A rapid, enzymatic assay for measurement of inorganic pyrophosphate in biological samples.
An enzymatic assay for measurement of inorganic pyrophosphate (PPi) in biological samples is described using the UDPG pyrophosphorylase reaction and coupling it with three other enzymes in a system of phosphorylation and double reduction to form NADPH, which is measured fluorometrically. The method is specific for PPi in the presence of inorganic orthophosphate and can be used on small volumes of deproteinized ultrafiltrates of fluids or cell extracts. The assay was used here to determine the concentration of PPi in human plasma and serum, and the intracellular content of PPi in human skin fibroblasts, chondrocytes, and red blood cells. The mean PPi concentration of plasma was found to be 2.72 +/- 0.14 muM (mumo/l +/- S.E.M.); for serum it was 6.09 +/- 0.36 muM. The range (95% confidence limits) was 2.14-3.30 muM for plasma and 4.62-7.51 muM for serum. The average intracellular PPi content (pmoles PPi per 10(6) cells +/- S.E.M.) was as follows: (a) skin fibroblasts, 332 +/- 66; (b) articular chondrocytes, 655 +/- 46; red blood cells, 1.74 +/- 0.28.
D000818:Animals; D002356:Cartilage / Q000032:analysis; D011756:Diphosphates / Q000032:analysis* / Q000097:blood; D004912:Erythrocytes / Q000032:analysis; D006801:Humans; D010710:Phosphates / Q000032:analysis* / Q000097:blood; D012867:Skin / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase
D011756:Diphosphates; D010710:Phosphates; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase