Measuring Lactate and Pyruvate in Body Fluids

Methods for Measuring Lactate and Pyruvate in Body Fluids 

Lactate is oxidized to pyruvate by lactate dehydrogenase in the presence of NADL The NADH formed in this reaction is measured by a spectrophotometer at 340nm and serves as a measure of the lactate concentration. 

The equilibrium of the reaction normally lies far to the left. However, by using a pH of 9,0 to 9.6, an excess of NAD, and trapping the reaction product pyruvate with hydrazine, the equihbrium can be shifted to the right. Pyruvate can also be removed by reacting it with L-glutamate in the presence of alanine aminotransferase. Use of tris (hydroxymethyl)-aminomethane (TRIS) buffer results in more rapid completion of a side reaction between NAD and hydrazine and prevents the creeping of blank values observed when glycine buffer is used.  

Because of its high specificity and simplicity, the enzymatic method is the method of choice for measuring lactate, although other methods may also be used (e.g., gas chromatography and photometry). 

The Vitros analyzer (Ortho-Cfinical Diagnostics, Raritan, N.J.) uses an assay in which lactic acid is oxidized to pyruvate by lactate oxidase. The H2O2 generated oxidizes a chromogen system, and the absorbance of the resulting dye complex, measured by a spectrophotometer at 540nm, is directly proportional to the lactate concentration in the specimen. Each mole oflactate oxidized produces O.Smol of dye complex. 

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