Detection of a new acetaldehyde-induced hemoglobin fraction HbA1ach by cation exchange liquid chromatography

We developed a sensitive cation exchange liquid chromatographic method for analysis of acetaldehyde binding with hemoglobin. When human hemolysates were incubated in vitro with micromolar concentrations of acetaldehyde without reducing agents, HbA0 was found to form two new fractions. One of them was eluated with HbA1c, whereas a novel, previously undescribed fraction was detected between HbF and HbA1c. This new fraction was termed HbA1ach. The mean within-assay variation for the different hemoglobin fractions varied from 0.45 (HbA0) to 8.2% (HbA1ach) and the between-assay variation from 0.6 to 19.3%, respectively. After incubation with [1,2(14)C]acetaldehyde the specific radioactivity of HbA1ach was about 10 times higher than that of HbA0 and twice as high as the activity of the HbA1c fraction. The reaction kinetics of the formation of hemoglobin adducts were studied in vitro by incubation of hemolysates with various acetaldehyde concentrations. Acetaldehyde caused both short-term and permanent changes in hemoglobin. Even low acetaldehyde concentrations from 10 to 100 mumol/liter caused detectable changes in hemoglobin. During incubation the amount of HbA1ach and HbA1c fractions increased rapidly within the first 10 min and decreased slowly during the next hr but did not return to the initial levels. This suggests that only the part of binding of acetaldehyde with hemoglobin is irreversible and the changes can be already detected after incubation with physiological acetaldehyde concentrations.