Alcohol Dehydrogenase Assay Kit from Biomedical Research Service at University of Buffalo

Alcohol dehydrogenase (ADH) is an enzyme present in most organisms, including humans. A number of studies on this enzyme have been done on yeast ADH. This enzyme is present in highest levels in mammalian liver and is primarily responsible for metabolizing the alcohol in our diets. ADH occurs in several different forms called isozymes and all of them are oxidoreductases. They serve to convert toxic alcohols into aldehydes and ketones in humans and other mammals.

The reaction of alcohol dehydrogenase involves protonation and de-protonation of co-enzyme NAD+ (Nicotinamide dinucleotide) in presence of Zn²⁺ ion which acts as a co-factor. ADH catalyzes the oxidation of ethanol to acetaldehyde (and concomitant reduction of NAD+ to NADH). Although this is a simple enzymatic reaction that could be coupled easily to a colorimetric reaction, there are not many non-radioactive assay kits available in the market for determining ADH activity in cell lysates.

The Biomedical Research Service Center (BRSC) at the University of Buffalo have developed the Alcohol Dehydrogenase Assay Kit to determine the activity of ADH from a variety of mammalian tissues and cell lysates. The kit is non-radioactive and colorimetric. The principle of the reaction in this kit involves the conversion of tetrazolium salt INT in a NADH-coupled enzymatic reaction to formazan by reduction. Formazan is water soluble and is red in color. The intensity of color increases in the presence of ADH activity. The increase in color intensity is linear to the ADH activity and can be read spectrophotometrically at 492 nm. The assay kit comes with 10X lysis buffer and assay solution. Phosphate buffered saline (PBS) for washing the cells and 0.5 M Acetic acid to stop the reaction are not supplied. The protocol is simple. The cells are first lysed in the lysis buffer and centrifuged at high speed. The debris is removed as a pellet and ADH assay solution is added to 10-20 µl of clear lysates. After 30 min of incubation at 37°C, the reaction is stopped with acetic acid. The color intensity is read at 492 nm in a spectrophotometer or a plate reader. If the intensity of the color is too strong or if the color develops too rapidly, the reaction mixture could be further diluted with 1X lysis buffer. The kit appears to be stable for more than a year since we were able to use it past the expiration date with good results; however, the manufacturer suggests using it within a year.

The ADH Assay Kit has been used in our laboratory for the past two years for estimation of ADH activity in human fetal brain derived neural stem cells. The procedure for this kit is simple and easy to perform. We have been able to get consistent results in our experiments using this kit.

In summary, the ADH Assay Kit from BRSC is a very convenient tool for estimating ADH activity in human tissues and cell culture lysates.