The occurrence of pyrophosphate in bone.

The conditions required for the calcification in vitro of slices of rachitic rat cartilage have been extensively investigated, and attempts have been made to analyse the process in biochemical terms. The study of the role of enzymes in the calcification was initiated by Robison (1923), who discovered high concentrations of alkaline phosphatase in the pre-osseous cartilage of young rats. Robison suggested that a local increase in the concentration of phosphate ions produced by the hydrolysis of ester phosphates by phosphatase might be a factor in calcification. A difficulty which Robison recognised was that in body fluids the concentration of ester phosphate available as substrate for phosphatase action was low. Harris (1932, 1933), however, correlating older observations on the presence of glycogen in cartilage (especially marked in the hypertrophic cells of calcifying cartilage) with the then more recent knowledge of the biochemistry of glycolysis, suggested that “ hypertrophic cartilage cells provide both the phosphatase enzyme described by Robison and glycogen which, on hydrolysis, yields hexose-phosphoric esters.” The link between glycogen and phosphatase in cartilage was strengthened by the demonstration by Gutman and his co-workers (Gutman and Gutman 1941 ; Gutman, Warrick and Gutman 1942) that many of the enzymes of the glycolytic sequence of reactions are present in cartilage. Gutman also showed that in vitro calcification could be inhibited wholly or partly by compounds known to block certain reactions in the glycolytic sequence. He concluded that his experiments offered “ an explanation for the accumulation and disappearance of glycogen at the site of calcification in cartilage and provide a potential source of phosphoric ester substrate for phosphatase.” The results of Robison and Gutman do not however provide a complete explanation of the calcification process, and several difficulties in the acceptance of the proposed mechanism have arisen. Cartilage slices have been shown to remain calcifiable in vitro after all enzymes have been destroyed. Rubin and Howard (1950) emphasised the importance in calcifiable cartilage matrix of material having properties resembling those of chondroitin sulphate. A local concentration of calcium ions in the matrix can be achieved by this material through a reversible ion-exchange process, and this may provide nucleation centres for the formation of crystals of bone salt. It is probably this portion of the calcification mechanism which survives in slices after the enzymes have been destroyed. Although the evidence gathered by Robison concerning the association of phosphatase activity and calcification was impressive, more detailed information on the localisation of phosphatase in bone and cartilage obtained by modern histochemical techniques does not suggest any direct relationship between the enzyme and the deposition of bone salt. Phosphatase activity is greatest at the pre-osseous stage before calcification has begun, and the occurrence of glycogenolysis and high phosphatase levels in pre-hypertrophic cartilage cells may simply be indications of the intense metabolic processes accompanying cell differentiation (Pritchard 1952). Indeed, McLean and Urist (1955), after reviewing the evidence, stated that “one must conclude that all efforts to implicate phosphatase in calcification have failed, in spite of a most promising and apparently self-evident start thirty years ago.” Gutman himself was not convinced that the substrate for phosphatase was one of the phosphoric esters found in the course of the glycolytic sequence of reactions, and he suggested as an alternative that the phosphate was transferred to some unidentified phosphate acceptor in cartilage matrix before being finally deposited as bone salt (Gutman and Yu 1950). It is