Resumen
The high-affinity (KM<1 μM) 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 μM. 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 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.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 19-24 |
| Número de páginas | 6 |
| Publicación | Alcohol |
| Volumen | 31 |
| N.º | 1-2 |
| DOI | |
| Estado | Published - 1 ene 2003 |
Huella dactilar
ASJC Scopus subject areas
- Health(social science)
- Biochemistry
- Toxicology
- Neurology
- Behavioral Neuroscience
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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. / Moncada, Claudio; Fuentes, Nelson; Lladser, Alvaro; Encina, Gonzalo; Sapag, Amalia; Karahanian, Eduardo; Israel, Yedy.
En: Alcohol, Vol. 31, N.º 1-2, 01.01.2003, p. 19-24.Resultado de la investigación: Article
TY - JOUR
T1 - Use of an "acetaldehyde clamp" in the determination of low-K M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells
AU - Moncada, Claudio
AU - Fuentes, Nelson
AU - Lladser, Alvaro
AU - Encina, Gonzalo
AU - Sapag, Amalia
AU - Karahanian, Eduardo
AU - Israel, Yedy
PY - 2003/1/1
Y1 - 2003/1/1
N2 - The high-affinity (KM<1 μM) 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 μM. 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 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.
AB - The high-affinity (KM<1 μM) 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 μM. 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 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.
KW - Acetate
KW - ALDH2
KW - Ethanol
KW - H4-II-E-C3
KW - Hepatoma
UR - http://www.scopus.com/inward/record.url?scp=0242438569&partnerID=8YFLogxK
U2 - 10.1016/j.alcohol.2003.06.004
DO - 10.1016/j.alcohol.2003.06.004
M3 - Article
C2 - 14615007
AN - SCOPUS:0242438569
VL - 31
SP - 19
EP - 24
JO - Alcohol
JF - Alcohol
SN - 0741-8329
IS - 1-2
ER -