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 .