Double bond in the side chain of 1α,25-dihydroxy-22-ene-vitamin D 3 is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney

Abstract

1α,25-Dihydroxyvitamin D 3 [1α,25(OH) 2D 3] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1α,25-dihydroxyvitamin D 2 [1α,25(OH) 2D 2] like 1α,25(OH) 2D 3, is also converted into the same final product, calcitroic acid. This finding indicated that 1α,25(OH) 2D 2 also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1α,25(OH) 2D 2 when compared to the side chain of 1α,25(OH) 2D 3, has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1α,25(OH) 2D 2. We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D 2 related compounds. We compared the metabolism of 1α,25-dihydroxy-22-ene-vitamin D 3 [1α,25(OH) 2-22-ene-D 3], a synthetic vitamin D analog whose side chain differs from that of 1α,25(OH) 2D 3 only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1α,25(OH) 2-22-ene-D 3 and 1α,25(OH) 2D 3 are converted into common metabolites namely, 1α,24( R),25-trihydroxyvitamin D 3 [1α,24( R),25(OH) 3D 3], 1α,25-dihydroxy-24-oxovitamin D 3 [1α,25(OH) 2-24-oxo-D 3], 1α,23( S),25-trihydroxy-24-oxovitamin D 3 and 1α,23-dihydroxy-24,25,26,27-tetranorvitamin D 3. This finding indicates that the double bond in the side chain of 1α,25(OH) 2-22-ene-D 3 is reduced during its metabolism. Along with the aforementioned metabolites, 1α,25(OH) 2-22-ene-D 3 is also converted into two additional metabolites namely, 1α,24,25(OH) 3-22-ene-D 3 and 1α,25(OH) 2-24-oxo-22-ene-D 3. Furthermore, we did not observe direct conversion of 1α,25(OH) 2-22-ene-D 3 into 1α,25(OH) 2D 3. These findings indicate that 1α,25(OH) 2-22-ene-D 3 is first converted into 1α,24,25(OH) 3-22-ene-D 3 and 1α,25(OH) 2-24-oxo-22-ene-D 3. Then the double bonds in the side chains of 1α,24,25(OH) 3-22-ene-D 3 and 1α,25(OH) 2-24-oxo-22-ene-D 3 undergo reduction to form 1α,24( R),25(OH) 3D 3 and 1α,25(OH) 2-24-oxo-D 3, respectively. Thus, our study indicates that the double bond in 1α,25(OH) 2-22-ene-D 3 is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1α,25(OH) 2-22-ene-D 3 metabolism indicating that prior C-24 hydroxylation of 1α,25(OH) 2-22-ene-D 3 is required for the double bond reduction to occur.