Extraction of vitamin D metabolites by bones of normal adult dogs.

Using the isolated perfused canine tibia we examined the extraction of [(3)H]25(OH)D(3), [(3)H]1,25(OH)(2)D(3) and [(3)H]24,25(OH)(2)D(3) by bone of normal adult dogs. The studies were performed with and without vitamin D binding protein (DBP) in the perfusate to examine the effect of protein binding on the extraction of the vitamin D metabolites. An average of 48+/-2% of [(3)H]25(OH)D(3) was extracted by bone, when no DBP was present. However, addition of only a small amount of DBP ( approximately 720 ng/ml of perfusate) nearly completely abolished the extraction of [(3)H]25(OH)D(3) by bone. No degradation and/or transformation of the labeled 25(OH)D(3) could be demonstrated during passage through the isolated perfused bone. The extraction of [(3)H]24,25(OH)(2)D(3) in a DBP-free medium averaged 33+/-5%. Addition of 720 ng of DBP/ml of perfusate completely inhibited the extraction of this metabolite. The extraction of [(3)H]1,25(OH)(2)D(3) averaged 30+/-3% in a DBP free medium and no inhibition of the extraction was demonstrated after addition of DBP (720 ng/ml of perfusate). However, addition of DBP in a concentration of 14.4 mug/ml of perfusate reduced the extraction of 1,25(OH)(2)D(3) to 8+/-2%, a value still significantly higher than that seen after addition of 20 times less DBP to perfusions with 25(OH)D(3) and 24,25(OH)(2)D(3). It is concluded that the isolated perfused bone of normal dogs can extract significant amounts of 25(OH)D(3), 1,25(OH)(2)D(3), and 24,25(OH)(2)D(3). Small concentrations of DBP (720 ng/ml) in the perfusate significantly inhibited the extraction of 25(OH)D(3) and 24,25(OH)(2)D(3). A carrier role for DBP is suggested and it is proposed that the levels of free vitamin D are important for extraction of the metabolites by bone. Therefore, due to the different affinities of DBP for the various metabolites of vitamin D, only 1,25(OH)(2)D(3) is extracted in vitro in significant amounts by bone of normal adult dogs, in the presence of DBP.