Separation of hemoglobin acetaldehyde adducts by high-performance liquid chromatography-cation-exchange chromatography.

A HPLC-based method was developed to provide a simple way to study changes to hemoglobin induced by acetaldehyde in vitro. This method distinguished 18 human hemoglobin fractions including a new acetaldehyde-induced fraction HbA1ach3. The method consists of a Poly CAT A cation-exchange column and a stepwise salt and pH gradient, with a total analysis time of 31 min. The formation of acetaldehyde adducts was studied by incubation of hemoglobin with different Ach concentrations (5-1000 microM) and different incubation times (0-48 h). Physiological (5-250 microM) Ach concentrations induced increases mainly in 3 known fractions: HbA1ach1, HbA1prec, and HbA1d3; plus, it caused the formation of a new fraction, HbA1ach3. The specificity of the changes to acetaldehyde was studied by incubation of hemoglobin with glucose and acetylsalicylic acid. HbA1ach3 was the only acetaldehyde-induced hemoglobin fraction which was not also increased by glucose and acetylsalicylic acid treatment. The formation of HbA1ach3 showed a dose and time dependence on acetaldehyde incubations. Dialyzation and reduction experiments showed that HbA1ach3 is a stable adduct of hemoglobin, and incubation with purified HbAO showed that HbA1ach3 is an adduct of HbAO. The within-run and between-run coefficients of variation for HbA1ach3 (0.83% of total hemoglobin) were 10.8 and 15.1%, respectively, and the analytical recovery was 82-97%. These results indicate that in addition to the new, acetaldehyde-specific fraction HbA1ach3, several other types of hemoglobin adducts were formed with acetaldehyde. The current method might be useful in clarifying the relationships between hemoglobin and acetaldehyde in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)