Calcergy and calciphylaxis: Timed appearance of γ-carboxyglutamic acid and osteocalcin in mineral deposits

γ-Carboxyglutamate (Gla), a calcium binding amino acid whose synthesis depends on vitamin K, has been found in association with pathologic calcifications. It is of interest therefore to examine the role of Gla-containing proteins in the formation of nonskeletal mineralized tissues. Calcergy and calciphylaxis, experimentally induced models of pathologic calcification, offer the opportunity to study the formation of mineral deposits in the absence of an endochondral sequence of bone formation. Before induction of subcutaneous calcinosis by topical treatment with the direct calcergen, KMnO4, or by challenging dihydrotachysterol-sensitized animals with FeCl2, control specimens contain no γ-carboxyglutamic acid. With the initial formation of cytoplasmic vesicles, calcium content of the tissues increases and Gla is detected. Gla levels are further elevated with the appearance of poorly crystalline apatite-like crystallites. Origin of protein bound Gla was established by positive identification of osteocalcin by radioimmunoassay. Gla and osteocalcin appear concomitant with the earliest mineral deposits observed by electron microscopy and micro X-ray analysis. The formation of organized extracellular mineral deposits allowed sufficient mineral accumulation for detection of an apatite-like pattern by X-ray diffraction with calcium, Gla, and osteocalcin increasing proportionately as mineral is deposited.