Synthesis of Hemoglobin AIc and Related Minor Hemoglobins by Erythrocytes

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°-37°C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4°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 ³²P-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.