Partition of inorganic ions and phospholipids in isolated cell, membrane and matrix vesicle fractions: evidence for Ca-Pi-acidic phospholipid complexes.

Electrolytes and phospholipids of cartilage fractions were partitioned by extraction with organic and aqueous solvents into six solubility groups: Electrolytes I, II and III, and Lipids I, II and III. Of the total Ca, only 4% was water soluble (Electrolytes I); 4-12% was complexed with lipids (Electrolytes II); while the majority (84-92%) was insoluble (Electrolytes III). In contrast, nearly half of the Mg and Pi were water soluble. Of the neutral phospholipid, 95% was not complexed with mineral ions (Lipids I), but 30-45% of the acidic phospholipid was (Lipids II). Ca/Pi ratios were extremely low in the water-soluble phase, but were in the range of amorphous calcium phosphate (ACP) in the insoluble. Molar ratios of the lipid-mineral complex were: Ca:Mg:Pi:acidic phospholipid, 4:3:2:2. Mg/Ca ratios in the soluble fraction were high (5.5-8.9), sufficient to stabilize ACP. Kinetic studies revealed rapid turnover of soluble Ca, insoluble turning over much more slowly. Labeling of lipid-complexed Ca was rapid in cells, but occurred later in matrix vesicles, suggesting transfer. While lipid-Ca-Pi complexes can nucleate apatite in vitro, those present in vivo inside matrix vesicles apparently do not because of the excess Mg. We conclude therefore, that in vesicle-mediated calcification, lysis of the membrane may be essential to allow release of internal Mg.