Partition of calcium, phosphate, and protein in the fluid phase aspirated at calcifying sites in epiphyseal cartilage

David S. Howell; Julio C. Pita; Juan F. Marquez; Juan E. Madruga

doi:10.1172/JCI105801

Partition of calcium, phosphate, and protein in the fluid phase aspirated at calcifying sites in epiphyseal cartilage

David S. Howell, Julio C. Pita, Juan F. Marquez, and Juan E. Madruga

Published May 1, 1968 - More info

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Abstract

A reproducible method, adapted from renal micropuncture techniques, was developed for sampling 10-40 mμl of a clear fluid from epiphyseal cartilage of normal or rachitic rats in vivo, either from the hypertrophic cell zone (C_f1) or surface resting cell cartilage (L_f1).

Characterization of this fluid depended upon quantitation of protein, total inorganic phosphate (P_{i_t}), total calcium (Ca_t), nucleotide, and hemoglobin in volumes of 20 mμl. Established methods for macroscale measurements of each of these parameters have been modified to permit direct spectrophotometric readings on samples of 10^-10-10^-11 g.

The fluid from hypertrophic and peripheral resting cell cartilage was of an extracellular nature as evidenced by a high chloride and sodium, as well as low potassium, protein, and nucleotide content.

The pH of fluid isolated from endochrondral plates in vivo was measured under oil as a function of P_CO₂ and the computed bicarbonate was elevated above concurrent serum levels.

After ultracentrifugation of C_f1 of normal, rachitic, and healing rachitic animals, nonprotein-bound calcium (Ca_f) and phosphate (P_{i_f}) were determined on supernatant fluids. The hypertrophic cell cartilage fluid of rachitic rats was distinguished by a high ratio C_f1/serum of P_{i_f}. This ratio returned to normal during treatment of rickets. The upper limit for ionic activity A¹ Ca⁺⁺ × A HPO₄⁼ was too low to initiate precipitation of brushite or dicalcium phosphate but was in a range of supersaturation in respect to crystalline apatites. Thus these data are consistent with initiation of calcification by heterogeneous nucleation of mineral in the septal matrix but can be reconciled alternately with a precipitation mechanism only if the site of initial mineral phase separation is outside the septal matrix.