Skeletal alkaline phosphatase activity as a bone formation index in vitro

Abstract

These studies were intended to examine the relationship between skeletal collagen formation and skeletal alkaline phosphatase (ALP) activity in vitro. Embryonic chick calvaria were exposed to skeletal effectors (including high and low pH, a range of [p i] and [Ca], PTH, NaF, etc), and collagen formation was assessed by the incorporation of 3[H]-proline as 3[H]-hydroxyproline ( 3[H]-hyp). ALP activity was measured in the serum-free conditioned medium and in 20% butanol extracts of the bones. We found that ALP activity and 3[H]-hyp incorporation were coordinately increased from pH 5.5 to pH 7.2 ( r = .99, P < 0.001). Calvarial ALP was not increased in response to low [P i], but low [Ca] increased ALP and coordinately decreased collagen formation ( r = −.81, P < 0.05). Although calvarial ALP and 3[H]-hyp incorporation were coordinately increased by NaF, vanadate, PGE 2, calcitonin, and insulin, the slopes of the correlations were not the same for all effectors (eg, NaF: r = .97, P < 0.01, slope = 0.90; vanadate, r = .95, P < 0.005, slope = 0.20), indicating differential actions on ALP and 3[H]-hyp incorporation. When a variety of effectors, including low [Ca], were used to treat different groups of calvaria, ALP activity was not correlated with 3[H]-hyp incorporation ( r = .35), but when the exposure to effectors was limited to a preincubation, or when the low [Ca] data were excluded, a correlation was observed ( r = .87, P < 0.001, and r = .64, P < 0.02, respectively). Although ALP activity in the serum-free medium generally reflected the amount of ALP activity in the bones, that correlation was less significant in the presence of effectors ( r = .60, P < 0.05) than in their absence ( r = .89, P < 0.01). Together, these data indicate that (1) skeletal ALP is generally useful as an index of in vitro 3[H]-hyp incorporation and (2) even under conditions in which that correlation is disrupted (ie, in the presence of specific skeletal effectors), skeletal ALP is still useful as a predictive index of the capacity for in vitro calvarial collagen synthesis (ie, the predicted rate of 3[H]-hyp incorporation if the effectors were removed).