VALIDATED FLUOROMETRIC HPLC ANALYSIS OF ACETALDEHYDE IN HEMOGLOBIN FRACTIONS SEPARATED BY CATION-EXCHANGE CHROMATOGRAPHY - 3 NEW PEAKS ASSOCIATED WITH ACETALDEHYDE

Stable hemoglobin-acetaldehyde adducts present in hemoglobin fractions separated by polyaspartic acid cation exchange chromatography were qu antified by fluorimetric HPLC. The fluorescent species eluted from the HPLC was confirmed by mass spectrometry to be consistent with the exp ected product from reaction of acetaldehyde, 1,3-cyclohexanedione (CHD ), and ammonium ion. Hemolysate (2.2 mM hemoglobin) was incubated in e quivalent volumes of either phosphate-buffered saline or 5 mM acetalde hyde at 37 degrees C for 30 min and washed three times with H2O to rem ove free acetaldehyde and labile adducts before the injection of 14.7 mg hemoglobin onto the cation exchange column, Fluorimetric HPLC analy sis of hemolysate samples either with or without in vitro reaction wit h acetaldehyde revealed that most acetaldehyde resides in the hemoglob in A(0) fraction, The reaction with acetaldehyde in vitro resulted in a significant increase in fast-eluting minor hemoglobin species on cat ion exchange chromatography concomitant with increased acetaldehyde in the HbA(1a+b), HbA(1c), and HbA(1-AcH) fractions. We report three new cation exchange chromatographic peaks after reaction with acetaldehyd e: HbA(1-AcH-3), HbA(1c-1), and Hb(0-1). Each new peak was found to as sociate with a significant quantity of CHD-reactive acetaldehyde. Thes e experiments provide additional evidence that stable adducts form bet ween acetaldehyde and hemoglobin and that these adducts occur in multi ple hemoglobin species separated by cation exchange chromatography. Fu rther characterization and structural assignment of these species are warranted in view of their potential utility as markers for ethanol in take.