Improved enzymic assay for serum formate with colorimetric endpoint.
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.
D003124:Colorimetry; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004355:Drug Stability; D005560:Formate Dehydrogenases / Q000097:blood; D005561:Formates / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D005561:Formates; D007202:Indicators and Reagents; D005560:Formate Dehydrogenases; D008058:Dihydrolipoamide Dehydrogenase