Procedure for the study of acidic calcium phosphate precursor phases in enamel mineral formation

Considerable evidence suggests that an acidic calcium phosphate, such as octacalcium phosphate (OCP) or brushite, is involved as a precursor in enamel and other hard tissue formation. Additionally, there is in vitro evidence suggesting that fluoride accelerates and magnesium inhibits the hydrolysis of OCP to hydroxyapatite (OHAp). As the amount of OCP or brushite in enamel cannot be measured directly in the presence of an excess of hydroxyapatite, a procedure was developed that allows for their indirect in vivo quantification as pyrophosphate. This permits study of the effects of fluoride and magnesium ions on enamel mineral synthesis. Rat incisor calcium phosphate was labeled by intraperitoneal injection of NaH2(32)PO4. The rats were then subjected to various fluoride and magnesium treatments with subcutaneous implanted osmotic pumps. They were then killed at predetermined intervals; the nascent sections of the incisors were collected, cleaned, and pyrolyzed at 500 degrees C for 48 hours to convert acidic calcium phosphates to calcium pyrophosphate; the pyrophosphate was separated from orthophosphate by anion-exchange chromatography; and the resulting fractions were counted by liquid scintillation spectrometry. The activities of the pyro- and orthophosphate fractions were used to calculate the amount of acidic calcium phosphate present in the nascent mineral. The results demonstrated that the percentage of radioactive pyrophosphate in nascent incisors decreased with time, with increasing serum F- concentration, and with decreasing serum magnesium content. The technique described here should prove to be a powerful new tool for studying the effects of various agents on biological mineral formation.