depletion of blood glutathione ( gsh)has been documented in many clinical situations such as malnutrition , severe burn injury , human immunodeficiency virus ( hiv ) infection , and diabetes , both type 1 and type 2 . glutathione depletion may have adverse consequences in diabetic patients , independent of glycemic control , and it may weaken the defense against oxidative stress . this could cause damage to protein , dna , or membrane lipids and thus potentially lead to cell dysfunction in various tissues . in diabetes , increased oxidative stress is known to play a decisive role in the pathogenesis of vascular complications . the role of glutathione peroxidase ( gpo ) in cellular defense against oxidant attack has been discussed for many years . the red cell has been a central focus because it is thought to undergo a high endogenous rate of h2o2 production from hemoglobin autoxidation , which can be markedly increased in cells with unstable hemoglobins . in addition , the red cell is probably exposed to h2o2 from stimulated macrophages and , under certain circumstances , from pathogenic bacteria or malarial parasites . it is believed that the glutathione peroxidase enzyme , protects the erythrocyte against peroxides that are generated intracellularly or exogenously . the relative importance of gpo in defending the cell has been debated since the discovery of glutathione peroxidase . glutathione reductase plays an important role in protecting hemoglobin , red cell enzymes , and biological cell membranes against oxidative damage by increasing the level of reduced glutathione ( gssgr ) in the process of aerobic glycolysis . the enzyme deficiency may result in mild to moderately severe hemolytic anemia upon exposure to certain drugs or chemicals . permission from the ethical committee of qassim university , buraidah central hospital management were taken and verbal consent from study subjects on explanation of study objectives . blood samples used in this study were drawn using vacutainer system into heparin and edta anticoagulant , also urine specimens were collected from 100 adult diabetic patients attending buraidah central hospital , while 30 adult non- diabetic subjects who are apparently healthy , were selected at random as a control . in this study , test samples were collected from diabetic patients with fasting blood glucose level > 140 mg / dl and hemoglobin a1c ( hb a1c ) percentage > 8% . it is important to state that adequate measures were taken in making sure that the study controls and diabetic patients were not suffering from nephropathy . however study subjects with blood urea < 45 mg / dl , serum creatinine below 1 mg / dl , and microalbuminuria level below 30 mcg / mg were used for this study . the heparinized , and edta samples were labeled by serial number and the date of collections were noted . samples were mixed well and tested within one hour for hb a1c estimation , gpo , gr measurement , and reticulocyte count . after which the samples were tested using coulter machine ( sysmex company , japan ) to determine the hemoglobin ( hgb ) , red cell ( rbcs ) count , hematocrit ( hct ) , mean cell volume ( mcv ) , mean cell hemoglobin(mch ) and mean cell hemoglobin concentration ( mchc ) . the heparinized samples were centrifuged at 1000  g for 10 min and plasma was harvested to be tested for blood glucose level , blood urea , plasma creatinine and blood total and direct bilirubin . all reagents were obtained from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . hba1c was assayed using the hemolysate preparation , where the labile fraction is eliminated , hemoglobins are retained by a cationic exchange resin . is specifically eluted after washing away the hemoglobin a1a+b fraction ( hba1a+b ) , and is quantified by direct photometric reading at 415 nm . gsh was estimated by reducing ( gssg ) in the presence of nadph , which is oxidized to nadp . microalbumiuria was measured as the ratio of albumin to creatinine in urine samples using commercial kits from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . data were analyzed statistically using student t - test to see if there is any significant difference between the mean of patients and the mean controls results and also to correlate between hematology parameters and gsh , gpo , and gr using pearson correlation with significance less or equal to 0.05 level . permission from the ethical committee of qassim university , buraidah central hospital management were taken and verbal consent from study subjects on explanation of study objectives . blood samples used in this study were drawn using vacutainer system into heparin and edta anticoagulant , also urine specimens were collected from 100 adult diabetic patients attending buraidah central hospital , while 30 adult non- diabetic subjects who are apparently healthy , were selected at random as a control . in this study , test samples were collected from diabetic patients with fasting blood glucose level > 140 mg / dl and hemoglobin a1c ( hb a1c ) percentage > 8% . it is important to state that adequate measures were taken in making sure that the study controls and diabetic patients were not suffering from nephropathy . however study subjects with blood urea < 45 mg / dl , serum creatinine below 1 mg / dl , and microalbuminuria level below 30 mcg / mg were used for this study . the heparinized , and edta samples were labeled by serial number and the date of collections were noted . samples were mixed well and tested within one hour for hb a1c estimation , gpo , gr measurement , and reticulocyte count . after which the samples were tested using coulter machine ( sysmex company , japan ) to determine the hemoglobin ( hgb ) , red cell ( rbcs ) count , hematocrit ( hct ) , mean cell volume ( mcv ) , mean cell hemoglobin(mch ) and mean cell hemoglobin concentration ( mchc ) . the heparinized samples were centrifuged at 1000  g for 10 min and plasma was harvested to be tested for blood glucose level , blood urea , plasma creatinine and blood total and direct bilirubin . all reagents were obtained from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . hba1c was assayed using the hemolysate preparation , where the labile fraction is eliminated , hemoglobins are retained by a cationic exchange resin . is specifically eluted after washing away the hemoglobin a1a+b fraction ( hba1a+b ) , and is quantified by direct photometric reading at 415 nm . gsh was estimated by reducing ( gssg ) in the presence of nadph , which is oxidized to nadp . microalbumiuria was measured as the ratio of albumin to creatinine in urine samples using commercial kits from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . data were analyzed statistically using student t - test to see if there is any significant difference between the mean of patients and the mean controls results and also to correlate between hematology parameters and gsh , gpo , and gr using pearson correlation with significance less or equal to 0.05 level . permission from the ethical committee of qassim university , buraidah central hospital management were taken and verbal consent from study subjects on explanation of study objectives . blood samples used in this study were drawn using vacutainer system into heparin and edta anticoagulant , also urine specimens were collected from 100 adult diabetic patients attending buraidah central hospital , while 30 adult non- diabetic subjects who are apparently healthy , were selected at random as a control . in this study , test samples were collected from diabetic patients with fasting blood glucose level > 140 mg / dl and hemoglobin a1c ( hb a1c ) percentage > 8% . it is important to state that adequate measures were taken in making sure that the study controls and diabetic patients were not suffering from nephropathy . however study subjects with blood urea < 45 mg / dl , serum creatinine below 1 mg / dl , and microalbuminuria level below 30 mcg / mg were used for this study . the heparinized , and edta samples were labeled by serial number and the date of collections were noted . samples were mixed well and tested within one hour for hb a1c estimation , gpo , gr measurement , and reticulocyte count . after which the samples were tested using coulter machine ( sysmex company , japan ) to determine the hemoglobin ( hgb ) , red cell ( rbcs ) count , hematocrit ( hct ) , mean cell volume ( mcv ) , mean cell hemoglobin(mch ) and mean cell hemoglobin concentration ( mchc ) . the heparinized samples were centrifuged at 1000  g for 10 min and plasma was harvested to be tested for blood glucose level , blood urea , plasma creatinine and blood total and direct bilirubin . all reagents were obtained from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . hba1c was assayed using the hemolysate preparation , where the labile fraction is eliminated , hemoglobins are retained by a cationic exchange resin . hemoglobin a1c ( hba1c ) is specifically eluted after washing away the hemoglobin a1a+b fraction ( hba1a+b ) , and is quantified by direct photometric reading at 415 nm . gsh was estimated by reducing ( gssg ) in the presence of nadph , which is oxidized to nadp . microalbumiuria was measured as the ratio of albumin to creatinine in urine samples using commercial kits from randox laboratories ltd , ardmore , diamond road , crumlin , co. antrim , united kingdom bt29 4qy . data were analyzed statistically using student t - test to see if there is any significant difference between the mean of patients and the mean controls results and also to correlate between hematology parameters and gsh , gpo , and gr using pearson correlation with significance less or equal to 0.05 level . as shown in table 1 the level of gsh , gpo , gr , fasting blood sugar , total bilirubin in diabetic patients are highly significant ( p 0.001 ) in comparison with healthy controls , while the direct bilirubin shows a significant increase ( p 0.05 ) . total glutathione ( gsh ) , glutathione peroxidas ( gpo ) , glutathione reductase ( gr ) , fasting blood sugar ( fbs ) , total bilirubin , direct bilirubin , blood urea , serum creatinine , microalbuminuria ( means  s.e.m ) in diabetic patients ( n=100 ) and healthy controls ( n=30 ) . there is a significant decrease ( p 0.001 ) in hgb concentration and mch value ( p 0.05 ) in all diabetic patients in comparison with controls also the table shows highly significant increase in hb a1c , and reticulocyte count . comparative hematological values ( means  s.e.m ) in diabetic patients ( n=100 ) , and control ( n=30 ) . the data in table 3 shows significant correlation between low hgb concentration , gsh , gpo and gr deficiency at level 0.01 ( two tailed ) . also the data indicates that hgb concentration have high significant correlation with rbcs count , mcv , mch , and mchc at level ( 0.01 ) , while the prevalence of anemia constitutes 22% among diabetic patients as shown in figure 1 . correlation between hematology values , total glutathione ( gsh ) glutathione peroxidase ( gpo ) , and glutathione reducrase ( gr ) glutathione can be used to  detoxify  reactive oxygen species such as hydrogen peroxide h2o2 , a process in which glutathione is oxidized to the dimer glutathione disulfide in a reaction catalyzed by glutathione peroxidase . reduced glutathione in turn is regenerated from glutathione disulfide by glutathione reductase in a reaction requiring nadph as a cofactor . high level of exogenous or endogenous free radicals could lead to destruction of major components of cellular structure , including nucleic acids , proteins , amino acids , lipids , and carbohydrates and would affect various cell functions such as membrane function , metabolism and even gene expression , and they can cause a number of other pathological conditions as such , they are an important antioxidant defense in nearly all cells exposed to oxygen . the red cell has been a central focus of this research , because it is thought to undergo a high endogenous rate of production h2o2 from hemoglobin autoxidation , which can be markedly increased in cells with unstable hemoglobins . in addition , the red cell is exposed to h2o2 , superoxide , and no in under certain conditions like diabetes . chronic hyperglycemia might result in abnormal red blood cells , oxidative stress , and sympathetic denervation of the kidney related to autonomic neuropathy . these factors promote a hypoxic environment in the renal interstitium , which leads to impaired production of erythropoietin by the peritubular fibroblasts . inappropriately low erythropoietin level is an important cause of early anemia in patients with diabetes mellitus . in this study the erythropoietin deficiency factor has been excluded , because all patients were selected have no nephropathy or any renal problem the present study demonstrates that red cell gsh , gpo and gr are significantly reduced in uncontrolled diabetic patients without nephropathy , compared with age and sex matched group of healthy volunteers . the reduction of these enzymes is due to a decrease in gsh concentration which leads to an increase in oxidative stress . however , it is important to note that elevation of blood glucose level generates oxidative stress1 , which could contribute to increase in glutathione utilization . the deficiency of most potent antioxidant gsh , and gpo , gr enzymes leads to increase free radical such h2o2 , superoxide , and no which are major oxidant in diabetic oxidative stress . in figure 1 , 22% of diabetic patients are suffering from anemia due decrease in the hgb , rbcs count and hct as shown in table 1 . the deficiency of antioxidants lead to increase free radical such h2 o2 , superoxide , and no which are major oxidant in diabetes oxidative stress which could further lead to oxidation of red cell components , such as hgb and the cell becomes functionless and unable to carry oxygen . these red cells will be taken by reticuloendothelial system , which decreases the number of circulating rbcs and hgb concentration , which results to hemolytic anemia . the lifespan of red blood cells might be decreased in patients with diabetes mellitus , so red blood cells are affected by various disturbances in the hematopoietic milieu , such as chronic hyperglycemia and hyperosmolarity , and these disturbances lead to elevated internal viscosity and increased membrane rigidity in these blood cells , so that the number of red blood cell is significantly reduced . in comparison with previous cross - sectional study done by thomas et al shows that approximately 25% of patients who attended a diabetes clinic were anemic , but in this present study only 22% of the diabetic patients are anemic . it can be concluded that there is strong significant impact of oxidative stress ( low glutathione ) on glutathione peroxidase , glutathione reductase level could reduce hemoglobin concentration in diabetic patients , that means oxidative stress of diabetes mellitus is the possible cause of anemia in diabetic patients without nephropathy .
background : the oxidative stress is considered as major consequence of diabetes mellitus affecting red cell antioxidant enzymesaim : the present study was conducted to assess the impact of oxidative stress ( reduced glutathione ) on glutathione peroxidase , and glutathione reductse and prevalence of anemia among diabetic patients.materials and methods : the study involved 100 adult patients attending buraidah central hospital and 30 healthy controls . 
 blood samples were collected and analyzed for glutathione ( gsh ) concentration , glutathione peroxidase ( gpo ) , glutathione reductase ( gr ) , fasting blood sugar ( rbs ) , hemoglobin ( hgb ) , red cell count ( rbcs ) hematocrit ( hct ) mean cell volume ( mcv ) mean cell hemoglobin ( mch ) and mean cell hemoglobin concentration ( mchc ) and hemoglobin a1c . 
 blood urea , serum creatinine , and microalbuminuria were measured to exclude diabetes mellitus nephropathy.results:were obtained showed significant correlation between deficiency of glutathione peroxidase , glutathione reductase and deficient of glutathione among diabetics , which has significant correlation between low hemoglobin concentration ( females < 120 
 g / l , males < 130 
 g / l ) , also there is low concentration of red cell count and red cell indices ( mcv , mch and mchc ) . 
 the prevalence of anemia was 22% in diabetes patients.conclusion:it can be concluded that there is strong significant effect of oxidative stress ( reduced glutathione ) on glutathione peroxidase , glutathione reductase level these may reduce hemoglobin concentration in diabetic patients . 
 this means oxidative stress of diabetes mellitus is the possible cause of anemia in diabetics without nephropathy .