Lactate enzymatic determination - by reacting with nicotinamide adenine dinucleotide in the presence of lactate dehydrogenase

摘要

Quantitative determination of L-lactate involves reacting with nicotinamide-adenine-dinucleotide (NAD) in the presence of lactate dehydrogenase (LDH) to form reduced nicotinamide-adenine-dinucleotide (NADH) and pyruvate. The latter is reacted with glutamate in the presence of glutamate-pyruvate transaminase (GPT) in alkaline buffered solution to form alanine and alpha-keto-glutarate. The NADH formed is measured. The novel feature is that the reaction is carried out in the presence of at least 0.1 M (bi)carbonate. The new reagent contains LDH, GPT, NAD, glutamate, and 0.45-0.60 mol/1. carbonate buffer pH 9.5-10.5. Determination of blood, serum or plasma lactate is important in the stabilization and therapeutic control of diabetics. Elevated serum L-lactate levels also occur in shock, myocardial infarct, uraemia, leukaemia and liver cirrhosis. L-Lactate determination is therefore an important diagnostic tool. (Bi)carbonate ions activate GPT, increasing the rate of removal of pyruvate from the reaction system. This shifts the equilibrium of the initial reaction and considerably reduces reaction time. The result is that lactate determinations can be carried out in 10 mins. or less. Results obtained with the procedure are accurate and reproducible. Additionally, the amount of relatively expensive GPT used can be reduced to 2-4 per ml.