Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1978-07-19
1981-03-03
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435805, 435810, C12Q 132
Patent
active
042542227
ABSTRACT:
There is provided a procedure for determining the concentration of a metabolic acid in a biological fluid. The method involves use of a tetrazolium salt, a pyridine nucleotide, an electron carrier, and an enzyme that is a dehydrogenase for the specific acid being assayed. These components are used to form an assay mixture; thereafter a quantity of the fluid to be assayed is combined with the assay mixture, so that there may commence a reaction in which the tetrazolium salt is changed to a formazan in an amount that is indicative of the concentration of the specific acid. In a preferred embodiment, the procedure permits assay of the concentration of beta-hydroxybutyrate, and the assay mixture includes components in the relative proportions of 0.8 micromoles 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), 0.32 micromoles phenazine methosulfate (PMS), 1.5 micromoles nicotinamide adenine dinucleotide (NAD), and 0.75 International Units beta-hydroxybutyrate dehydrogenase, in a water solution buffered at a pH of approximately 8.5 with hydrogen phosphates of potassium together with glycine and sodium hydroxide and also containing alkylphenoxypolyethyoxyethanol. In another preferred embodiment, the procedure permits assay of lactic acid by means of a similar assay mixture utilizing 27.5 International Units of lactic dehydrogenase in place of the beta-hydroxybutyrate dehydrogenase used in the previously discussed embodiment. In the latter procedure a preferred embodiment of the assay mixture is buffered at a pH of approximately 9.6 in a glycine-sodium hydroxide buffer and also contains phenoxypolyethyoxyethanol.
REFERENCES:
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patent: 3539453 (1970-11-01), Deutsch
patent: 3867258 (1975-02-01), Forgione
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Sunstein Bruce D.
Wiseman Thomas G.
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