Stimulatory effect of 1 alpha, 25-dihydroxyvitamin D3 on the formation of skin tumors in mice treated chronically with 7,12-dimethylbenz[a]anthracene.

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) and its 24,24-difluoro analog on the formation of skin tumors in mice was evaluated in a complete carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the carcinogen. Twice weekly topical application of 0.25-0.50 nmol of 1 alpha, 25-(OH)2D3 or 0.05-0.10 nmol of the difluoro analog of 1 alpha, 25-(OH)2D3 1 hour prior to treatment with 50 nmol DMBA stimulated tumor formation several fold compared to animals receiving DMBA alone. Topical application of 0.50 nmol of 1 alpha, 25-(OH)2D3 24 hours after treatment with DMBA, or half of this dose of the vitamin D3 metabolite, applied 1 hour before and 24 hours after treatment with DMBA, also stimulated tumor formation several fold. These results are in marked contrast to the potent inhibitory effect of 1 alpha, 25-(OH)2D3 and its difluoro analog on the formation of skin tumors in mice promoted by 12-O-tetradecanoylphorbol-13-acetate.

19-nor-10-ketovitamin D derivatives: unique metabolites of vitamin D3, vitamin D2, and 25-hydroxyvitamin D3.

Three metabolites were isolated after incubation of vitamin D3, vitamin D2, or 25-hydroxyvitamin D3 with bovine rumen microbes. They are identified as 5(E)-19-nor-10-ketovitamin D3, 5(E)-19-nor-10-ketovitamin D2, and 5(E)-19-nor-10-keto-25-hydroxyvitamin D3, respectively. The identifications were based on ultraviolet absorbance, mass spectroscopy, and chemical reactivity. All 5(E)-19-nor-10-ketovitamin D derivatives examined had an absorbance maximum at 312 nm and a characteristic fragment in their mass spectra corresponding to loss of 43 amu from their molecular ions. The vitamin D3 metabolite was identical in all essential spectral and chromatographic aspects with authentic synthetic 5(E)-19-nor-10-ketovitamin D3. These metabolites represent a unique pathway of vitamin D metabolism and the first characterized products of microbial vitamin D metabolism. The conversion of vitamin D and its metabolites to their 19-nor-10-keto forms likely represents a detoxification mechanism.

Plasma concentrations of 1,25-dihydroxyvitamin D, 1,24R,25-trihydroxyvitamin D3, and 2,25,26-trihydroxyvitamin D3 after their administration to dairy cows.

Intramuscular administration of 1,25 (R or S),26-trihydroxyvitamin D3 to dairy cows resulted in significant elevation of 1,25-dihydroxyvitamin D3 concentration in blood plasma. The increase of 1,25-dihydroxyvitamin D3 in plasma paralleled the increase of 1,25,26-trihydroxyvitamin D3. We also found that intramuscular 1,25-dihydroxyvitamin D3 administration to dairy cows significantly elevated plasma 1,24,25-trihydroxyvitamin D3. These data suggest possible impairment of 1,25-dihydroxyvitamin D3 metabolism during the presence of pharmacologic concentrations of 1,25,26-dihydroxyvitamin D3 in plasma. In addition, the 24-hydroxylation of 1,25-dihydroxyvitamin D3 to 1,24,25-trihydroxyvitamin D3 appears to be an important route for further metabolism of 1,25-dihydroxyvitamin D3 in dairy cows.

23S,25-dihydroxyvitamin D3 as a circulating metabolite of vitamin D3. Its role in 25-hydroxyvitamin D3-26,23-lactone biosynthesis.

23,25-Dihydroxyvitamin D3 was isolated from vitamin D-toxic pig plasma by sequential chromatography through two gravity columns and three high performance liquid chromatography systems. Two of the high performance liquid chromatography systems separated the R and S diastereomers of 23,25-dihydroxyvitamin D3 and demonstrated that the metabolite has the 23S configuration. Ultraviolet absorbance and mass spectroscopy of the pure metabolite and mass spectroscopy of its trisilylated derivative confirmed the structural assignment. 23S,25-Dihydroxyvitamin D3, 23R,25-dihydroxyvitamin D3, 25S,26-dihydroxyvitamin D3, and 25R,26-dihydroxyvitamin D3 were assessed for their ability to produce 25-hydroxyvitamin D3-26,23-lactone in vitamin D2-toxic rats. On 23S,25-dihydroxyvitamin D3, of the naturally occurring compounds, was able to increase the plasma lactone concentration. This metabolite was a more efficient precursor than 25-hydroxyvitamin D3, suggesting that 23S-hydroxylation is a rate-limiting step in 25-hydroxyvitamin D3-26,23-lactone formation. 23S,25-Dihydroxyvitamin D3 was not detected in 25-hydroxyvitamin D3-dosed rats, indicating that the former is rapidly metabolized. Nephrectomized rats had a diminished but significant ability to synthesize 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3. Nephrectomy did not affect synthesis of 25-hydroxyvitamin D3-26,23-lactone from 23S,25-dihydroxyvitamin D3. These results demonstrate that vitamin D3 23S-hydroxylase(s) are also located extrarenally and that extrarenal tissues are quantitatively important to 25-hydroxyvitamin D3-26,23-lactone synthesis.

1 Alpha-25,26-trihydroxyvitamin D3: an in vivo and in vitro metabolite of vitamin D3.

A new metabolite of vitamin D3 has been isolated from the plasma of vitamin D3 treated cows and has been generated from 25(S),26-dihydroxyvitamin D3 with homogenates of vitamin D deficient chick kidney. This metabolite has been identified as 1,25,26-trihydroxyvitamin D3 by comigration with synthetic 1,25(S),26-trihydroxyvitamin D3 in four chromatographic systems, ultraviolet spectroscopy, mass spectrometry, and high-pressure liquid chromatography and mass spectrometry of derivatives. 1,25(S),26-Trihydroxyvitamin D3 is one-tenth as effective as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol 1,25-dihydroxyvitamin D receptor. Either 25(S),26-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 can serve as precursor for in vitro production of 1,25,26-trihydroxyvitamin D3 by chick kidney tissue.