Abstract
Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.