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Journal of analytical toxicology

Improved enzymic assay for serum formate with colorimetric endpoint.


PMID 3754027

Abstract

The fluorometric assay for formate in serum was modified by pretreating samples with acetonitrile (1:1) precipitation; substituting p-iodonitrotetrazolium violet (INT) for resazurin; and by combining the cofactor (NAD), coupled enzyme (diaphorase), and secondary substrate (INT) into one reagent. Formate is oxidized by formate dehydrogenase producing NADH which reduces INT via diaphorase to a visible red-colored endpoint that can be measured on a spectrophotometer at 500 nm. Previous problems with fluorometric endpoint methods are eliminated when using this modified procedure: calibration is linear rather than nonlinear; blanking is rarely needed due to the acetonitrile sample preparation; dynamic range is expanded up to 10-fold; a simple spectrometer rather than a fluorometer is used; and the number of steps is reduced. The method is demonstrated to be linear, specific, sensitive, precise, and accurate.