Application of isoelectric focusing in immobilized pH gradients to the study of acetaldehyde-modified hemoglobin

We applied the technique of isoelectric focusing (IEF) on immobilized pH gradients (LKB) to determine whether acetaldehyde-modified hemoglobins (Hb) prepared in vitro with unphysiological acetaldehyde concentrations have clinical relevance. This technique separates proteins with pl less than 0.01 and provides detail about hemoglobins not otherwise separable. We performed two kinds of studies. In one kind of study, we incubated red cells from control subjects with acetaldehyde. Products of these incubations were applied to IEF gels either directly or after reduction with sodium cyanoborohydride. Incubation of red cells with acetaldehyde in 1-150 mM concentration without cyanoborohydride reduction yielded hemoglobin bands of decreasing pl the appearance of which coincided with the disappearance of Hb A and Hb A1c. When the products of incubation were reduced with cyanoborohydride before IEF, an additional acidic Hb band appeared which we call the "anodal CNBH band." In a second kind of study, we compared IEF patterns of hemolysates from control subjects and alcoholism detoxification patients, without adding acetaldehyde. Again, samples were applied to IEF gels either directly or after reduction with cyanoborohydride. When samples were run on IEF without reduction, no differences were seen between patients and controls. When samples were reduced before IEF, an anodal CNBH band appeared having the same mobility as the band seen after in vitro incubations with acetaldehyde. These bands were often stronger in samples from patients, but not consistently so. Several experiments, including the use of glycolysis inhibitors, indicated that the anodal CNBH band is an adduct of fructose 1,6-diphosphate with hemoglobin. We suggest that in millimolar concentrations, acetaldehyde may function as an inhibitor of glycolysis at or below the aldolase step.