Review: Development of a fluid phase measure of acetaldehyde for biological samples

Abstract

The measurement of acetaldehyde is difficult in part because of volatility at room temperatures, the small amounts in biological samples, and reactivity with proteins. During studies of adduct formation with hemoglobin it became apparent that an assay based on modification of charged groups would be difficult for acetaldehyde adducts. The changes induced by physiologic concentrations of acetaldehyde were small and overshadowed by changes induced by increased levels of minor hemoglobin species resulting from the inhibition of glycolysis at or below the aldolase step. This inhibition produces increased glucose and phosphorylated glycolytic intermediates that react with hemoglobin resulting in fast eluting components on ion exchange. Studies of radioactive acetaldehyde-reacted hemoglobin documented that dilution of hemoglobin, separation into dimers, monomers, peptides, or amino acids, increased loss of radioactivity with potential for loss and inter- and intrachain exchange. We therefore developed a fluid phase assay that relies on the reaction of free or the liberation of bound acetaldehyde in the presence of heat, HCl, and NH 4+ with 1,3-cyclohexanedione to form a fluorophore (1,2,3,4,6,7,8,9,10-decahydro-9-methylacridine-1,8-dione) that can be separated from other reacted aliphatic aldehydes on a C18 column. Measurement of hemoglobin- or whole blood-associated acetaldehyde levels (WBAA) by high performance liquid chromatography can distinguish between drinking and nondrinking humans, mice, and pigs well after the last drink. We have recently applied the assay to samples of tissue and breath.