In vitro action of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol on matrix organization and mineral distribution in rabbit growth plate.

Growth plates of 18-day-old rabbits were incubated in a protein-free synthetic medium, either without any additive, with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] (10(-10) M), with 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] (10(-10) M and 10(-9) M), with both metabolites, or with the ethanol solvent alone. Cartilages, before and after 5 days of incubation, were studied by light and electron microscopy. The intracellular calcium distribution was analyzed by the potassium pyroantimonate method, and the calcium content was verified by x-ray microprobe analysis. When compared to nonincubated samples the cartilages incubated for 5 days without any additive as well as the cartilages incubated with the solvent alone showed excessive hydratation and hypertrophy of the chondrocytes, which had lost their columnar arrangement. The matrix and the cells were devoid of mineral. The ultrastructure of the cells was well preserved. These changes were largely prevented by the presence of both vitamin D3 metabolites. With regard to calcium distribution, 1,25-(OH)2D3 maintained calcium in mitochondria and crystals in matrix vesicles, whereas 24,25-(OH)2D3 only partly maintained mitochondrial mineral. In the chondrocytes incubated with this latter metabolite, small calcium granules were seen in the cytoplasm; most vesicles were devoid of crystals, and amorphous precipitates were seen in the matrix. These data demonstrate the in vitro influence of vitamin D3 metabolites on the organization and mineralization of the cartilage matrix and on the distribution of intracellular calcium in chondrocytes. Furthermore, they support the hypothesis that the in vitro action of 1,25-(OH)2D3 is different from that of 24,25-(OH)2D3 in that 1,25-(OH)2D3 may influence calcium storage in mitochondria and matrix vesicles, whereas 24,25-(OH)2D3 is likely to be involved in calcium transport and release.

The in vitro production and activity of 24, 25-dihydroxycholecalciferol in cartilage and calvarium.

Previously reported results from our laboratory have indicated that 24, 25-dihydroxycholecalciferol can be formed in vitro during incubations of cartilage tissue or cartilage cells with 25-hydroxycholecalcified. They have also demonstrated the high potency of this dihydroxymetabolite of vitamin D3 in stimulating in vitro the sulfate incorporation into proteoglycans of cartilage cells in culture and in decreasing in vitro the parathyroid hormone action on rat calvarium phosphatases activities. The present report shows that 24, 25-)OH)2 D3 can also be produced during rat calvarium incubations with 25-hydroxycholecalciferol and that therefore calvarium as well as cartilage might be both a site of formation and a site of action for 24, 25-(OH)2 D3. The review of recent experimental and clinical investigations strongly suggests that this metabolite has a physiological significance and may be specifically active on some parameters of bone mineralization. Further studies on the cartilage and calvarium abilities to convert 25-hydroxycholecalciferol into 24, 25-(OH)2 D3 shows that this transformation occurs in the mitochondrial fraction but that it does not seem modified by factors known to control the 25-hydroxycholecalciferol metabolism in the kidney. Finally the analysis of experimental and clinical results published so far does not yet bring enough information to understand the significance of this extrarenal metabolism of 25-hydroxycholecalciferol.

Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage.

In the present work, the capacity of cartilage to metabolize 25-hydroxycholecalciferol was investigated. Cartilage preparations from growth plate, articular surface, rib, scapula, and ear were isolated from 3-week-old normal rabbits and chickens. Each tissue was separately incubated with tritiated 25-hydroxycholecalciferol (, x 10(-9) M) for 1-24 h. Incubations of kidney and muscle were performed simultaneously for comparison. Similarly, cultured chondrocytes isolated from rabbit growth plate and articular cartilage were incubated for 1 or 20 h in medium free of fetal calf serum. After methanol-chloroform extraction of tissues, cells, and their respective media, chloroform phases were chromatographed on Sephadex LH-20 columns. The results show that kidney and cartilage are able to convert 25-hydroxycholecalciferol into a derivative which migrates in the 24,25-dihydroxycholecalciferol region. Cartilage tissue previously boiled is unable to metabolize 25-hydroxycholecalciferol. The conversion of 25-hydroxycholecalciferol occurs with all types of cartilage and is also observed in incubations of cultured chondrocytes. In the latter, the polar 25-hydroxycholecalciferol derivative is detected as early as 1 h after addition of 25-hydroxycholecalciferol. Two findings suggest that the polar derivative of 25-hydroxycholecalciferol produced by cartilage is 24,25-dihydroxycholecalciferol: 1) the cartilage derivative and 24,25-dihydroxycholecalciferol (synthetic and biosynthetic) comigrate during Sephadex LH-20 and high liquid pressure chromatography; and 2) both the cartilage derivative and 24,25-dihydroxycholecalciferol are sensitive to periodate treatment.