the ratio of copper ( cu ) to other dietary components ( e.g. , zinc ( zn ) , iron , sulfate , and molybdenum ) may be as important as the actual cu levels in the diet .
cu / zn ratios may be important to the adequate metabolism of cholesterol , with low ratios resulting in hypercholesterolemia .
previous studies have focused on acute severe cu deficiency , which is relatively rare in humans and animals on typical varied diets .
the determination of cu needs and marginal deficiency is complicated by the fact that while cu deficiency does not necessarily lower the level of cu - dependent enzymes , it does significantly lowers their activity .
superoxide dismutase ( sod ) functions as an antioxidant by catalyzing the conversion of superoxide radicals ( free radicals or reactive oxygen species ( ros ) ) to hydrogenperoxide , which can subsequently be reduced to water by other antioxidant enzymes .
superoxide radicals may react with other ros such as nitricoxide to form highly toxic species like peroxynitrite , in addition to its direct toxic effects .
peroxy nitrite reacts with the tyrosine residues in proteins resulting with the nitrotyrosine production in plasma proteins , which is considered as an in direct evidence of peroxynitrite production and increased oxidative stress .
although nitrotyrosine was not detectable in the plasma of the healthy controls , nitrotyrosine was found in the plasma of all type 2 diabetic patients ( type 2 diabetes mellitus ( t2 dm ) ) examined .
previous studies correlated plasma nitrotyrosine values with plasma glucose concentrations and found a significant positive correlation .
furthermore , exposure of endothelial cells to high glucose level leads to an augmented production of superoxide anion , which may quench nitric oxide level resulting in impaired endothelial functions , vasodilation , and delayed cell replication .
alternatively , superoxide can be dismutated to a much more reactive hydrogen peroxide , which through the fenton reaction can then lead to a highly toxic hydroxyl radical formation .
two forms of sod contain cu : i ) cu / zn - sod is found within most cells of the body , including red blood cells , and ii ) extracellular ( ec)-sod is a cu - containing enzyme found in high levels in the lungs and low levels in blood plasma . almost all of the cu in our bodies is bound either to transport proteins ( ceruloplasmin and cu - albumin ) , storage proteins ( metallothioneins ) , or cu containing enzymes .
intracellular metallothionine normally stores little cu providing protection from the harmful effects of free cu .
ceruloplasmin may function as an antioxidant in two different ways : by binding to cu , ceruloplasmin prevents free cu ions from catalyzing oxidative damage .
the other way is through the oxidation of ferrous iron by ceruplasmin , facilitating iron load into its transport protein , transferrin , and preventing free ferrous ions from participating in harmful free radical generating reactions .
major reason for the decreased sod activity is the glycosylation of cu / zn - sod which has been shown to lead to enzyme inactivation both in vivo and in vitro .
also cu / zn - sod cleavage and release of cu 
 in vitro resulted intransition metalcatalyzed ros formation .
erythrocyte cu / zn - sod activity correlated inversely with indices of glycemic control in dm patients .
however , red cell cu / zn - sod activity has also been found to be decreased in dm patients .
glycation may decrease cell - associated ec - sod , which could predispose to oxidative damage .
earlier reports found decreased red cell cu / zn - sod activity in dm patients with retinopathy compared to dm patients without microvascular complications and nondiabetic control subjects .
this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university .
ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) .
type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study 
 patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . 
 
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) .
patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital .
the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs .
all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity .
the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92  8.9 years ) .
the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy .
a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting .
two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay .
the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection .
all assays were obtained by running duplicates for the test , control , and the standard .
random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio .
no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements .
a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution .
urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve .
urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution .
cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame .
serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) .
serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) .
glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) .
statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression .
this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university .
ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) .
type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study 
 patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) . 
 
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) .
patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital .
the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs .
all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity .
the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92  8.9 years ) .
the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy .
a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting .
two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay .
the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection .
all assays were obtained by running duplicates for the test , control , and the standard .
random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio .
no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements .
a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution .
urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve .
urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution .
cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame .
serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) .
serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) .
glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) .
statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression .
all groups were closely age - matched , and the two diabetes groups were well - matched for duration of disease [ table 1 ] .
microabuminuric diabetic group shows a significant increase in the mean urinary cu / creatinine ratio when compared with controls ( p < 0.05 ) , while normoal buminuric diabetic patients shows an insignificant increase , p > 0.05 in mean urine cu / creatinine ratio when compared with controls [ table 2 ] .
urinary copper excretion and serum superoxide dismutase enzyme levels in microal buminurics , normoal buminurics , and control subjects serum sod was significantly decreased in the diabetes microal buminuric group compared to the control group ( p < 0.05 ) , such a significant correlation was not seen with the diabetes normoal buminuric group [ figure 1 and table 2 ] .
studies showed a reduction in erythrocyte sod and catalase ( cat ) activities in subjects with impaired glucose tolerance ( igt ) , early hyperglycemia , and type 2 dm patients .
however , other studies showed that the activities of these enzymes were within normal range in t2 dm patients in poor glycemic control .
ec - sod activity was found to be similar in t1 dm patients despite some what higher plasma ec - sod levels .
red cell cu / zn - sod activity was similar in t1 dm and t2 dm patients compared to normal subjects , irrespective of microvascular complications .
leukocyte sod activity was similar between type 2 dm patients and healthy control subjects , despite increased lipidperoxidation and decreased ascorbate levels . while the role of adequate cu levels in maintaining cardiovascular health is well - established , there are still inconsistent data about the correlation of cu with persisting hyperglycemia .
some studies showed an elevation of serum cu , while other studies showed a significant reduction of cu in diabetes .
many researchers have considered this elevation of serum cu to play a role in the pathogenesis of cardiovascular disease , although other researchers have strongly disagreed with this hypothesis .
an animal study , however , seems to have explained this relationship between cu levels and cardiovascular disease .
this study examined the effects of diet - induced atherosclerosis on the cu levels and status of numerous tissues .
it was found that serum cu levels increase significantly , while aorta and liver cu levels decrease significantly , in rats with experimental atherosclerosis .
so instead of assuming that these elevated cu levels contribute to the formation of atherosclerosis , these researchers examined the effects of increasing the dietary cu levels in these animals .
administration of additional cu resulted in a further increase in serum cu , a significant decrease in serum cholesterol , and an increase and normalization in aorta and liver cu levels .
however , instead of increasing the incidence of atherosclerosis , additional cu significantly decreased the incidence of atherosclerosis in the aorta and coronary arteries .
further , it has been shown that excess dietary cholesterol causes cardiovascular disease by lowering the absorption of cu , an effect that is preventable by increasing the cu level in the diet .
however , increased urinary albumin loss has been postulated to be a marker of a generalized increase in vascular permeability , which might predispose to greater penetration into the arterial wall of atherogenic lipoprotein particles .
urinary cu concentrations significantly increased only in microal buminuric patients , similar results were found by , but with macroal buminuric diabetic patients . in diabetic patients with advanced nephropathy
, urinary cu excretion may be due to dissociations from both cu - albumin and ceruloplasmin - cu complexes filtered through the damaged glomerulus .
overloading of urinary cu to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy .
therefore , regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy .
however , there is still a lot to know about the mechanism of cu homeostasis at the cellular level .
if reduction of serum cu can be shown to have a protective effect against oxidative stress in dm , this may have a direct impact on the use of cu chelators as a safe therapeutic modality in diabetes .
 background : copper ( cu ) is essential both for its role in antioxidant enzymes , like cu / zinc ( zn ) superoxide dismutase ( sod ) and ceruloplasmin , as well as its role in lysyl oxidase , essential for the strength and integrity of the heart and blood vessels . with such a central role in cardiovascular health , cu has been generally overlooked in the debate over improving our cardiovascular health . 
 cu deficiency has produced many of the same abnormalities present in cardiovascular disease . 
 it seems almost certain that cu plays a large role in the development of this killer disease , not because of its excess in the diet , but rather its deficiency.aim:this study was undertaken to investigate the cardiovascular effects of cu deficiency on the activity of sod in patients with type 2 diabetes mellitus ( t2 dm ) with and without diabetic nephropathy.materials and methods : fifty - five patients with t2 dm were recruited in this study which were divided into two subgroups based on the presence of microalbuminuria , the first group ( microal buminuric group , n = 31 ) had a microalbuminuria between 30 and 299 g / mg . 
 the second group ( normoal buminuric group , n = 29 ) had an albumin level less than 30 g / mg . 
 the two diabetic groups were compared to the control group ( n = 37).results : the results of our study showed a significant reduction in the levels of sod enzyme associated with an increased urinary cu excretion in microalbuminuric group compared to the control group at p < 
 0.05.conclusions:the current study illustrates that the regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy . 
Introduction
Materials and Methods
Study protocol and participants
Blood samples
Urine samples
Parameters of the study
Statistical analysis
Results
Discussion
Conclusion