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 cha nges to hemoglobin induced by acetaldehyde in vitro. This method disti nguished 18 human hemoglobin fractions including a new acetaldehyde-in duced fraction HbA1ach3. The method consists of a Poly CAT A cation-ex change column and a stepwise salt and pH gradient, with a total analys is time of 31 min. The formation of acetaldehyde adducts was studied b y incubation of hemoglobin with different Ach concentrations (5-1000 m u M) and different incubation times (0-48 h). Physiological (5-250 mu M) Ach concentrations induced increases mainly in 3 known fractions: H bA1ach1, 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 w hich was not also increased by glucose and acetylsalicylic acid treatm ent. The formation of HbA1ach3 showed a dose and time dependence on ac etaldehyde incubations. Dialyzation and reduction experiments showed t hat HbA1ach3 is a stable adduct of hemoglobin, and incubation with pur ified HbAO showed that HbA1ach3 is an adduct of HbAO. The within-run a nd between-run coefficients of variation for HbA1ach3 (0.83% of total hemoglobin) were 10.8 and 15.1%, respectively, and the analytical reco very was 82-97%. These results indicate that in addition to the new, a cetaldehyde-specific fraction HbA1ach3, several other types of hemoglo bin adducts were formed with acetaldehyde. The current method might be useful in clarifying the relationships between hemoglobin and aceteta ldehyde in vitro. Moreover, the measurement of HbA1ach3 may form the b asis for an improved method for the clinical monitoring of alcohol abu sers. (C) 1995 Academic Press, Inc.