Diabetic Erythrocytes Research Articles

Oxidation of Spectrin and Deformability Defects in Diabetic Erythrocytes

We reasoned that de novo oxidative damage, as a result of increased protein glycosylation, could participate in the mechanisms whereby diabetic erythrocytes acquire membrane abnormalities. To examine this hypothesis, the extent of erythrocyte membrane protein glycosylation and the oxidative status of spectrin, the major component of the erythrocyte membrane skeleton, were examined. Labeling erythrocyte membranes with [3H]borohydride, which labels glucose residues bound to proteins, revealed that several proteins were heavily glycosylated compared with nondiabetic erythrocyte membranes. In particular, the proteins beta-spectrin, ankyrin, and protein 4.2 were the most glycosylated. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis of diabetic erythrocyte membranes did not reveal any quantitative or qualitative abnormalities in spectrin or other membrane proteins, examination of spectrin oxidative status by amino acid analysis and with cis-dichlorodiammineplatinum(II) (cDDP), a chemical probe specific for protein methionine and cysteine residues, demonstrated that the diabetic spectrin was oxidatively damaged: spectrin from diabetic subjects contained 35% less methionine (P less than 0.002), 15% less histidine (P less than 0.006), and a twofold increase in cysteic acid (P less than 0.001) compared with normal spectrin. Diabetic spectrin bound 32% less cDDP than normal spectrin (P less than 0.001); the lowest cDDP binding was observed with spectrin from insulin-dependent diabetic subjects. The extent of cDDP binding to diabetic spectrin correlated moderately and inversely with glycosylated hemoglobin (GHb) levels (n = 12, r = -0.727). Erythrocyte deformability, measured by ektacytometry, was decreased between 5 and 23% of control measurements (average of approximately 10%) in 21 of 32 diabetic subjects surveyed.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of spectrin and deformability defects in diabetic erythrocytes

Oxidation of spectrin and deformability defects in diabetic erythrocytes

Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics.

A remarkable increase in the permeability of erythrocyte ghosts and liposomal membranes composed of erythrocyte lipids from aged diabetics was revealed by measuring [14C]glucose leakage. There were no significant differences in the contents of free cholesterol or phospholipids, or in the cholesterol/phospholipid ratio between diabetic and normal erythrocyte membranes, but significantly higher amounts of unsaturated fatty acids, arachidonic acid and docosahexaenoic acid were observed in the erythrocyte membranes of diabetics. Reconstituted liposomes prepared from aged diabetic erythrocyte lipids were highly susceptible to superoxide-induced oxidative stress. Vitamin E was highly effective in suppressing the peroxidative lysis of liposomes composed of diabetic erythrocyte lipids. The effect of superoxide dismutase (SOD) on the inhibition of peroxidation of unsaturated lipids within liposomal membranes was less than that of vitamin E.

Erythrocyte membrane lipid peroxidation and glycosylated hemoglobin in diabetes.

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vivo membrane lipid peroxidation in erythrocytes of diabetic subjects and its possible relationship with hyperglycemia. Lipid peroxidation was assessed in fresh, untreated erythrocytes by quantitating thiobarbituric acid reactivity and an adduct of phospholipids and malonyldialdehyde (MDA), an end product of lipid peroxidation, with thin-layer chromatography of lipid extract of diabetic erythrocytes. There was a significantly increased membrane lipid peroxidation in diabetic erythrocytes compared with nondiabetic erythrocytes. The degree of membrane lipid peroxidative damage in erythrocytes was significantly correlated with the level of glycosylated hemoglobin, an index of mean glucose level for the preceding 3-4 mo. This suggests that peroxidation of membrane lipids and accumulation of MDA occurs in erythrocytes of diabetic patients.

Impairment of glutathione metabolism in erythrocytes from patients with diabetes mellitus

The metabolism of glutathione and activities of its related enzymes were studied in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM). A decrease in the levels of the reduced form of glutathione and an increase in the levels of glutathione disulfide were found in erythrocytes of diabetics. To elucidate these changes in the levels of glutathione, synthetic and degradative processes were studied. The activity of γ-glytamylcysteine synthetase was significantly lower in diabetics than in normal controls. The activity of glutathione synthetase of each group was the same. The rate of outward transport of glutathione disulfide in diabetics decreased to approximately 70% of that of normal controls. The activity of glutathione reductase decreased in diabetics. These data suggest that the decrease in the levels of reduced form of glutathione in erythrocytes of diabetics is brought about by impaired glutathione synthesis and that the increase in the levels of glutathione disulfide is brought about by the decreased transport activity of glutathione disulfide through the erythrocyte membrane together with a decrease in the activity of glutathione reductase. These data also suggest that the impairment of glutathione metabolism weakens the defense mechanism against oxidativve stress in erythrocytes of diabetics.

Reversible sodium pump defect and swelling in the diabetic rat erythrocyte: effects on filterability and implications for microangiopathy.

We have found a defect in the ouabain-sensitive Na+, K+-ATPase (Na+ pump, EC 3.6.1.37) of erythrocytes from streptozocin diabetic rats. This defect was accompanied by an increase in cell volume and osmotic fragility and a decrease in the cytosolic K+/Na+ ratio. There was also a doubling in the time needed for diabetic erythrocytes to pass through 4.7-micron channels in a polycarbonate filter. Our data are consistent with a primary defect in the erythrocyte Na+ pump and secondary changes in cell volume, osmotic fragility, K+/Na+ ratio, and cell filterability. All were reversed or prevented in vivo by insulin or the aldose reductase inhibitor Sorbinil. Protein kinase C agonists (phorbol ester and diacylglycerol) and agonist precursor (myoinositol) reversed the Na+ pump lesion, suggesting that protein kinase C-dependent phosphorylation of the 100-kDa subunit regulates Na+ pump activity and that insulin can influence erythrocyte protein kinase C activity. Ouabain inhibition of the erythrocyte Na+ pump also produced increases in cell size and reductions in rates of filtration. Theoretical treatment of the volume changes also predicts reduction in filterability as a consequence of cell swelling. We suggest that enlarged erythrocytes could play a role in the evolution of the microvascular changes of diabetes mellitus.

Decrease of the inhibition of lipid peroxidation by glutathione-dependent system in erythrocytes of non-insulin dependent diabetics.

The inhibition of lipid peroxidation of erythrocyte membranes by glutathione-dependent protection was studied in patients with non-insulin dependent diabetes mellitus. Incubation of red cells from diabetics with 1.5 mM t-butyl hydroperoxide resulted in a lipid peroxidation increase greater than that of normal controls. Glutathione-dependent and glutathione-independent protection against oxidative damage was examined using an artificial system, in which erythrocyte ghosts were incubated with t-butyl peroxide and dialysed hemolysate in the presence or the absence of 2 mM glutathione. The glutathione-dependent protection of hemolysate from diabetics was approximately 70% of that from normal controls. The results suggest that decrease in glutathione-dependent protection against lipid peroxidation, along with decrease in glutathione levels, increases oxidative damage in erythrocyte membranes taken from diabetic patients.

1-anilino-8-naphthalenesulphonate binding parameters in red cell membranes. Does diabetes mellitus affect cell membrane dynamics?

In this study we report the use of 1-anilino-8-naphthalenesulphonate as a fluorescence probe to investigate the properties of plasma membranes derived from normal and diabetic red blood cells. The binding of 1-anilino-8-naphthalenesulphonate to diabetes-affected erythrocyte membranes, as compared with controls, was measured by means of fluorescence polarization and fluorescence titration techniques. These measurements demonstrated anomalous 1-anilino-8-naphthalenesulphonate binding to pathological red cell membranes. The amount of 1-anilino-8-naphthalenesulphonate bound to diabetic erythrocyte membranes was greater than that of controls. This fluorometric study indicated that the outer monolayer binding of 1-anilino-8-naphthalenesulphonate was markedly augmented in the erythrocyte membranes of diabetic patients. Significant correlations were found between 1-anilino-8-naphthalenesulphonate binding parameters and membrane lipid composition. The correlation between these binding parameters and non-enzymatic protein glycation was poor or moderate. Though the fluorescence intensity and emission maximum were quite similar in both groups investigated, binding studies revealed that there were approximately 1.2 times the number of 1-anilino-8-naphthalenesulphonate binding sites and a 33% increase in the KD value in diabetic membranes, suggesting significant differences in the environment of the 1-anilino-8-naphthalenesulphonate binding sites in these two groups of patients. The results presented in this report indicate that (a) 1-anilino-8-naphthalenesulphonate is a sensitive probe of membrane architecture alterations, and can be used to elucidate the perturbating effects of sterols in membrane systems, and (b) that significant differences in membrane dynamics exist between normal and diabetic red cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased acylation of phosphatidylcholine in diabetic rat erythrocytes

Decreased acylation of phosphatidylcholine in diabetic rat erythrocytes

Alterations in organization of phospholipids in erythrocytes as factor in adherence to endothelial cells in diabetes mellitus.

Erythrocytes from patients with diabetes mellitus exhibit increased adherence to cultured human vascular endothelial cells. We investigated the alterations in erythrocyte surface characteristics that may contribute to their abnormal adherence. The organization of phospholipids in the lipid bilayer, as determined by phospholipase A2 treatment and chemical labeling with fluorescamine and trinitrobenzene sulfonic acid (TNBS), is altered in erythrocytes from diabetic patients. Specifically, 12-18% of phosphatidylserine in diabetic erythrocytes (n = 25) is accessible to phospholipase A2 hydrolysis and TNBS labeling, compared to none in normal subjects. These results suggest either a loss in lipid asymmetry or in vivo destabilization of erythrocyte membranes in diabetic patients, causing increased accessibility to phospholipase A2 degradation. The dye merocyanine 540 (MC-540), which is sensitive to the packing of lipids in the bilayer of the membrane, revealed more binding and fluorescence in erythrocytes from diabetic patients than in those from normal subjects. On flow cytometric analysis, 64.5 +/- 17.0% red blood cells (RBCs) in diabetic patients, compared to 35.1 +/- 25.9% RBCs in normal subjects, showed positive MC-540 binding, indicating significant (P less than .001) differences in the packing of lipids in the external leaflet of the bilayer. The results of our study suggest that a loss of lipid asymmetry and/or less ordered packing in the outer leaflet of the diabetic erythrocyte membrane may be responsible for the increased propensity of erythrocytes to adhere to vascular endothelium.

Sodium Transport in Erythrocytes in Postpartum Gestational Diabetes

Sodium Transport in Erythrocytes in Postpartum Gestational Diabetes

Correction by pentoxifylline of the abnormal fluorescence polarization of erythrocyte membranes from diabetic patients.

Erythrocytes from diabetic patients show abnormal rheology. Pentoxifylline, a methylxanthine, improves the abnormal deformability of diabetic erythrocytes, but its mechanism of action remains unclear. We have studied the effect of pentoxifylline on the lipid order of erythrocyte membranes from controls and patients with Type I diabetes. We studied the structural organization of membrane lipids in individual erythrocyte ghosts by fluorescence polarization using a cell sorter. Fluorescence polarization values (P) for 17 controls (P = 0.244) and 20 diabetic patients (P = 0.215) were significantly different. Pentoxifylline added in vitro had no effect on normal membranes, but significantly increased at 10(-5) mol X l-1 (P = 0.233), and normalized at 10(-4) mol X l-1 (P = 0.243), the P value of membrane ghosts from diabetics.

Reduction of erythrocyte (Na+-K+)ATPase activity in type 1 (insulin-dependent) diabetic subjects and its activation by homologous plasma.

The (Na+-K+)ATPase and (Mg2+)ATPase activities of erythrocyte membranes of Type 1 (insulin-dependent) diabetic patients were found to be significantly reduced compared to matched controls (p less than 0.005). On the contrary, erythrocyte Na+ and K+ contents were similar in diabetic patients and in normal subjects. When erythrocyte membranes from diabetic patients were incubated with their own plasma, a significant increase was observed in sodium-potassium ATPase activity (p less than 0.005), whereas (Mg2+)ATPase activity was not affected. The plasma stimulatory effect showed saturation kinetics. Maximum average stimulation was 96% (+/- 21.3). A similar stimulation pattern, although more limited in extent (maximum 48.3% +/- 12.2), was found when erythrocyte membranes from normal subjects were incubated with diabetic plasma. Normal plasma exhibited a modest stimulatory effect on erythrocyte (Na+-K+)ATPase activity. Similar stimulatory effects by diabetic plasma were observed on a (Na+-K+) ATPase preparation from beef heart. It is proposed that diabetic plasma contains a specific (Na+-K+)ATPase activator in a higher concentration than normal plasma. This may explain why a normal cellular electrolyte content was found in diabetic erythrocytes in spite of a reduced Na+-K+ pump activity. Purification experiments indicate that the plasma activator is a protein with a molecular weight greater than 50,000. Both the (Na+-K+)ATPase activity and the stimulatory effect of diabetic plasma were not influenced by the metabolic control, since they did not correlate significantly with fasting blood glucose and daily insulin dosage. Moreover, no correlation was found with duration of diabetes or age at diagnosis of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of trace and bulk elements in plasma and erythrocytes of diabetic pregnant women by pixe method

Proton-Induced X-ray Emission (PIXE) analysis of blood samples from diabetic pregnant women was carried out. Elements S, Ca, P, K, Cl, Fe, Zn, Cu, Rb and Br were detected in red blood cells, while S, Ca, P, K, Cl, Fe, Zn, Cu, Ni, Br in the plasma. The concentrations of P, S, Ni, Cu were found to be higher, while those of K, Fe, and Zn were lower in diabetic plasma than in controls. Significantly higher concentrations were measured for P, S, Cl, Fe, Zn and Rb in diabetic erythrocytes compared to normals. Statistical evaluation of the results also indicated significant alteration in the changes of concentrations throughout the pregnancy. Diabetes also resulted in changes in most of the correlations observed in normal pregnancy between the concentrations of elements.

Evaluation of sodium and potassium pump activity and number in diabetic erythrocytes.

The Na+,K+-ATPase (= Na pump), which produces the concentration gradient of Na+ and K+ across the cell membrane, was studied in diabetic erythrocytes. The activity and number of Na pumps, which are functional and quantitative expression of the Na+,K+-ATPase in erythrocytes, were measured by ouabain-sensitive 42K or 43K influx and by [3H]ouabain binding, respectively. The turnover rate of the pumps was calculated from the two measurements to evaluate in situ activity of the pump. The Na pump activity was found to be higher in the diabetic (193 +/- 12 nmol K+/h . 10(9) cells) than in the normal group (164 +/- 6) (P less than 0.05), though the affinities of the pump for extracellular K+ were not different. The number of Na pumps and the Kd of the pumps for ouabain in the diabetic group were not significantly different from those in the normal group (354 +/- 12 site/cell and 4.33 +/- 0.20 nM). The turnover rate of the diabetic group tended to be higher than that of the normal group. The rate was about one third of the molecular activity reported for Na+,K+-ATPase under optimum conditions. These results indicate that the enzymatic properties as well as the number of Na pumps were not altered in diabetic erythrocytes despite the increased Na pump activity. Therefore the increased activity of the erythrocyte Na pump in diabetes mellitus suggests an increase of cation permeability associated with a possible disorder in the diabetic membrane.

Changes in fluidity and composition of erythrocyte membranes and in composition of plasma lipids in type I diabetes.

Changes in the fluidity and composition of human erythrocyte membranes and in the composition of plasma in Type I (insulin-dependent) diabetes were investigated. The increased microviscosity of diabetic erythrocyte membranes provide unambiguous proof of the structural deterioration of erythrocyte membranes in diabetes. It seems most likely that enhancement of the membrane cholesterol/phospholipid ratio is the main reason for decreased membrane fluidity in diabetes. A distinct correlation between membrane cholesterol/phospholipid ratio, plasma cholesterol content and membrane fluidity was found. Composition and structural changes in erythrocyte membranes and compositional changes in plasma lipids may contribute to the development of diabetic complications in diabetes.

Possible participation of adenosine 5′-diphosphate, arachidonic acid and paf-acether in the platelet pro-aggregating effect of uncontrolled insulin-dependent diabetic erythrocytes in vitro

The red blood cells (RBC) from uncontrolled insulin-dependent diabetics (U.IDD) induce aggregation of platelets from control subjects. This effect was not observed when using platelets made unsensitive to adenosine 5'-diphosphate (ADP), arachidonic acid (AA) or paf-acether. Since RBC from U.IDD are less deformable than those from control subjects, we treated normal RBC in vitro with glutaraldehyde. These rigid RBC were used to study aggregation of normal platelets and of those unsensitive to ADP, AA or paf-acether. Results obtained with glutaraldehyde-treated RBC were comparable to those obtained with RBC from U.IDD, i.e. aggregation was obtained with untreated platelets but not with platelets unsensitive to ADP, AA or paf-acether. It is hypothesized that aggregation of control platelets induced by RBC from U.IDD is purely mechanical at its origin but is ultimately mediated by ADP, AA or paf-acether.

High glucose concentrations partially release hexokinase from inhibition by glucose 6-phosphate.

The phosphorylation of glucose by human erythrocyte hexokinase follows classical Michaelis-Menten kinetics; hexokinase manifests maximum activity at 5 mM glucose, and no further increase in activity can be measured at higher glucose concentrations. However, the erythrocytes of diabetics and normal erythrocytes incubated with high concentrations of glucose contain increased concentrations of glucose 6-phosphate. To elucidate the mechanism of accumulation of glucose 6-phosphate when erythrocytes are exposed to high glucose concentrations, hexokinase activity was examined in the presence of naturally occurring inhibitors, such as glucose 1,6-bisphosphate, 2,3-diphosphoglycerate, ADP, and glucose 6-phosphate at physiological concentrations. Without inhibitors or in the presence of glucose 1,6-bisphosphate,2,3-diphosphoglycerate, and ADP, maximum hexokinase activity was observed at 5 mM glucose concentration. On the contrary, in the presence of glucose 6-phosphate, hexokinase activity increased at glucose concentrations greater than 5 mM; inhibition by glucose 6-phosphate was partially competitive with glucose. The relief by glucose of glucose 6-phosphate inhibition of hexokinase is a possible explanation of the increased glucose 6-phosphate level in diabetic erythrocytes.

Nonuniform loss of membrane glycoconjugates during in vivo aging of human erythrocytes: Studies of normal and diabetic red cell saccharides

Changes occurring in membrane saccharides during the in vivo aging of normal human erythrocytes have been evaluated after the fractionation of the red cells into five age groups by density gradient centrifugation. The glycoconjugate fractions studied included sialoglycoproteins, macroglycolipids, low-molecular-weight glycolipids, and Band 3 glycoproteins. All of the carbohydrate constituents of the membrane were found to decrease relative to the total ghost protein as a function of cell age, with the most substantial losses occurring in the macroglycolipids (50%) and Band 3 glycoprotein (30%); the smallest changes were observed in the sialoglycoproteins (13%). No preferential loss of sialic acid or other peripheral sugars was found, making unlikely the importance of glycosidase action in the removal of sugars from the membrane. It is suggested that the changes observed in the composition of the ghosts during aging are best explained by a loss of membrane segments enriched in glycoproteins and glycolipids and deficient in internally located molecules such as spectrin. Analyses were also performed on the glycoconjugate fractions from diabetic erythrocytes separated according to cell age. These erythrocytes, which had glycosylated hemoglobin values twice those of normals, had somewhat smaller amounts of membrane-bound carbohydrate. The difference between diabetic and normal erythrocytes was greatest when young cells were examined (diabetic to normal = 0.93), suggesting that the known increased turnover of red cells in diabetes leads to an early loss of membrane constituents.

Evidence for increased peroxidative activity in muscles from streptozotocin-diabetic rats.

The ability of cardiac and skeletal muscles from diabetic rats to metabolize superoxide and hydrogen peroxide was determined by the activities of superoxide dismutase (SOD) and catalase, respectively. Male and female Sprague-Dawley rats, 43 days old, were made diabetic with a single intravenous injection of streptozotocin (70 mg/kg body weight). On the 80th day after injection the blood glucose concentration of these rats was increased fourfold, and the plasma insulin concentration was decreased four- to fivefold compared to controls. Body weights of male diabetic rats were 61% and those of female diabetic rats were 66% of their ad libitum-fed controls. The seven different skeletal muscles examined weighed less in the diabetic rats than in controls of the same age and body weight. The hearts of the diabetic rats weighed more than those of controls of the same age and body weight. Comparison to the body weight controls allowed the distinction of specific effects due to lack of insulin from effects due to retardation in muscle growth. Increased catalase activity in all muscles examined from diabetic rats (plantaris, gastrocnemius, and heart) suggested a response in catalase activity similar to that of starved rats. SOD activity was not altered in the diabetic rat skeletal muscles and erythrocytes, but was somewhat decreased in the heart.