Alcohol is a very widely consumed drink in the human diet. . It is often necessary to quantify the amount of alcohol in body fluids such as plasma and serum as well as in dietary and nutritional products. Measurement of alcohol concentration is essential in several applications including clinical and basic research, drug development, as well as in the fields of viticulture and enology
In the biological systems, ethanol is metabolized by the enzyme alcohol dehydrogenase and is converted to acetaldehyde. In the lab, this enzymatic reaction is generally coupled with a colorimetric reaction that is used to determine the ethanol concentration. The enzymatic reaction of alcohol dehydrogenase utilizes co-enzyme NAD+ (Nicotinamide dinucleotide) and the cofactor Zn2+ ion. There are a few assay reagents and kits available in the market for quantification of ethanol concentration in various kinds of samples, both biological and inorganic.
The Biomedical Research Service Center (BRSC) at the University of Buffalo has developed the Ethanol Assay Kit to determine the concentration of ethanol from a variety of samples including, serum, plasma, and cell lysates. The kit is non-radioactive and colorimetric. The principle of the reaction in this kit involves formation of red colored Formazan by the conversion of tetrazolium salt INT in a NADH-coupled enzymatic reaction. The intensity of the color is proportional to the concentration of ethanol in the sample which 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 are to be prepared by the scientists. The kit is stable for one year.
The protocol is easy to follow yet the assay is precise and accurate. The cells are lysed in the lysis buffer and centrifuged at high speed. Ethanol assay solution is added to 10-20 ul of clear lysate. 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.
The Ethanol Assay Kit has been used in our laboratory for the past four years for estimation of ethanol concentration in media supplemented with ethanol. In our experiments, various concentrations of ethanol ranging from 1mM to 100mM are added to the media and human fetal brain derived stem and progenitor cells are grown in this media to study the mechanism of brain damage occurring in fetal alcohol syndrome. We find that the assay is sensitive both at low and high concentrations of alcohol. However, we found that the linearity was not maintained if other drugs or metabolites were added to the media.
In summary, the Ethanol Assay Kit from BRSC is a very convenient tool for estimating ethanol concentration in biological samples.
Scientist
Children’s Research Center of Michigan
Children’s Hospital of Michigan