An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling.
A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.
D000818:Animals; D002358:Cartilage, Articular / Q000032:analysis*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D011756:Diphosphates / Q000032:analysis*; D010710:Phosphates / Q000032:analysis; D010761:Phosphorus Radioisotopes; D011817:Rabbits
D011756:Diphosphates; D010710:Phosphates; D010761:Phosphorus Radioisotopes