Automated Analysis of Cellular Metabolites at Nanomolar to Micromolar Concentrations Using Bioluminescent Methods

Automated bioluminescent assays for lactate, oxoglutarate, pyruvate, ammonia, NAD, glutamate, ATP, phosphocreatine, creatine, and NADH were developed from existing spectrophotometric and fluorometric assays. A key feature was the development of a luminescent reagent that was not only relatively cheap and stable, but also could be stored frozen. With this reagent it was possible to measure NADH concentrations as low as 0.1 nM in a 25-μl sample. The sensitivity of the other assays was limited by contamination of the enzymes and cofactors used in the assays and ranged from 0.05 to 25 μM with sample volumes of 0.75-10 μl.