Diabetic Erythrocytes Research Articles

Doublet formation of diabetic erythrocytes as a model of impaired membrane viscous deformation

Erythrocyte deformation involves both viscous dissipation in the cell interior and viscoelastic motion of the cell membrane. Reports that describe reduced filterability of diabetic erythrocytes, altered response to oscillatory motion in a capillary-sized pipet, and impaired packing during centrifugation indicate a disturbance of red cell rheology in diabetes. We have selected conditions that minimize the macromolecule-mediated energy of attraction between erythrocytes and studied erythrocyte motion during doublet formation. Under such conditions, doublet formation frequency is strikingly reduced in diabetes. For nondiabetic erythrocytes the formation rate is 0.73 doublets per minute, whereas for diabetic erythrocytes the rate is 0.23 doublets per minute. In addition, mean velocity of doublet formation was found to be decreased to half of normal in diabetes. Completeness of doublet formation, regularly diminished when cell size of the two component cells was similar, was the same for diabetic and nondiabetic erythrocytes. Observation of several features of doublet formation gave a picture of the mechanical process. The initial cell making contact with the glass microscope slide was observed to remain fixed in position. The late arriving cell's ability to form a doublet was seen to decrease rapidly, apparently because it came to adhere to the glass surface. The attractive force between the cells overcomes the force of gravity, but cell deformation resistance slows doublet formation by balancing the tendency for cell-cell contact area to increase. An integral equation combining strain energy and viscous dissipation was applied to the doublet formation process. Slowing of doublet formation in diabetes appears to be produced by a doubling of resistance to rate of change of curvature of diabetic erythrocytes.

Characteristics of labile HbA1 in erythrocytes from normals and diabetics.

Incubation of erythrocytes from diabetic patients and non-diabetic persons in 0.15 mol/l saline at 37 degrees C was followed by a decrease in ion-exchange microcolumn determined HbA1 only during the first 4 h. The fraction removed was denoted as labile HbA1 and the remaining fraction as stable HbA1. Incubation in saline overnight at 20 degrees C did not completely remove the labile fraction. Incubation in saline overnight at 4 degrees C yielded only a slight decrease in labile HbA1. Incubation in saline containing various glucose concentrations (up to 100 mmol/l) produced increasing amounts of HbA1 during the first 3-4 h after which an equilibrium was reached. In both normal and diabetic erythrocytes incubated for 3-4 h we found a linear relationship between changes in labile HbA1 and glucose concentration. The degree of increase in labile HbA1 was the same in normals and diabetics and not dependent on pre-existing normal or moderately increased stable HbA1. In the blood from non-diabetics (n = 30) labile HbA1 was 0.5 +/- 0.3% (mean +/- SD) and in diabetics (n = 80) 1.6 +/- 0.8%. The correlation between labile HbA1 and simultaneously determined blood glucose was r = 0.72.

Electrophoretically determined haemoglobin A1 concentrations during short-term changes in glucose concentration.

In our experience, electrophoresis on agar gel is a very satisfactory alternative to the more widely used chromatographic methods for the determination of haemoglobin A1 (HbA1). Like the chromatographic method, the electrophoretic method is unable to detect any difference between the labile intermediate form of HbA1, which changes rapidly with acute changes in blood glucose level, and the more stable end-product, which reflects long-term glucose levels. In vitro at 37 degrees C the electrophoretically determined HbA1 concentration increases with increasing glucose concentration and with time in both normal and diabetic erythrocytes, but decreases to the preincubation concentration during further incubation of the erythrocytes in a glucose-free medium at 37 degrees C. Similarly, if normal or diabetic erythrocytes are incubated with isotonic saline before the HbA1 assay, the labile fraction is eliminated. In diabetics, the decrease in HbA1 concentration correlates with both the blood glucose level and the preincubation HbA1 concentration. Thus for HbA1 to be an accurate indicator of long-term glucose control in diabetic patients saline incubation of the erythrocytes may be necessary before HbA1 assay by the electrophoretic method, otherwise the assay results will also reflect recent changes in the blood glucose level.

Erythrocyte Spectrin Glucosylation in Diabetes

Because reduced erythrocyte deformability in diabetes might be mediated by increased noneniymatic glucosylation of membrane proteins, we have isolated and studied the degree of glucosylation of spectrin, the major protein of the inner membrane surface. Spectrin was recovered from the erythrocytes of seven age-and sex-matched diabetic and nondiabetic individuals. The thiobarbituric acid (TBA) method was used to measure the glucosylation of spectrin and of 10 mg of hemoglobin from the same ceils. Increased glucosylation of both spectrin and hemoglobin was found in diabetes. Glucosylation, expressed as a ratio of TBA absorbance to protein weight was nearly three times as high for spectrin as for hemoglobin. The ratio of TBA absorbance to hemoglobin content was examined and found to be curvilinear. A reaction model with both first and second order components was fitted to the data to obtain a reaction constant. When this model and constant were used to adjust TBA ab-sorbances, spectrin's glucosylation became less than double that of hemoglobin. Spectrin forms an inner membrane network responsible for returning deformed erythrocytes to their original shape. The reported normal response to stretching of diabetic erythrocytes suggests that increased glucosylation of spectrin fails to stiffen the membrane. Some other mechanism, such as reduced phosphorylation of spectrin and other membrane proteins, may be responsible for reduced deformability of diabetic erythrocytes. But spectrin's increased glucosylation might be responsible for the increased number of poorly deformable erythrocytes found among aged diabetic erythrocytes. It may produce this effect by inhibiting the membrane protein transglutamidation responsible for removal of aged erythrocytes.

The importance of determining irreversibly glycosylated hemoglobin in diabetics.

The concentration of glycohemoglobins (HbA1(a+b+c), HbA1,) was measured before and after incubation of normal and diabetic erythrocytes for 6 h at 37°C in saline. This procedure removes as much as 80-90% of the labile glucose-HbA0 adduct (labile HbA1,), thus allowing accurate estimation of irreversibly glycosylated hemoglobin (stable HbA1,). The concentration of HbA1 measured before such an incubation is total HbA1, (stable + labile). We determined the concentration of total, stable, and labile HbA1, in the same blood samples used to measure fasting plasma glucose (FPG) every day, for 4 consecutive days, in two groups of hospitalized insulin-treated diabetics. Group A subjects (N = 7) were type I, C-peptide negative, unstable diabetics, while group B subjects (N = 15) were type II, C-peptide positive, stable diabetics. Individual day-to-day variations of total HbA1, were wide in group A (Δ = 1.58 ± 0.14%), and slight in group B (Δ = 0.12 ± 0.01%; P < 0.001), paralleling similar plasma glucose fluctuations. Day-to-day variations of stable HbA1, were virtually absent not only in group B subjects with stable glycemic values (Δ = 0.08 ± 0.01%), but also in those of group A with marked glycemic instability (Δ = 0.07 ± 0.01%; P = NS). Day-to-day variations of labile HbA1, were marked in group A (Δ = 1.31 ± 0.14%), but negligible in group B (Δ = 0.15 ± 0.03%; P < 0.001). On admission, FPG correlated with labile HbA1, in group A (r = 0.89) and B (r = 0.71). FPG correlated with stable HbA1, in group B (r = 0.73) but not in group A subjects and with total HbA1, more closely in group B (r = 0.73) than in A (r = 0.61). A very close correlation was found between the concentration of total and labile HbA1 in subjects of group B (r = 0.82). In group B, fasting, post-breakfast, and mean daily plasma glucose values, determined every 3-6 days during the 2 mo before admission, significantly correlated both with total and stable HbA1 determined on admission, while in group A they did not. In group A, the correlation was significant when stable instead of total HbA1 was considered. We conclude that the significant fluctuations of total HbA1 reduce its value as an index of long-term control in unstable diabetics. On the other hand, a single determination of stable HbA1, totally independent of simultaneous blood glucose values, closely reflects blood glucose control over the previous 2 mo. We propose routine estimation of stable HbA1, which is simple and straightforward, to carry out follow-up studies of unstable diabetics.

Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine [ε-N(L-furoylmethyl)-L-lysine

Non-enzymatic glycosylation of erythrocyte membranes was studied using a non-radioactive and sensitive procedure for specific quantitation of lysine-bound glucose in proteins. About 2 nmol lysine-bound glucose/mg protein were found in ghosts from normal erythrocytes, and this value was about doubled in diabetic patients. In vitro incubation of normal ghosts with glucose gave rise to levels of lysine-bound glucose similar to those found in diabetics. There was a linear correlation between the amount of lysine-bound glucose of total hemoglobin and of membrane proteins. Membrane glycosylation also depended on the age of erythrocytes displaying significantly higher values in old cell populations.

Hematologic alterations in diabetes mellitus

Several hematologic abnormalities have been defined in patients with diabetes mellitus, despite the lack of classic hematologic pathologic findings in this condition. Studies of the erythrocyte and the formation of hemoglobin A1c have provided a means of documenting glycemia and a model reaction for diabetic sequelae through postsynthetic protein modification. Oxygen affinity has been noted to be abnormal in the diabetic erythrocyte, concomitant with a decreased concentration of inorganic phosphorus, glycosylation of the 2,3-diphosphoglycerate binding site or preexisting vascular disease. Red cell membrane viscosity has also been documented to be increased in the hyperglycemic subject. Abnormalities in the polymorphonuclear leukocyte have been described, involving the properties of adherence, random migration, chemotaxis, phagocytosis and killing. Certain metabolic abnormalities are also present in this cell type. The lymphocyte has been shown to have abnormal metabolic properties, mitogen responses and cell surface properties in diabetes both in animals and human subjects. Certain subpopulations of lymphocytes appear to be especially vulnerable to changes concomitant with diabetes mellitus. In vitro abnormalities of platelet behavior have been widely studied, although the in vivo significance of these findings remains controversial. Studies of the fluid phase of coagulation have suggested the existence of a hypercoagulable state in hyperglycemic subjects. The clinical significance of most of these findings remains to be defined. Nevertheless, the observation that many of the abnormalities described are reversible when hyperglycemia is corrected has given impetus to the development of improved systems of glucose “control” for diabetic patients.

Rapid changes in chromatographically determined haemoglobin A1c induced by short-term changes in glucose concentration.

Chromatographically determined haemoglobin A1c concentration was measured during short-term (1-24h) changes in glucose concentration in vitro and in vivo. In vitro at 37 degrees C the HbA1c concentration increased with glucose concentration and time both in normal and diabetic erythrocytes. In normal erythrocytes incubated in 20--100 mmol/l glucose, the increases in the HbA1c concentration were maximal after 4--6 h and then stable for the next 18--20 h. During the first hour, increases in the HbA1c concentration were linear with time and on average 0.034% HbA1c x h-1 x mmol/l glucose-1. In erythrocytes, after a rapidly produced increase (2 h), HbA1c decreased to preincubation concentrations during a further incubation of the erythrocytes in a glucose-free medium at 37 degrees C for 4--6 h. The mean rate of linear decrease was 0.017% x h-1 x mmol/l glucose-1. After incubation of erythrocytes in 100 mmol/l glucose for 24 h, 1.3% HbA1c remained stable for 6 h in saline. The rapid increase in HbA1c concentration, as determined by chromatography, was not due to stable HbA1c (ketoamine linked glucose) as no increase was found in the HbA1c concentrations determined by the thiobarbiturate method. In juvenile diabetics controlled by an artificial beta-cell, rapid changes of blood glucose concentration (up to 20 mmol/l) resulted in increases in HbA1c concentration of as much as 1.9% within 12 h (mean 1.1%). Rapid in vivo increases in HbA1c concentration were reversible by normalization of the blood glucose concentration. That rapid changes in HbA1c may occur in daily diabetic life was evidenced by differences in HbA1c concentration between blood samples from out-patient diabetics incubated in saline for 16 hours at 4 degrees C and 37 degrees C (range of differences 0.2--1.4% HbA1c). The differences correlated to the blood glucose concentration at the time of sampling blood for HbA1c determination. Thus, incubation of blood at a low glucose concentration prior to determination of the glycosylated haemoglobin concentration may overcome interference from rapidly produced HbA1c.

Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes.

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.

La Microangiopathy Diabetique

Diabetic microangiopathy is morphologically defined as a thickening of the basement membrane of the capillaries. Its clinical manifestations are essentially the retinopathy (the four evolutionary stages of which are described) and the glomerular nephropathy, extending from the diffuse type to the classical nodular one. Various etiopathogenic hypotheses have been proposed, the most important of which are: 1. The diabetic erythrocytes deliver less easily oxygen to the tissues; this is due to a deficiency in organic phosphate compounds (mainly diphosphoglycerate) regulating the hemoglobin dissociation curve, and to an ab normal accumulation of the dysfunctional glycosylated hemoglobin A1c. 2. The diabetic endothelial cell may suffer from an excessive sorbitol accumulation and from inositol (which is a growth factor) deficiency. 3. Basement membrane of diabetics is chemically and functionally altered, particularly in its glycoprotein component. Prolonged hyperglycernia in itself can explain these three alterations. Consequently, the major aim of the treatment of diabetes is to prevent the development of the microangiopathy by ensuring continuously an optimal blood glucose con tro1

Red cell age-related changes of hemoglobins AIa+b and AIc in normal and diabetic subjects.

The minor hemoglobin components, hemoglobin AIa+b and hemoglobin AIc, were measured in the 10% youngest and 10% oldest erythrocytes of 15 normal and 14 diabetic subjects. Erythrocyte fractions were obtained by centrifugation in isopyknic concentrations of dextran: 28.5% of 40,000-mol wt dextran yeilded the 10% lightest of young cells, and 30.5% dextran provided the 10% heaviest or old erythrocytes. Both normal and diabetic erythrocytes contain increased amounts of Hb AIa+b and Hb AIc in old as compared to young cells. In normal subjects, young cells contained 1.2+/-0.2%, and old cells contained 1.8+/-0.4% Hb AIa+b. Corresponding values for diabetic cells were 1.7+/-0.6 and 2.6+/-0.9%. Hb AIc increased from 3.1+/-0.8 to 6.0+/-1.1% in normals and from 5.1+/-2.1 to 10.1+/-3.7% in diabetics. The results indicate that both cell age and diabetes are significant determinants of the amounts of Hb AIa+b and Hb AIc.