Evaluation of potassium pyroantimonate/sucrose/glutaraldehyde concentration and incubation time as essential variables for localizing calcium bound to organic compounds in epiphyseal chondrocytes.

The solution chemistry of potassium pyroantimonate (PA) was clarified, enabling preparation of stable concentrated PA solutions. The soluble conjugate base of PA determines the pH of the solution; therefore, the pH reflects the maximum amount of conjugate base dissolved ("saturated") in solution. Spontaneous precipitation occurs just below the "saturation" pH. Sucrose (S) stabilizes the conjugate base exponentially, with precipitation of the sucrose-stabilized PA occurring far below the saturation pH in alkaline solution and at the saturation pH in neutral solution. Using glutaraldehyde (G) at 4% concentration to preserve the cells and using 2-7% PASG solutions at pH 8.5, calcium bound to organic compounds was localized in the hypertrophied chondrocytes at the zone of calcification with optimal reaction. The minimal incubation time for optimal reaction increased with decreasing PA concentration; from 2 hr for 7% PA to 36 hr for 2% PA. With optimal reaction, electron-dense granules were localized in the nucleus, nuclear membrane, endoplasmic reticulum cisterns, mitochondria, Golgi apparatus, intercisternal matrix, plasma membrane, intercellular matrix, and calcospherules. Ethyleneglycoltetraacetic acid immersion and X-ray microprobe studies of the electron-dense granules indicated calcium as the predominant cation. Control tissues with implanted tricalcium phosphate crystals, and reaction kinetic between PA and cellular calcium, indicated that the calcium localized was not due to translocation but to calcium bound to organic compound that is synthesized by the hypertrophied cells in the zone of calcification.