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 β-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 < 0.002), 15% less histidine (P < 0.006), and a twofold increase in cysteic acid (P < 0.001) compared with normal spectrin. Diabetic spectrin bound 32% less cDDP than normal spectrin (P < 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 ∼10%) in 21 of 32 diabetic subjects surveyed. Diabetic subjects with decreased erythrocyte deformability also had higher GHb levels than diabetic subjects with normal erythrocyte deformability (P < 0.01). Although there was only a modest inverse correlation between these two parameters (n = 28, r = −0.566), the fact that 61% of the diabetic subjects studied exhibited both decreased deformability and increased GHb levels suggests that deformability abnormalities may become apparent once a threshold GHb level is reached. We conclude that spectrin in diabetic erythrocytes is oxidatively damaged. Protein glycosylation may be responsible for oxidative damage, although other factors in addition to or in concert with protein glycosylation may also be involved. Oxidation of spectrin may participate in the mechanisms resulting in diabetic erythrocyte deformability abnormalities.