A radioenzymatic method suitable for the estimation of nanomole amounts of a wide range of metabolites is described. The method involves the conversion of [ l-'4C]a-ketoglutarate to [ l-'4C]glutamate by NADH or NADPH generated stoichiometrically in preliminary reactions specific for the metabolite under assay. The labeled glutamate is then decarboxylated with glutamate decarboxylase and the liberated "'CO2 trapped and measured by scintillation counting. Examples of the variety of substrates that can be estimated by this method include procedures for the determination of lactate, glucose, ammonia, pyruvate, malate, sorbitol, and NAD(P)H. However, most enzymatic spectrophotometric or fluorimetric metabolite assays should be readily adaptable for radioenzymatic assay. In validation of the method, glucose and lactate were measured in whole blood or plasma, and the values obtained by the radioenzymatic method were found to correlate well with established clinical procedures. The method lends itself to both clinical and basic biochemical applications, can be used over a very wide range of metabolite concentrations, and is sufficiently sensitive to allow the measurement of an amount of metabolite as low as 0.25 nmol.
MATERIALS AND METHODS
Enzymes and Cofactors
Glutamate decarboxylase (EC 4.1.1.15) (20 U/mg) was either purchased from Worthington Biochemical Corporation, Freehold, New Jersey, or prepared from a crude