Synthesis of hemoglobin Aic and related minor hemoglobin by erythrocytes. In vitro study of regulation.

Factors that influence hemoglobin (Hb)Aic 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. HbAic 
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 HbAic increased linearly with time for 6 
wk; after incubation at the highest glucose concentration, HbAic comprised 50% 
of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect 
HbAic synthesis in vitro. In addition to glucose, galactose and mannose, 
but not fructose, served as precursors to HbAic. A good substrate 
for hexokinase (2-deoxy-glucose) and a poor hexokinase substrate (3-O-methyl-glucose), were better 
precursors for HbAic synthesis than glucose, suggesting that enzymatic phosphorylation of 
glucose is not required for HbAic synthesis. Autoradiography after erythrocyte incubation 
with 32P-phosphate showed incorporation of radioactivity into HbAia1 and HbAia2, but 
not HbAib, Aic, or A. Acetylated HbA, generated during incubation with 
acetylsalicylate, migrated anodal to HbAic and clearly separated from it. Erythrocytes 
from patients with insulinopenic diabetes mellitus synthesized HbAic at the same 
rate as controls when incubated with identical glucose concentrations. Likewise, the 
rate of HbAic 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. 