Extraction ofVitamin D Metabolites byBonesofNormalAdultDogs

Using theisolated perfused canine tibia we examined theextraction of(3H)25(OH)D3, (3H)1,25(OH)2D3 and(3H)24,25(OH)2D3 byboneof normal adult dogs. Thestudies wereperformed with andwithout vitamin D binding protein (DBP)inthe perfusate toexamine theeffect ofprotein binding on theextraction ofthevitamin D metabolites. Anav- erage of48±2%of(3H)25(OH)D3 wasextracted by bone, whennoDBPwaspresent. However, addition ofonlyasmall amountofDBP(-720ng/mlofper- fusate) nearly completely abolished theextraction of (3H)25(OH)D3 bybone. Nodegradation and/or trans- formation ofthelabeled 25(OH)D3 could bedemon- strated during passage through theisolated perfused bone. Theextraction of(3H)24,25(OH)2D3 inaDBP- free mediumaveraged 33±5%.Addition of720ngof DBP/mlofperfusate completely inhibited the extraction ofthismetabolite. Theextraction of (3H)1,25(OH)2D3 averaged 30±3%inaDBPfree me- diumandnoinhibition oftheextraction wasdemon- strated after addition ofDBP(720 ng/ml ofperfusate). However, addition ofDBPinaconcentration of14.4 jig/ml ofperfusate reduced theextraction of 1,25(OH)2D3 to8±2%,avalue still significantly higher thanthat seenafter addition of20times less DBPto perfusions with25(OH)D3 and24,25(OH)2D3. Itiscon- cluded thattheisolated perfused boneofnormal dogscanextract significant amounts of25(OH)D3, 1,25(OH)2D3, and24,25(OH)2D3. Small concentrations ofDBP(720ng/ml) intheperfusate significantly in- hibited theextraction of25(OH)D3 and24,25(OH)2D3. Acarrier role forDBPissuggested anditisproposed thatthelevels offreevitamin D areimportant for extraction ofthemetabolites bybone. Therefore, due tothedifferent affinities ofDBPforthevarious me-