Enzymatic measurement of ethanol or NAD in acid extracts of biological samples

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 N HClO4 is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mM in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.