Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl4 intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca2+-binding sites in mitochondria and to the general problems of soft tissue calcification.

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