Improved separation of acetaldehyde-induced hemoglobin.

A fast-eluting minor variant of hemoglobin A, designated as HbA1-AcH, appears elevated after the incubation of red blood cell hemolysates with acetaldehyde (AcH) and has been proposed as a diagnostic marker for alcoholism or as an indicator for heavy drinking. We have developed an improved HPLC separation of this peak and others elevated by AcH using a polyaspartic acid column (PolyCAT A, PolyLC, Inc.) and a nonlinear buffer gradient with pH changes from 6.6 to 6.8. Saline-washed red blood cells were treated with sodium acetate buffer (pH 5.5) to remove unstable Schiff bases, and then hemolyzed by addition of an equal volume of H2O and 0.4 volumes of CCI4. HbA1-AcH and several others, including two peaks in the HbA1a+b cluster, Hb Pre-A1c, and HbA1d3 were significantly increased by AcH incubation, and the changes were only partially reversible with time. Improved resolution of these peaks allows more accurate quantitation of AcH adducts of hemoglobin.