The coupling of NAD(P)(+)-producing reactions to a semiautomated bioluminescent reaction

Many cellular metabolites can be measured with high sensitivity using biolu minescent techniques. These metabolites are coupled to an appropriate enzym e to produce NAD(P)H, which can then be coupled to the bioluminescent react ions. The sensitivity of bioluminescence cannot be readily applied to metho ds in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration o f NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to mea sure the production of NAD(P)(+). Assays for creatine/creatine phosphate, p yruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensiti ve, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests tha t NAD(P)(+) bioluminescence has a wide potential for application. (C) 1999 Academic Press.