Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Abstract Two new metabolites of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], namely 1,25(OH) 2 -24-oxo-vitamin D 3 and 1,23,25(OH) 3 -24-oxo-vitamin D 3 , have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH) 2 -[23- 3 H]-24-oxo-D 3 and 1,23,25(OH) 3 -[23- 3 H]-24-oxo-D 3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH) 2 -[23,24- 3 H]-D 3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH) 2 D 3 . The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH) 2 D 3 . Whereas the RCI obtained for 1,25(OH) 2 -24-oxo-D 3 was 98 ± 2 (SE), the RCI for 1,23,25(OH) 3 -24-oxo-D 3 was only 28 ± 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH) 2 -24-oxo-D 3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH) 3 -24-oxo-D 3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH) 2 D 3 is metabolized to 1,24 R ,25(OH) 3 D 3 ) 1,25(OH) 2 -24-oxo-D 3 , and then 1,23,25(OH) 3 -24-oxo-D 3 .