Stimulatory Effect of Beta-Alanyl-<i>L</i>-Histidinato Zinc on Bone Formation in Tissue Culture

Abstract

The present investigation was undertaken to clarify the in vitro effect of beta-alanyl-L-histidinato zinc (AHZ) on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 96 h in Dulbecco's modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8) to 10(-4) mol/l AHZ. The bone cellular zinc content was significantly increased in cultures with concentrations of AHZ greater than 10(-6) mol/l. With 10(-5) mol/l zinc sulfate, the bone cellular zinc content was significantly elevated. Bone calcium content was significantly increased by the presence of 10(-7) to 10(-4) mol/l AHZ. This increase was blocked by the presence of 10(-7) mol/l cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of AHZ (10(-7) to 10(-4) mol/l), whereas it did not significantly alter acid phosphatase activity Bone collagen and DNA contents were significantly increased by 10(-7) to 10(-5) mol/l AHZ, while they were not significantly elevated by zinc sulfate (10(-7) and 10(-6) mol/l). The AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity and DNA content were prevented by 10(-4) mol/l dipicolinate, a chelator of zinc. Furthermore, the AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity, collagen and DNA contents were blocked by 10(-7) mol/l cycloheximide. These findings indicate that AHZ had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. The AHZ effect was more intensive than that of zinc sulfate.