10-Keto or 25-hydroxy substitution confer equivalent in vitro bone-resorbing activity to vitamin D₃

The biological activities of 10-keto derivatives of vitamin D₃ and 25-hydroxyvitamin D₃ were determined in bone organ culture. Fetal rat limb bones prelabeled with ⁴⁵Ca were incubated for 60 h with 10-keto-25-hydroxyvitamin D₃, 10-keto-vitamin D₃, 1,25-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃, or vitamin D₃. Resorption was quantified by release of ⁴⁵Ca. Substitution of a keto group in the 10 position of the vitamin D₃ molecule resulted in a compound equal in potency to 25-hydroxyvitamin D₃. When a 10-keto group was substituted in the 25-hydroxy vitamin D₃ molecule, the potency was increased 20- to 40-fold. In contrast, 1,25-dihydroxyvitamin D₃ was 7500-fold more potent than 25-hydroxyvitamin D₃. Since 10-keto-25-hydroxyvitamin D₃ has a retention time close to that of 1,25-dihydroxyvitamin D₃ on normal-phase HPLC eluted with isopropanol:hexane, it is a possible artifact in the assay of 1,25-dihydroxy-vitamin D₃. Based upon the observed relative activities of the two compounds, the concentration of 10-keto-25-hydroxyvitamin D₃ would have to be greater than 0.8 ng/ml for it to interfere in the bioassay of 1,25-dihydroxyvitamin D₃.