IN VITRO STUDIES OF BONE RESORPTIVE MECHANISMS 559 



mail and Neuman, 1953) liad shown that the Ca ion concentration 

 in tlie extracelkilar fluid is liigher than could be expected to exist at 

 equihbrinm between lione mineral and extracellular fluid at pH 7.4, 

 and had proposed that bone cells produce an acid metabolite — 

 citric acid — which not onl\' lowers the pH locally at the bone crystal 

 surface, but also forms a soluble complex with Ca ions, allowing 

 them to be swept away in the circulation. Furthermore, with in- 

 genious experiments in intact dogs ( Neuman et ah, 1956; Firschein 

 et al., 1958) he and his coworkers produced evidence in favor of his 

 hypothesis and showed that parathyroid hormone appeared to en- 

 hance bone citrate production. Thus, our first steps were to deter- 

 mine the nature and amount of acid produced by bone fragments 

 in vitro and examine the influence of factors such as parathyroid 

 hormone on the system. 



Incubating fragments of cartilage-free fresh bone at 37 °C in 

 Krebs-Ringer media buffered with bicarbonate at pH 7.4, we ( Borle, 

 Nichols, and Nichols, 1960tt) were able to show^ that normal bone 

 from mice produced H+ ion (measured manometrically by the 

 evolution of COl> from the bicarbonate medium) from a glucose 

 substrate in considerable amounts even under aerobic conditions. 

 Moreover, acid production was increased by previous treatment of 

 the animals with parathyroid extract. These experiments are illus- 

 trated in the left-hand part of Fig. 1. However, chemical analysis 

 of the medium after incubation revealed that the acid anions which 

 accumulated were almost entirely lactate, citrate accumulation being 

 only about 1 70 as great. Two other observations of importance 

 were made : First, both lactate and citrate production were increased 

 by parathyroid treatment ( Borle, Nichols, and Nichols, 1960Z? ) ; and, 

 second, acid production measured as lactate and citrate accumula- 

 tion was always considerably larger than that which could be meas- 

 ured manometrically as free H+ ion (Borle, Nichols, and Nichols, 

 1960a). 



These experiments confirmed with direct evidence Neuman's find- 

 ing of acid production bv bone and its enhancement by parathyroid 

 hormone, but indicated that lactic rather than citric acid was the 

 chief end product. Since May 1959, when these experiments were 

 performed, these observations have been confirmed many times 



