Use of an “acetaldehyde clamp” in the determination of low-KM aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells

Abstract The high-affinity (K M M aldehyde dehydrogenases. To maintain both low and constant concentrations of acetaldehyde we used an "acetaldehyde clamp," which keeps acetaldehyde at a concentration of 4.2±0.4 μM. The clamp is attained by addition of excess yeast alcohol dehydrogenase, 14 C-ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD + ) to the hepatoma cell culture medium. The concentration of 14 C-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction. Thus, 14 C-acetate is generated virtually by the low-K M aldehyde dehydrogenase activity. 14 C-acetate is separated from the culture medium by an anionic resin and its radioactivity is determined. We showed that (1) acetate production is linear for 120 min, (2) addition of 160 μM cyanamide to the culture medium leads to a 75%–80% reduction of acetate generated, and (3) ALDH2 activity is dependent on cell-to-cell contact and increases after cells reach confluence. The clamp system allows the determination of ALDH2 activity in less than one million H4-II-E-C3 rat hepatoma cells. The specificity and sensitivity of the "acetaldehyde clamp" assay should be of value in evaluation of the effects of new agents that modify Aldh2 gene expression, as well as in the study of ALDH2 regulation in intact cells.