0 2 \200\223 0 1 i / g m 0 5 \200\223 5 2 y r e v e o t j h 2 1 \200\223 8 y a d / g 4 \200\223 0 5 2 \200\223 0 0 2 / g m 0 0 5 m u m x a m h 4 2 g n d r o c c a g k / g m 4 \200\223 3 d n a h 8 i , g m 0 0 5 g m 0 0 5 y a d / g k s l e v e y r e v e y r e v e y r e v e : l a r O : l a r O e s o d i y l i a d R F G e o t : : h 6 V V I I l t s u d a / g k / g m 0 5 \200\223 5 2 h 6 y r e v e g m 0 0 5 e c n o g k / g m 5 \200\223 3 n m L m 0 5 \200\223 0 2 m u m x a m i o t j h 2 1 \200\223 8 y a d / g 4 e s o d \200\223 0 0 2 / \200\223 0 5 2 g m 0 0 5 h 2 1 g n d r o c c a g n d r o c c a g k / g m 6 \200\223 5 i d n a h 8 i i g m 0 0 5 s l e v e y r e v e y r e v e y r e v e : l a r O : l a r O y l i a d , y a d : : V V I I l h 8 s l e v e , s e s o d t n e m / g 5 1 m u m x a m g m 0 0 5 \200\223 0 5 2 g m 0 0 5 \200\223 0 0 2 y r e v e e c n o g k / g m 7 \200\223 5 n . l i / g m 0 5 \200\223 5 2 t s u d a f o t y a d / g k / g m 5 1 i h 2 1 \200\223 8 y a d / g 4 o m u m x a m d e d v d o w t s u d a g m 0 0 5 g n d r o c c a j h 6 j l a m r o N i i , i y a d / g k e s o D y r e v e y r e v e : l a r O : l a r O , e s o d i y l i a d y a d : : V V t I I e o z a d n o r t e M n c y m o r h t y r E l i n c m a t n e G i n c a k m A i i i i l a i b o r c i m a d m o r t i s e d i s o c y l g i o n m A i s e o z i t n A i l N n a c e s u d n a o t s i h t n a m d e r \200\230 d e d n e m m o c e r f o k s i r o t s e t a l e r e t a r s t n e m m o C s t n e m m o C e u r t n c y m o c n a V n c y m o t p a d s i x a l y h p a n a g n i r o t i n o M ; \200\231 i i e m o r d n y s y h t a p o y m s i n o i s u f n a K C t o n e s u a c n e t f o h t i e u d f w s i o i y t i l a d o m y t i l a d o m n m / L m 0 2 \200\223 0 1 i r o t i n o m d n a e g n a h c o N R F G e y l i a d g 1 h 8 4 g k / g m 6 \200\223 4 o N H V V C H V V C e g n a h c y r e v e e s o D s l e v e : l a r O s i s y l a i d o t g n d r o c c a t n e m s i s y l a i d o t g n d r o c c a t n e m n i : V s a I l R F G e e s o d n m / L m 0 1 < n m / L m 0 1 < n m / L m 0 1 < e g n a h c o N R F G e n n i i i i i i i s a s a : V D P A C D P A C I / l a r O R F G e e s o D e s o D n i s a ) s l e v e e s o d t s u d a e s o D t s u d a e s o D n m / L m 0 1 < n m / L m 0 1 < n m / L m 0 1 < e g n a h c o N R F G e n n l i i i i i j j r o t i n o m s a s a : V I / l a r O R F G e R F G e e s o D e s o D n D H D H i s a ( ) s l e v e h 2 7 \200\223 8 4 h 2 7 \200\223 8 4 r o y r e v e e c n o g k / g m 4 e c n a n e t n a M g m 0 0 4 n m L m 0 1 < s e s o d y l i a d f i i \200\223 0 0 2 l h 2 1 g 1 \200\223 5 0 r o t i n o m o n / / g n d a o L L e g n a h c g m 0 0 6 g m 0 0 4 y r e v e y r e v e y r e v e i e e r h t : : : l a r O . e s o d e s o d y l i a d 0 1 < h 2 1 i : V ( I ) s l e v e n m L m 0 2 \200\223 0 1 h 8 4 \200\223 4 2 r o i i y r e v e y r e v e e c n o g k / g m 4 e c n a n e t n a M g m 0 0 4 s e s o d f \200\223 0 0 2 / / l L h 2 1 g 1 \200\223 5 0 r o t i n o m o n g n d a o L h 8 4 \200\223 4 2 e g n a h c g m 0 0 6 g m 0 0 4 0 2 y r e v e y r e v e i e e r h t : : : l a r O . e s o d e s o d y l i a d h 2 1 i R F G e o t R F G e o t : 0 1 V ( I ) s l e v e e c n o g k / g m 6 \200\223 4 n m L m 0 5 \200\223 0 2 h 4 2 \200\223 2 1 r o i i y r e v e e c n a n e t n a M g m 0 0 4 s e s o d y l i a d f \200\223 0 0 2 / / l L h 2 1 g 1 \200\223 5 0 g n d r o c c a g n d r o c c a r o t i n o m o n g n d a o L i i e g n a h c g m 0 0 6 g m 0 0 4 0 5 y r e v e y r e v e i e e r h t : : : l a r O . e s o d e s o d y l i a d h 2 1 i : 0 2 V ( I t n e m t n e m g m 0 0 4 \200\223 0 0 2 g m 0 0 5 \200\223 5 2 1 h 2 1 h 2 1 y r e v e g m 0 0 4 ) s l e v e h 2 1 e c n o g k / g m 6 \200\223 4 s e s o d e e r h t : t s u d a t s u d a y r e v e e s o d g n d a o L e c n a n e t n a M y r e v e l j j r o t i n o m h 6 l a m r o N l g m 0 0 6 e s o D e s o D g 1 y r e v e : i : l a r O e s o d y l i a d y l i a d i r o : V N ( I f ) d e u n i t n o C n c y m o t p a D e d i t p e p o p L n c y m o c n a V n n a l p o c e T i i i d i l o z e n L i ( l a i b o r c i m l a i b o r c i m i 2 2 1 . i t p e p o c y l G i d i l e l b a T o z a x O e n o n i t n A i t n A s e d i ) d e u n i t n o C ( , m e n e p a t r e m e n e p m r o s e r u z i e s y t i c x o t o r u e n d e e n f s t n e m m o C m e n e p o r e M o t r o t i n o m o t i y l e k i l l i t s i e s u a c i l n a h t s s e d n a t u b l y t i l a d o m n m / L m 0 2 \200\223 0 1 n m / L m 0 2 \200\223 0 1 h 2 1 i i e g n a h c o N R F G e y r e v e R F G e s a H V V C e s o D R F G e e s o D R F G e s i s y l a i d o t g n d r o c c a t n e m n i g 1 s a n i n m / L m 0 1 < n m / L m 0 1 < n m / L m 0 1 < e g n a h c o N n n n i i i i i i i s a s a s a D P A C R F G e e s o D e s o D e s o D / L m 0 1 < R F G e / L m 0 1 < R F G e t s u d a e s o D n m / L m 0 1 < e g n a h c o N n n n i i i i j s a s a s a R F G e e s o D e s o D e s o D D H n m n m i i , s y a d 3 r o n n g 1 n / g k / g m 0 3 n e h t i i a i l a n e r l a n e r n m L m 0 1 < e s o d e s o d : s i x a l y h p o r P y a d / g k / g m 0 6 r o s e m d e d v d o w d e d v d o w i e g n a h c o N : t n e m t a e r T g 1 \200\223 g m 0 0 5 h 4 2 , / n o i t c n u f n o i t c n u f i i i t l a m r o n l a m r o n f f f i i o % 0 5 o % 0 5 n s e s o d , s y a d s e s o d y r e v e e e r h t k e e w i y a d t t l a m r o n n n n m L m 0 2 \200\223 0 1 n n o i t c n u f i i i i l a n e r y a d / g k / g m 0 6 y a d / g k / g m 0 3 e s o d : s i x a l y h p o r P d e d v d o w d e d v d o w f 3 : t n e m t a e r T o % 0 0 1 \200\223 0 5 e g n a h c o N / g 1 \200\223 g m 0 0 5 h 2 1 r o n o i t c n u f i i n l a m r o n f f i i o % 0 5 i s e s o d s e s o d y r e v e l a n e r e s o d n e h t R F G e o t t t n m L m 0 5 \200\223 0 2 i e g n a h c o N e g n a h c o N / g 2 \200\223 g m 0 0 5 h 2 1 g n d r o c c a y l i a d i y r e v e g 1 4 \200\223 2 h 8 t n e m y r e v e e t a n r e t l a n f h 2 1 o e s o d i r o y l i a d y a d / g k / g m 0 2 1 s e s o d t s u d a n e h t : s i x a l y h p o r P y r e v e : t n e m t a e r T g 1 \200\223 g m 0 0 5 j l a m r o N g n d a o L y l i a d d e d v d , g m 0 0 1 g m 0 8 4 g m 0 6 9 e s o D i g m 0 5 s y a d i g 1 i e o z a x o m l m e n e p o r e M e n m e n e p a t r E i l c y c e g T i r t - o C l a i b o r c i m i n e p a b r a C ) m a t c a l - l y c y l G s r e h t O e n i t n A i l c y c s m e ( e n i r u e t a u q e d a n s a i s a r c s y d d o o b d n a , s t c e f f e e s r e v d a y a m e n n i t a e r c a m e a l a k r e p y h d n a ; n g n i s u a c n o i t a r t n e c n o c i g u r d e h t s t n e m m o C s t n e m m o C i R F G e n e h n o i t i t e p m o c a m s a l p c x o t e v i t c e f f e n n o m m o c y h t a p o r u e n o t y l r a l u c i t r a p , / L m 0 2 < s i n o i t e r c e s n a c l a n e r 5 / 4 D K C e u d i m u r e S o t e u d , r u c c o s l e v e , i n m e s i r r o W i i l s i s i f l i y t i l a d o m y t i l a d o m e g n a h c o N n a r t n o C i d e t a c d H V V C H V V C s i s y l a i d o t g n d r o c c a t n e m s i s y l a i d o t g n d r o c c a t n e m i d e t a c d n a r t n o C e g n a h c o N i i i i D P A C D P A C d e t a c d n a r t n o C d e t a c d n a r t n o C t s u d a e s o D t s u d a e s o D e g n a h c o N i j j i D H D H n m L m 0 1 < n m L m 0 1 < i i e g n a h c o N i / / i d e t a c d n a r t n o C n m L m 0 2 \200\223 0 1 n m L m 0 2 i i e g n a h c o N / / i i R F G e o t R F G e o t 0 1 n m L m 0 5 \200\223 0 2 n m L m 0 5 i i e g n a h c o N / / g n d r o c c a g n d r o c c a h t i n o i t u a c i i w e s U 0 2 t n e m t n e m g m 0 0 2 t h g n i t s u d a t s u d a t a : s i x a l y h p o r P : s i x a l y h p o r P y l i a d : t n e m t a e r T : t n e m t a e r T g m 0 0 1 \200\223 0 5 g m 0 0 1 \200\223 0 5 h 2 1 j j h 6 l a m r o N l a m r o N g m 0 0 1 e s o D e s o D y r e v e y r e v e ) d e u n i t n o C n o t n a r u f o r t i m i i r p o h t e m i r T ( l a i b o r c i m l a i b o r c i m N 2 2 1 . e l b a T i t n A i t n A ) d e u n i t n o C ( h t i d e d n e m m o c e r s g u r d y t i c x o t o r u e N k s i r p u s o e y m n o i t a n b m o c s t n e m m o C w r u c c o n a c r o s s e n i s w o r d n i r e h t o h t i / n o i s u f n o c p u s o e y m m u m x a m f i d e s a e r c n I n o i s s e r p y l l o s r o t i n o m e v i s s e r p l i u f e r a c , e s o d l i f w o y t i l a d o m n m / L m 0 2 \200\223 0 1 n m / L m 0 2 \200\223 0 1 n m / L m 0 2 \200\223 0 1 i i i e g n a h c o N e g n a h c o N h 4 2 g k / g m 5 2 R F G e R F G e R F G e s a s a s a H V V C y r e v e e s o D e s o D e s o D s i s y l a i d o t g n d r o c c a t n e m n n n . i i i / L m 0 1 < R F G e / L m 0 1 < R F G e n m / L m 0 1 < n m / L m 0 1 < e g n a h c o N e g n a h c o N n n n n i i i i i i i s a s a s a s a D P A C R F G e R F G e e s o D e s o D e s o D e s o D n m n m i i / L m 0 1 < R F G e / L m 0 1 < R F G e / L m 0 1 < R F G e t s u d a e s o D n m / L m 0 1 < e g n a h c o N e g n a h c o N n n n n i i i i i j s a s a s a s a R F G e e s o D e s o D e s o D e s o D y r e v e n m n m n m D H i i i h \200\223 0 0 4 / x 3 \200\223 2 y r e v e n m L m 0 1 < 2 1 \200\223 8 : s i x a l y h p o r P 3 \200\223 2 g k / g m 5 \200\223 5 2 : t n e m t a e r T i k e e w / x 2 \200\223 1 e g n a h c o N e g n a h c o N g k / g m 5 2 1 h 4 2 r o / s k e e w 3 : r e t s o Z r o f g m 0 0 2 g m 0 0 8 g m 0 5 4 g m 0 5 4 s k e e w y r e v e k e e w f h 2 1 h 4 2 . . s k e e w 3 r o y r e v e n m L m 0 2 \200\223 0 1 g m 0 0 8 i y r e v e g m 0 5 4 e c w 3 \200\223 2 : s i x a l y h p o r P e g n a h c o N g k / g m 0 1 \200\223 5 : t n e m t a e r T / e g n a h c o N i h 4 2 y a d / x 4 \200\223 3 g k / g m 5 2 t : r e t s o Z y l k e e w r o g m 0 0 2 g m 0 5 4 s k e e w h 2 1 \200\223 8 y r e v e f f h 8 4 h 4 2 R F G e o t . s k e e w 3 r o h 8 4 y r e v e g m 0 5 y r e v e n m L m 0 5 \200\223 0 2 g m 0 0 8 i 4 y r e v e g m 0 5 4 3 \200\223 2 : s i x a l y h p o r P g m 0 0 4 \200\223 0 0 2 e g n a h c o N e g n a h c o N g k / g m 0 1 \200\223 5 : t n e m t a e r T / h 2 1 g n d r o c c a g k / g m 5 2 : r e t s o Z r o i y a d / 5 x y a d s k e e w y r e v e f f h 4 2 h 2 1 x 5 . t n e m g m 0 0 9 h 2 1 h 4 2 y l i a d , 1 g m 0 0 8 s k e e w 3 \200\223 2 y a d \200\223 0 0 2 y a d / 5 x r o y r e v e t s u d a y r e v e : s i x a l y h p o r P g m 0 0 1 f e g n a h c o N g k / g m 0 1 \200\223 5 : t n e m t a e r T n o g m 0 0 2 h 2 1 j h 8 l a m r o N : s k e e w 3 x e p m S : r e t s o Z g k / g m 5 g m 0 0 4 g m 0 0 9 y a d e s o D y r e v e y r e v e l n e h t r o x 5 i f ) V I ( ) r i v o l c c n a g l a V O P ( ) V I ( e n n c y m a d n r i v o l c c n a G i i l c y c y x o D r i v o l c c A r i v o l c c A i i ) O P ( i i i l l a i b o r c i m C e d i s o e l c u n s e u g o l a n a c i t e h t n y S i t n A , y t i c x o t o r h p e n r e b m u n s e s a e r c n f d n a n s t n e m m o C o y l r a l u c i t r a P e s a e s i d i s t n e m m o C h t i f i d e s u n o i t a c d e m n o i t c a r e t n n i r u e n c l a c o y l l a i t n e t o P d e t a c o s s a w s r o t i b h n l r e h t o n i r a f r a w e v e o t s u o i r e s t n e i t a p a i i y l e r a r l a n e r i e t o n l h t i i e u D i e h t f w o i i y t i l a d o m y t i l a d o m n m / L m 0 2 \200\223 0 1 i g m 0 0 8 \200\223 0 0 4 e g n a h c o N e g n a h c o N e g n a h c o N h 4 2 H V V C R F G e s a H V V C s i s y l a i d o t g n d r o c c a t n e m y r e v e e s o D s i s y l a i d o t g n d r o c c a t n e m n i n m / L m 0 1 < n m / L m 0 1 < e g n a h c o N e g n a h c o N e g n a h c o N n n i i i i i i D P A C s a s a D P A C R F G e R F G e e s o D R F G e e s o D R F G e n y l i a d n m / L m 0 1 < l a m r o n % 0 0 1 r o t s u d a e s o D t s u d a e s o D s e m r e t f a n m / L m 0 1 < e g n a h c o N e g n a h c o N e g n a h c o N n i j i i j i i t 3 e s o d s a s a e s o D k e e w a s i s y l a d e s o D D H D H i n m L m 0 1 < y r e v e n m L m 0 1 < i n e h t y a d / g k / g m 6 i e g n a h c o N e g n a h c o N e g n a h c o N 3 \200\223 2 g m 0 0 2 \200\223 0 5 / / g k / g m 6 , s k e e w r o y l i a d f h 8 n m L m 0 2 \200\223 0 1 n m L m 0 2 \200\223 0 1 y r e v e i i y a d / g k / g m 5 1 n e h t / e g n a h c o N e g n a h c o N e g n a h c o N / 3 \200\223 2 g m 0 0 4 \200\223 0 5 g k / g m 5 1 , s k e e w r o y l i a d R F G e o t f R F G e o t h 8 n m L m 0 5 \200\223 0 2 n m L m 0 5 \200\223 0 2 y r e v e i i y a d / g k / g m 8 2 n e h t / e g n a h c o N e g n a h c o N e g n a h c o N g n d r o c c a / 3 \200\223 2 g m 0 0 4 \200\223 0 5 g n d r o c c a g k / g m 8 2 i i , s k e e w r o y l i a d f h 8 n e h t / g m 4 \200\223 3 n e h t t n e m y l r u o h - 2 1 g k / g m 6 t n e m h 8 y r e v e , e c n o g m 0 0 4 \200\223 0 0 2 h 4 2 r o g m 0 0 3 \200\223 0 0 2 n e h t y l i a d , s k e e w 3 \200\223 2 y r e v e t s u d a t s u d a y a d / g k / g m 0 6 y l r u o h - 2 1 g k g m 0 0 4 g m 0 0 2 \200\223 0 0 1 j f g m 0 0 4 \200\223 0 5 j l a m r o N y l r u o h - 2 1 y l r u o h - 2 1 l a m r o N g k / g m 0 6 , h 4 2 \200\223 2 1 h 4 2 r o e s o D e s o D : l a r O y l i a d r o : V I f f ) d e u n i t n o C e o z a n o c o t e K e o z a n o c i r o V l e o z a n o c a r t I l e o z a n o c u F l l t e n r a c s o F l a i b o r c i m ( l a i b l o a r g c n i m u f i i t t n n A A l 2 2 1 . c n o h p s o h P i s e o z a d m e v i t a v i r e d s e o z a i r T l e l b a T l i t n A d c a d n a i i i d n a , s i s o d c a n o m m o c l a t s i d e s u a c n a c r o h t i s i w o r r a m e n o B , c i l , a m e a l o v o p y h n c i r e t o h p m A a m e a l a k o p y h e s a e s i d n c i r e t o h p m a f s t n e m m o C h t i e s a e s i d i i i n o i s s e r p p u s w c x o t o r h p e n o b a t e m d n a i d e r r e f e r p s i i i w s t n e i t a p t n e i t a p l a m o s o p , ; s i s o d c a a i r u y l o p y l h g h s i r a l u b u t e r o m l a n e r l a n e r i i n i l i y t i l a d o m n m / L m 0 2 \200\223 0 1 i e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N R F G e s a H V V C e s o D s i s y l a i d o t g n d r o c c a t n e m n i y r e v e s e s o d r u o e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N i g k / g m 0 5 R F G e f d e d v d D P A C n i h 4 2 i i t s u d a e s o D n m / L m 0 1 < e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N n i i j s a e s o D D H o t n m L m 0 1 < e s o d e r i e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N g n d r o c c a , g k / g m 0 5 / i s l e v e n e h t l n m L m 0 2 \200\223 0 1 y r e v e i e g n a h c o N e g n a h c o N e g n a h c o N e g n a h c o N / g k / g m 0 5 h 4 2 R F G e o t n m L m 0 5 \200\223 0 2 y r e v e i e g n a h c o N e g n a h c o N e g n a h c o N e s o d e g n a h c o N / g n d r o c c a g k / g m 0 5 i h 2 1