Past work has shown that the acidic phospholipids are involved in tissue mineralization ( 1-3). Calcium-phospholipid-phosphate (Ca-PL-P04) complexes, containing the acidic phospholipids, phosphatidyl inositol (PI) and phosphatidyl serine (PS), were isolated from the long bones of developing animals (4). These complexes induced hydroxyapatite (HA) formation in metastable calcium phosphate solutions (5). The same study also showed that the uncomplexed acidic phospholipids when placed in metastable calcium phosphate solution, first formed a Ca-PL-P04 complex and then nucleated HA. The fact that more complexed lipids were found in the bones of developing as opposed to more mature animals further supports the thesis that such compounds are related to the onset of mineralization. The purpose of this study was to analyze further for complexed lipids in a number of hard and soft tissues in order to enlarge our view of the role of the Ca-PL-PO4 complexes in mineralization.Materials and methods. This investigaton was carried out on white New Zealand rabbits of ages 1 4 days (50-70 g), 4-6 weeks (0.9-1.4 kg), 12-16 weeks (1.5-1.8 kg), and older than 100 weeks (3.6-5.4 kg). The tissues studied were: cortex of long bones, epiphyseal regions of long bones (including articular cartilage, growth plate, and secondary ossification centers), skull bones (cranium and mandible), scapulae, marrow, adipose tissue, heart muscle, pooled leg muscles, ear cartilage, and incisors. All bones were scraped to remove the periosteum and marrow, ground in a liquid nitrogen-cooled colloid mill, washed in 0.05 M tris-hydroxy-methyl amino methane-HC1 (Tris) buffer at pH 7.4 to remove blood, and then were lyophilized. Noncalcified tissues were frozen and minced with dry ice and then lyophilized. The ash weight

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The diphosphonates disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and disodium dichloromethylene diphosphonate (Cl2MDP) prevent hydroxyapatite (HA) formation in metastable calcium phosphate solutions, induced by calcium-phospholipid-phosphate complexes and by the acidic phospholipids phosphatidyl serine and phosphatidyl inositol. The diphosphonates appear to act not only as HA crystal poisons but also as surfactants which probably change the nature of the lipid micelle and the charge and conformational properties of the lipid molecules. The surfactants sodium dodecyl sulfate (SDS) and Non-Idet P-40 (NP-40), like the diphosphonates, prevent HA formation by the acidic phospholipids and complexed lipids, but do not act as HA surface poisons. The lipid surfactant lyso-phosphatidyl serine did not induce HA formation from solution. The relevance of the ability of the diphosphonates to act as lipid surfactants to the in vivo use of these agents is discussed.