Specificity of the Redox Complex between Cytochrome P450 24A1 and Adrenodoxin Relies on Carbon-25 Hydroxylation of Vitamin-D Substrate.

Metabolic deactivation of 1,25(OH)2D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. CYP24A1 function relies on electron transfer from the soluble ferredoxin protein adrenodoxin (Adx). Recent structural evidence suggests that regioselectivity of the CYP24A1 reaction may correlate with distinct modes of Adx recognition. Here we used nuclear magnetic resonance (NMR) spectroscopy to monitor the structure of 15N-labeled full-length Adx from rat while forming the complex with rat CYP24A1 in the ligand-free state or bound to either 1,25(OH)2D3 or the vitamin-D supplement 1α(OH)D3. Although both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a crosslinking analysis suggested that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key finding is that, whereas the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site α-helix 3 of rat adrenodoxin, the interaction in the presence of 1α(OH)D3, which is lacking the carbon-25 hydroxyl, results in disruption of the NMR peak broadening pattern, thus indicating a ligand-induced nonspecific protein interaction. These findings provide a structural basis for the poor substrate turnover of side-chain-modified vitamin-D analogs, while also confirming that specificity of the CYP24A1-ligand interaction influences specificity of CYP24A1-Adx recognition. SIGNIFICANCE STATEMENT: Mitochondrial cytochrome P450 enzymes, such as CYP24A1 responsible for catabolizing vitamin-D and its analogs, rely on a protein-protein interaction with a ferredoxin in order to receive delivery of the electrons required for catalysis. In this study, we demonstrate that this protein interaction is influenced by the enzyme-ligand interaction that precedes it. Specifically, vitamin-D missing carbon-25 hydroxylation binds the enzyme active site with high affinity but results in a loss of P450-ferredoxin binding specificity.

Relative biological value of 1α-hydroxycholecalciferol to 25-hydroxycholecalciferol in broiler chicken diets.

This study was conducted to evaluate the relative biological value (RBV) of 1α-hydroxycholecalciferol (1α-OH-D3) to 25-hydroxycholecalciferol (25-OH-D3) in one- to 21-day-old broiler chickens fed calcium (Ca)- and phosphorus (P)-deficient diets. On the d of hatch, 450 male Ross 308 broiler chickens were weighed and randomly allotted to 9 treatments with 5 replicates of 10 birds per replicate. The basal diet contained 0.50% Ca and 0.25% non-phytate phosphorus (NPP) but was not supplemented with cholecalciferol (vitamin D3). The levels of Ca and NPP in basal diets were lower than those recommended by NRC (1994). 25-OH-D3 was fed at zero, 1.25, 2.5, 5.0, and 10.0 µg/kg, and 1α-OH-D3 was fed at 0.625, 1.25, 2.5, and 5.0 µg/kg. The RBV of 1α-OH-D3 to 25-OH-D3 based on vitamin D intake was determined by the slope ratio method. Results showed that 25-OH-D3 or 1α-OH-D3 improved the growth performance and decreased the mortality in one- to 21-day-old broilers. A linear relationship was observed between the level of 25-OH-D3 or 1α-OH-D3 and mineralization of the femur, tibia, or metatarsus. The RBV of 1α-OH-D3 to 25-OH-D3 were 234, 253, and 202% when the weight, ash weight, and Ca percentage of femur were used as criteria. The corresponding RBV of 1α-OH-D3 to 25-OH-D3 were 232 to 263% and 245 to 267%, respectively, when tibia and metatarsus mineralization were used as criteria. These data indicate that when directly feeding a hormonally active form of vitamin D as 1α-OH-D3 proportionally less is needed than when using the precursor (25-OH-D3) in diets deficient in Ca and P.

Conversion of oral alfacalcidol to oral calcitriol in the treatment of secondary hyperparathyroidism in chronic hemodialysis patients.

PURPOSE: The optimal vitamin D3 therapy for the treatment of secondary hyperparathyroidism (SHPT) in chronic hemodialysis patients is still controversial. Recent studies suggest that uremia in end-stage renal disease is associated with enzymatic hepatic dysfunction altering 25-hydroxylation of vitamin D3. The goal of our study was to compare the efficacy of calcitriol, the fully hydroxylated active form of vitamin D3, to alfacalcidol which needs 25-hydroxylation to be effective, for the treatment of SHPT in chronic hemodialysis patients. METHODS: We retrospectively reviewed 45 chronic hemodialysis patients who were switched from oral alfacalcidol to oral calcitriol for the treatment of SHPT. Parathyroid hormone (PTH), serum calcium and serum phosphorus levels were compared pre- and post-conversion using paired Student's t tests. RESULTS: The mean dose of active vitamin D3 decreased from 3.50 mcg/week at baseline to 2.86 mcg (P < 0001) after the switch from alfacalcidol to calcitriol. PTH significantly decreased from 94.4 to 82.6 pmol/L (-11.8 pmol/L, P = 0.02). The mean corrected calcium increased from 2.17 to 2.25 mmol/L (+0.08 mmol/L, P < 0.001) without any clinically significant hypercalcemia, and phosphorus levels were stable. Results were similar in a subgroup of patients (n = 17) for whom the medication was administrated during the hemodialysis session, ensuring a complete compliance. CONCLUSIONS: According to our study, calcitriol in equal dosage is more effective than alfacalcidol in lowering serum PTH level in chronic hemodialysis patients. This suggests that calcitriol may be the optimal active vitamin D3 for the treatment of SHPT in chronic hemodialysis patients.

Comparative effects of 1α-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(-/-) mice.

Previous studies have shown that 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P-gp expression. The present study compared the equimolar efficacies of 1α-hydroxyvitamin D3 [1α(OH)D3 ] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half-life that is converted to 1,25(OH)2 D3 , and 1,25(OH)2 D3 on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)-directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(-/-)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D3 and 1,25(OH)2 D3 , 1α(OH)D3 treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(-/-) mice compared to 1,25(OH)2 D3 treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P-gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D3 treatment. The higher activity of 1α(OH)D3 was explained by its rapid conversion to 1,25(OH)2 D3 in tissue sites, furnishing higher plasma and tissue 1,25(OH)2 D3 levels compared to following 1,25(OH)2 D3 -treatment. In conclusion, 1α(OH)D3 exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)2 D3 in mice.

Human pharmacokinetics of orally administered (24 R)-hydroxycalcidiol.

To gain an insight in the regulation of (24R)-hydroxycalcidiol, we studied the pharmacokinetics of orally administered (24R)-hydroxycalcidiol in 6 healthy subjects without calcium supplementation, in 4 healthy subjects with calcium supplementation and in 6 patients with primary hyperparathyroidism. Various quantities related to calcium and vitamin D metabolism were also monitored. In the healthy subjects without calcium supplementation, the basal (24R)-hydroxycalcidiol concentration (Cb) in serum was 2.4 +/- 0.8 nmol/l (mean +/- SD, n = 5), the terminal serum half-time (t 1/2) 7.2 +/- 1.4 days, the production rate 0.05 +/- 0.01 nmol/kg.day, and the production rate/[calcidiol] ratio (1.5 +/- 0.4 x 10(-3) l/kg.day). In the healthy subjects studied, the serum concentration vs time curves exhibited a second maximum after administration, possibly due to binding by intestinal cells or (partial) uptake by the lymph system. In the calcium-supplemented healthy subjects, the pharmacokinetic quantities were not significantly different while the area under the serum concentration-time curve and the estimated bioavailability were significantly decreased. Basal concentration (Cb), production rate and the production rate/[calcidiol] ratio were significantly lower in patients with primary hyperparathyroidism but t 1/2 was unchanged. Exogenous (24R)-hydroxycalcidiol had no clear effect on calcium and vitamin D metabolism. In conclusion, a) exogenous (24R)-hydroxycalcidiol has no clear effect on calcium and vitamin D metabolism, b) clearance and production rate of (24R)-hydroxycalcidiol are not affected by calcium supplementation, c) bioavailability is lower in the calcium-supplemented state, d) basal concentration (Cb) and production rate are significantly decreased in patients with hyperparathyroidism.