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.

Phosphatase content of rat calvaria after in vivo administration of vitamin D3 metabolites.

The effects of acute or chronic administration of small doses (130 pmol) of 25-hydroxycholecalciferol, 24,25-dihydroxycholecalciferol, and 1,25-dihydroxycholecalciferol on rat calvaria acid and alkaline phosphatase activities were investigated in weanling male albino Wistar rats raised on a vitamin D-deficient, low-calcium diet. The results indicate that each of these active metabolites has a different effect on calvarial phosphatase activities. 25-hydroxycholecalciferol causes a significant increase, and 24,25-dihydroxycholecalciferol a decrease in the enzymatic activity. In animals treated with 1,25-dihydroxycholecalciferol these activities are lower after one injection, but after seven daily doses they are not different from those of ethanol-injected control rats. The observed changes do not seem to be related to changes in serum calcium and/or phosphorus concentrations.

Binding of cholecalciferol metabolites to rat duodenal mucosa cytosol.

In vitro binding of 25-hydroxycholecalciferol (25-(OH)D3) and 1alpha,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the duodenal mucosa cytosol of rachitic rats: both 25-(OH)D3 and 1,25-(OH)2D3 bind to a macromolecule (sedimentation coefficient 5.5 to 6 S in low or high ionic strength) with a high affinity (KD at 4 degrees C = 1.2 X 10-9M and 2 x 10-9M, respectively). In addition, it is concluded from competition and chase experiments that 25-(OH)D3 binding sites differ from that for 1,25-(OH)2D3.

1,25-Dihydroxycholecalciferol effect on serum phosphorus homeostasis in rats.

It has recently been shown that 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) increases the serum phosphorus concentration of rats on a low-phosphorus diet. While studying the biological activity of 1,25(OH)2D3, we observed that under certain circumstances 1,25-(OH)2D3 would decrease the serum phosphorus concentration. The analysis of all data obtained in rat experiments during the past 3 years revealed highly significant linear correlations (P less than 0.001) between changes of serum phosphorus concentrations after the administration of 1,25-(OH)2-D3 (130 pmol/d for 1 or 5 days) and serum phosphorus or calcium levels in the animals before injection. Similar correlations could only be found with the higher dose of 25-hydroxycholecalciferol (130 pmol/d for 5 days). Another vitamin D3 metabolite, 24,25-dihydroxycholecalciferol, had no effect on serum phosphorus concentrations under our experimental conditions. The 1,25-(OH)2D3 effect on serum phosphorus concentration does not require the presence of circulating parathormone and/or calcitonin. We suggest that 1,25-(OH)2D3 might be an important factor in serum phosphorus homeostasis.