Phosphate determination by enzymatic colorimetric assay.
A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.
D003124:Colorimetry; D005189:False Positive Reactions; D006801:Humans; D010430:Pentosyltransferases; D010710:Phosphates / Q000097:blood*; D011683:Purine-Nucleoside Phosphorylase; D013053:Spectrophotometry; D014969:Xanthine Oxidase
D010710:Phosphates; D014969:Xanthine Oxidase; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase