26-Hydroxylation of 1α,25-Dihydroxyvitamin D3 Does Not Occur under Physiological Conditions

The 26-hydroxylation of 1α,25-dihydroxyvitamin D3 in rats in vitro and in vivo was studied under physiological conditions. Incubation of 1α,25-dihydroxy-[26,27-3H]vitamin D3 with rat kidney or rat liver homogenate showed formation of a metabolite that was identified as 1α,25(S),26-trihydroxy-[26,27-3H]vitamin D3 by comigration on three different HPLC systems and a periodate cleavage reaction. This metabolite was not generated by hydroxylation of 1α,25-dihydroxy-[26,27-3H]vitamin D3 itself but by an enzymatic conversion of a precursor that was formed nonenzymatically in substantial amounts upon storage of 1α,25-dihydroxy-[26,27-3H]vitamin D3 in ethanol at −20°C under argon for more than three weeks. An in vivo metabolism study in rats dosed with a physiological dose of 1α,25-dihydroxy-[26,27-3H]vitamin D3 confirmed the absence of 26-hydroxylation of the hormone. As expected at 6 h postinjection of purified 1α,25-dihydroxy-[26,27-3H]vitamin D3, 1α,24(R),25-trihydroxy-[26,27-3H]vitamin D3, as well as traces of (23S,25R)-1α,25-dihydroxy-[3H]vitamin D3-lactone were detected and identified on straight phase and reverse phase HPLC in serum, kidney, and liver.