An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD' and NADH are assayed by the same procedure, which is also used to measure NADP' and NADPH after these nucleotides have been converted to NAD' and NADH, respectively.
The principle of this assay is based upon the finding of Gupta and Robinson (1) that liver alcohol dehydrogenase (LADH, EC 1.1.1.1) catalyzes a coupled oxidoreduction between an alcohol and an aldehyde in the presence of NAD. Acetaldehyde, formed in the presence of ratelimiting amounts of NAD and excess ethanol and lactaldehyde, is flushed into a vessel containing semicarbazide. Acetaldehyde semicarbazone is measured by its absorbance at 224 nm and is directly related to the NAD concentration. This procedure provides a quick and reliable measure of either NAD+ or NADH and offers several advantages over existing methods. NADP+ and NADPH content in tissues may also be determined by using the same assay on tissue samples previously digested with alkaline phosphatase (EC 3.1.3.1).
Methods
Substrates. NAD+, NADH, NADP', and NADPH (approx 98% pure), were obtained from Sigma Chemical Co. All chemicals were of reagent grade quality.
Lactaldehyde was synthesized by decarboxylating threonine according