20-Hydroxyvitamin D3 [
20(OH)D3], the major product of
CYP11A1 action on
vitamin D3, is biologically active and like
1,25-dihydroxyvitamin D3 [
1,25(OH)2D3] can inhibit proliferation and promote differentiation of a range of cells, and has anti-inflammatory properties. However, unlike
1,25(OH)2D3, it does not cause toxic
hypercalcemia at high doses and is therefore a good candidate for
therapeutic use to treat hyperproliferative and autoimmune disorders. In this study we analyzed the ability of mouse liver microsomes to metabolize
20(OH)D3. The two major products were identified from authentic standards as 20,24-dihydroxyvitamin D3 [20,24(
OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(
OH)2D3]. The reactions for synthesis of these two products from
20(OH)D3 displayed similar Km values suggesting that they were catalyzed by the same
cytochrome P450. Some minor metabolites were produced by reactions with higher Km values for
20(OH)D3. Some metabolites gave mass spectra suggesting that they were the result of hydroxylation followed by dehydrogenation. One product had an increase in the wavelength for maximum absorbance from 263nm seen for
20(OH)D3, to 290nm, suggesting a new double bond was interacting with the
vitamin D-triene chromophore. The two major products, 20,24(
OH)2D3 and 20,25(
OH)2D3 have both previously been shown to have higher potency for inhibition of colony formation by
melanoma cells than
20(OH)D3, thus it appears that metabolism of
20(OH)D3 by mouse liver microsomes can generate products with enhanced activity.