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

Authordc.contributor.authorMoncada, Claudio 
Authordc.contributor.authorFuentes, Nelson es_CL
Authordc.contributor.authorLladser, Álvaro es_CL
Authordc.contributor.authorEncina, Gonzalo es_CL
Authordc.contributor.authorSapag, Amalia es_CL
Authordc.contributor.authorKarahanian, Eduardo es_CL
Authordc.contributor.authorIsrael Jacard, Yedy es_CL
Admission datedc.date.accessioned2007-06-04T16:22:06Z
Available datedc.date.available2007-06-04T16:22:06Z
Publication datedc.date.issued2003-08
Cita de ítemdc.identifier.citationALCOHOL 31 (1-2): 19-24 AUG-OCT 2003en
Identifierdc.identifier.issn0741-8329
Identifierdc.identifier.urihttp://repositorio.uchile.cl/handle/2250/120426
Abstractdc.description.abstractThe high-affinity (K-M < 1 muM) 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 muM. The clamp is attained by addition of excess yeast alcohol dehydrogenase, C-14-ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD(+)) to the hepatoma cell culture medium. The concentration of C-14-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction. Thus, C-14-acetate is generated virtually by the low-K-M 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 muM 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 cell.en
Lenguagedc.language.isoenen
Publisherdc.publisherPERGAMON-ELSEVIER SCIENCE LTDen
Keywordsdc.subjectALCOHOL-METABOLISMen
Títulodc.titleUse of an "acetaldehyde clamp" in the determination of low-K-M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cellsen
Document typedc.typeArtículo de revistaen


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