Composition of Bone. II. Analytical Results.

Abstract

The micro-technique which we 1 have devised for analyzing small specimens of calcified tissue has been subjected to further test and study. The crushed bones are extracted with ether and alcohol immediately after removal from the body; they are then dried and pulverized. About 10 mg. of bone powder are digested with HCl, trichloracetic acid is added, the solution is made up to 10 cc. and then filtered. Calcium is determined on aliquots of this filtrate. To another aliquot molybdic acid reagent (Briggs-Bell-Doisy) is added, the volume is made up to 10 cc. and then filtered. Inorganic phosphorus is determined on aliquots of this second filtrate. Digestion with HCl of different samples of the same specimen of bone powder gives solutions whose content of calcium and inorganic phosphorus agree as well as duplicate determinations on aliquots of the same solution.

CO₂ is determined on about 20 mg. of bone powder in Van Slyke's manometric gas apparatus.

A specimen of crushed bone was divided into two portions. One portion was extracted with ether and alcohol immediately, the other was allowed to stand unextracted during the summer. After standing for 5 months it was extracted and analyzed. The results for calcium, inorganic phosphorus and CO₂ were the same for both portions. Similar results were obtained with a second specimen of normal bone. The second precipitation with the molybdic acid reagent does not interfere with the accuracy of the method; the values for the ratio residual Ca/P so obtained are normal within the experimental error. When phosphorus values obtained from the first filtrate are employed, low ratios result.

In studying the method 11 specimens of normal calcification and 7 specimens of pathological calcification were analyzed. In all cases the value of the ratio was 1.96 within the experimental error; the theoretical value from Ca₃ (PO₄)₂ is 1.94.