Use of an acetaldehyde clamp" in the determination of low-K-M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells"

Moncada, C; Fuentes N.; Lladser, A; Encina G.; Sapag, A; Karahanian, E; Israel, Y

Abstract

The high-affinity (KM<1 ?M) mitochondrial class 2 aldehyde dehydrogenase (ALDH2) metabolizes most of the acetaldehyde generated in the hepatic oxidation of ethanol. H4-II-E-C3 rat hepatoma cells have been found to express ALDH2. We report a method to assess ALDH2 activity in intact hepatoma cells that does not require mitochondrial isolation. To determine only the high-affinity ALDH2 activity it is necessary to keep constant low concentrations of acetaldehyde in the cells to minimize its metabolism by high-KM 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 ?M. The clamp is attained by addition of excess yeast alcohol dehydrogenase, 14C- ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD +) to the hepatoma cell culture medium. The concentration of 14C-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction. Thus, 14C-acetate is generated virtually by the low-KM aldehyde dehydrogenase activity. 14C-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 ?M 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. © 2003 Elsevier Inc. All rights reserved.

Más información

Título según WOS: Use of an "acetaldehyde clamp" in the determination of low-K-M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells
Título según SCOPUS: Use of an "acetaldehyde clamp" in the determination of low-K M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells
Título de la Revista: ALCOHOL
Volumen: 31
Número: 01-feb
Editorial: Elsevier Science Inc.
Fecha de publicación: 2003
Página de inicio: 19
Página final: 24
Idioma: English
URL: http://linkinghub.elsevier.com/retrieve/pii/S0741832903001630
DOI:

10.1016/j.alcohol.2003.06.004

Notas: ISI, SCOPUS