Determination by Radioimmunoassay of the Sum of Oxidized and Reduced Forms of NAD and NADP in Picomole Quantities from the Same Acid Extract

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Abstract
The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5′‐AMP. NAD was converted to 5′‐AMP via ADP‐ribose by alkaline treatment while NADH was converted first to ADP‐ribose by incubation of the acid extract at 25°C followed by alkaline conversion to 5′‐AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1–10 pmol.

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