An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

Abstract

A method is described for determination of inorganic pyrophosphate ( PP i) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate ( 32 P i). Intra- and extracellular 32 PP i formed was measured using high-performance liquid chromatographic (HPLC) separation of the PP i from orthophosphate ( P i) 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 Mg 2+ 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 Mg 2+ ions and ionic strength. In this case separation of small amounts of PP i from a large excess of P i was possible without prior removal of P i or extraction of the PP i 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 32 P i 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 PP i which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.