Simplified luciferase assay of NAD+ applied to microsamples from liver,kidney and pancreatic islets

A new single-step procedure for the bioluminescence assay of NAD⁺, permitting measurements on the pmol level (10^?12 mol) is described. Acid extracts of NAD⁺ were prepared in different tissues. The acidification destroys reduced pyridine nucleotides and most enzymes which are present in the tissue sample. After neutralization the extract is added to a light-yielding solution, and the luminescence is measured with a photomultiplier. The maximal height of the signal is measured by means of a digital voltmeter. The light yielder is bacterial luciferase with appropriate additives and supplemented with malate and malate dehydrogenase.The modified light-yielding solution provides for continuous formation of NADH resulting in a durable level of light emission. The cycle involved was shown not to operate with NADP⁺. The slow fading of the emission permits simplification of the measuring procedure. Rapid injection in front of the phototube can thus be omitted and replaced by ordinary mixing before the reaction cell is positioned for the measurement. Furthermore, the instrumentation required is less elaborate than in photokinetic assay, since it is not necessary to record and integrate the time course of the emission. To test the applicability of the method, analyses of pmol amounts were performed in the islets of Langerhans and in tissue samples of much smaller size than fine needle biopsies.