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

Claudio Moncada, Nelson Fuentes, Alvaro Lladser, Gonzalo Encina, Amalia Sapag, Eduardo Karahanian, Yedy Israel

Resultado de la investigación: Article

5 Citas (Scopus)

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 originalEnglish
Páginas (desde-hasta)19-24
Número de páginas6
PublicaciónAlcohol
Volumen31
N.º1-2
DOI
EstadoPublished - 1 ene 2003

Huella dactilar

Aldehyde Dehydrogenase
Acetaldehyde
Clamping devices
Rats
Hepatocellular Carcinoma
media culture
Acetates
Culture Media
Alcohol Dehydrogenase
alcohol
radioactivity
NAD
Ethanol
Cyanamide
social isolation
Equilibrium constants
Radioactivity
contact
Cell culture
Metabolism

ASJC Scopus subject areas

  • Health(social science)
  • Biochemistry
  • Toxicology
  • Neurology
  • Behavioral Neuroscience

Citar esto

Moncada, Claudio ; Fuentes, Nelson ; Lladser, Alvaro ; Encina, Gonzalo ; Sapag, Amalia ; Karahanian, Eduardo ; Israel, Yedy. / Use of an "acetaldehyde clamp" in the determination of low-K M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells. En: Alcohol. 2003 ; Vol. 31, N.º 1-2. pp. 19-24.
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title = "Use of an {"}acetaldehyde clamp{"} in the determination of low-K M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells",
abstract = "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.",
keywords = "Acetate, ALDH2, Ethanol, H4-II-E-C3, Hepatoma",
author = "Claudio Moncada and Nelson Fuentes and Alvaro Lladser and Gonzalo Encina and Amalia Sapag and Eduardo Karahanian and Yedy Israel",
year = "2003",
month = "1",
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doi = "10.1016/j.alcohol.2003.06.004",
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Moncada, C, Fuentes, N, Lladser, A, Encina, G, Sapag, A, Karahanian, E & Israel, Y 2003, 'Use of an "acetaldehyde clamp" in the determination of low-K M aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells', Alcohol, vol. 31, n.º 1-2, pp. 19-24. https://doi.org/10.1016/j.alcohol.2003.06.004

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

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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 -