Physiological Significance of C-28 Hydroxylation in the Metabolism of 1α,25-Dihydroxyvitamin D2

In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1α,25-dihydroxyvitamin D2 [1α,25(OH)2D2] by identifying 1α,24(S),25,28-tetrahydroxyvitamin D2 [1α,24(S),25,28(OH)4D2] as a major renal metabolite of 1α,25(OH)2D2 [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328–5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1α,25(OH)2D2. We perfused rat kidneys in vitro with 1α,25(OH)2[26,27-3H]D2 (5 × 10−10M) and demonstrated that 1α,24(R),25-trihydroxyvitamin D2 [1α,24(R),25(OH)3D2] and 1α,24(S),25,28(OH)4D2 are the only two major physiological metabolites of 1α,25(OH)2D2. In the same perfusion experiments, we also noted that there is no conversion of 1α,25(OH)2D2 into 1α,25,28-trihydroxyvitamin D2 [1α,25,28(OH)3D2]. Moreover, 1α,24(S),25,28(OH)4D2 is not formed in the perfused rat kidney when synthetic 1α,25,28(OH)3D2 is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1α,25(OH)2D2 occurs only after 1α,25(OH)2D2 is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1α,24(R),25(OH)3D2 in rat kidney is not known. Recently, it was found that 1α,25(OH)2D3-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D3 compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1α,24(R),25(OH)3D2 to form 1α,24(S),25,28(OH)4D2. The biological activity of 1α,24(S),25,28(OH)4D2, determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1α,24(S),25,28(OH)4D2 exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1α,25(OH)2D2 in kidney under physiological conditions.