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.

Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-α-Hydroxycholecalciferol.

In the context of the global prevalence of vitamin D insufficiency, we compared two key determinants of the bioavailability of 3 vitamin D forms with significant biopotencies: cholecalciferol, 25-hydroxycholecalciferol and 1-α-hydroxycholecalciferol. To this aim, we studied their incorporation into synthetic mixed micelles and their uptake by intestinal cells in culture. Our results show that 1-α-hydroxycholecalciferol was significantly more solubilized into mixed micelles compared to the other forms (1.6-fold and 2.9-fold improvement compared to cholecalciferol and 25-hydroxycholecalciferol, respectively). In Caco-2 TC7 cells, the hydroxylated forms were taken up more efficiently than cholecalciferol (p < 0.05), and conversely to cholecalciferol, their uptake was neither SR-BI(Scavenger-Receptor class B type I)- nor NPC1L1 (NPC1 like intracellular cholesterol transporter 1)-dependent. Besides, the apical membrane sodium-bile acid transporter ASBT (Apical Sodium-dependent Bile acid Transporter) was not involved, at least in vitro, in the uptake of any of the three vitamin D forms. Further investigations are needed to identify the uptake pathways of both 1-α-hydroxycholecalciferol and 25-hydroxycholecalciferol. However, considering its high bioavailability, our results suggest the potential interest of using 1-α-hydroxycholecalciferol in the treatment of severe vitamin D deficiency.

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.

Randomized trial comparing pulse calcitriol and alfacalcidol for the treatment of secondary hyperparathyroidism in haemodialysis patients.

AIM: Calcitriol and alfacalcidol are used extensively for the treatment of secondary hyperparathyroidism. Unfortunately, there is limited published data comparing the efficacy and tolerability of both active vitamin D sterols. This study was undertaken to determine whether calcitriol provides a therapeutic advantage to alfacalcidol. METHODS: This was a randomized, active controlled study. Patients with intact parathyroid hormone (iPTH) >32 pmol/L were randomized to receive orally calcitriol or alfacalcidol after each haemodialysis for up to 24 weeks. Reduction of PTH, changes of plasma albumin-corrected calcium and phosphorus were analysed. The initial dose of alfacalcidol was twice that of calcitriol. RESULTS: Sixteen patients were randomized into each group. At baseline, plasma albumin-corrected calcium, phosphorus and PTH were no different between groups. At 24 weeks, PTH changes were -50.8 ± 31.8% and -49.4 ± 32.5% from the baseline in the calcitriol and alfacalcidol groups, respectively (P = 0.91). The patients who achieved target PTH of 16-32 pmol/L were 82% in the calcitriol and 67% in the alfacalcidol group (P = 0.44). Plasma albumin-corrected calcium and phosphorus were not significantly different but showed trends toward gradually increasing from baseline in both groups (calcium, 6.0 ± 7.2% vs 10.9 ± 6.5% (P = 0.10); phosphorus, 13.0 ± 29.4% vs 16.7 ± 57.2% (P = 0.83) in calcitriol and alfacalcidol, respectively). The mean dose of calcitriol and alfacalcidol were 4.1 and 6.9 µg/week, respectively (P < 0.0001). CONCLUSION: Alfacalcidol can be used to control secondary hyperparathyroidism at doses of 1.5-2.0 times that of calcitriol. The two drugs are equally efficacious and lead to similar changes in calcium and phosphorus.

Hepatic activation and inactivation of clinically-relevant vitamin D analogs and prodrugs.

Like most pharmaceutical agents, vitamin D analogs are subject to hepatic metabolism by a variety of cytochrome P450 (CYP)-based systems. Metabolism can involve activation as well as inactivation of the vitamin D analog and one of the more successful families includes the 1alpha-hydroxyvitamin D prodrugs (1alpha-OH-D2, 1alpha-OH-D3, 1alpha-OH-D4, 1alpha-OH-D5), that all require a step of activation. Some of these prodrugs are in use or clinical trial because they have a therapeutic advantage over calcitriol. However, the nature of the activation of these molecules is poorly understood, particularly with regard to the CYP isoform involved. Various transfected CYPs and hepatic cell lines combined with tandem LC-MS analysis were used to investigate the metabolism of a spectrum of vitamin D analogs, including 1alpha-OH-Ds and the topical analog, calcipotriol. In the case of the 1alpha-OH-Ds, evidence was found of multiple sites of side-chain hydroxylation consistent with the generation of more than one active form. The potential involvement of CYP27A and other putative 25-hydroxylases in 1alpha-OH-D activation was also shown, as well as the potential for CYP24 activation and inactivation. In the case of calcipotriol, the respective roles of non-vitamin D-related CYPs and CYP24 in the catabolism of this anti-psoriatic drug were dissected out using cell lines with or without CYP24 expression, allowing us to demonstrate the potential contribution of CYP24 to "vitamin D resistance". The implications of hepatic metabolism in the context of other facets thought to play a role in the mechanism of action of anticancer and antiproliferative vitamin D analogs are discussed.

Gluten-induced enteropathy (coeliac disease) revealed by resistance to treatment with levothyroxine and alfacalcidol in a sixty-eight-year-old patient: a case report.

We report the case of a female patient in whom gluten-induced enteropathy was revealed at the age of 68 years by resistance to treatment with levothyroxine and alfacalcidol. This case report shows that malabsorption without major digestive symptoms led to reduction of absorption of levothyroxine (LT(4)) and alfacalcidol, although the patient had normal free thyroxine (T(4)) levels (which increased by 45.8% after a loading dose of 250 microg of levothyroxine), high thyrotropin (TSH) and slightly elevated 1.25 dihydroxy-vitamin D(3) levels, low free triiodothyronine (T(3)) and calcium levels were found. Malabsorption had additional effects by reducing T(3) and calcium levels. Because minor forms of gluten-induced enteropathy are not rare in patients with thyroid autoimmune diseases, the cost-effectiveness of using antigliadin antibodies as a first-line test (instead of an LT(4) loading test) in patients requiring daily doses of levothyroxine above 2 microg per kg of body weight, whatever their age may be, is discussed.

Pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) in normal and uraemic men.

BACKGROUND: The therapeutic equivalence of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) on the suppression of PTH synthesis and secretion has not clearly been established. The aim of the present study was to evaluate the pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) after oral and i.v. administration in healthy volunteers and uraemic patients. METHODS: Six healthy volunteers and 12 uraemic patients were included in the study. With an interval of 2 weeks, 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, 4 microg of 1alpha(OH)D(3) i.v. and 4 microg of 1alpha(OH)D(3) orally were administered. Blood samples for analysis of plasma-Ca(2+), plasma-1,25(OH)(2)D(3), and plasma-PTH were drawn at time 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h. The healthy volunteers were studied in all four protocols and the uraemic patients in either the 1alpha(OH)D(3) (n=6) or the 1,25(OH)(2)D(3) (n=6) protocol. RESULTS: After oral administration of 1,25(OH)(2)D(3) the bioavailability of 1,25(OH)(2)D(3) was 70.6+/-5.8/72.2+/-4.8% in healthy volunteers/uraemic patients (n.s.). After i.v. administration the volume of distribution of 1,25(OH)(2)D(3) was similar, 0.49+/-0.14 vs 0.27+/-0.06 l/kg in healthy volunteers vs uraemic patients (n.s.), while the metabolic clearance rate of 1,25(OH)(2)D(3) was 57% lower in the uraemic patients, 23.5+/-4.34 vs 10.1+/-1.35 ml/min in healthy volunteers vs uraemic patients, respectively (P<0.03). The bioavailability of 1,25(OH)(2)D(3) after i.v. administration of 1alpha(OH)D(3) was 42.4+/-11.0/42.0+/-2.0% in healthy volunteers/uraemic patients (n.s.); and after oral administration of 1alpha(OH)D(3) 42.0+/-2.0/29.8+/-3.1% in healthy volunteers/uraemic patients (n.s.). A small, but significant increase in plasma-Ca(2+) was seen after administration of 1,25(OH)(2)D(3) to the uraemic patients, while no increase was seen after administration of 1alpha(OH)D(3). PTH levels were significantly suppressed in the healthy volunteers 24 h after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) orally by 35+/-7, 30+/-8, and 35+/-4%, respectively (all P<0.03). In the uraemic patients, PTH levels were significantly suppressed after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) i.v. by 30+/-10, 45+/-7, and 40+/-7%, respectively (all P<0.04). The effect was transitory in the healthy volunteers and lasted for at least 72 h in the uraemic patients. CONCLUSION: The present study found a 57% lower metabolic clearance rate of 1,25(OH)(2)D(3) in uraemic patients, as compared with that of healthy volunteers (P<0.03). The bioavailability of 1,25(OH)(2)D(3) following administration of 1alpha(OH)D(3) i.v. and orally in both healthy volunteers and uraemic patients was markedly lower than after administration of oral 1,25(OH)(2)D(3) (P<0.03). In spite of lower plasma-1,25(OH)(2)D(3) levels after administration of 1alpha(OH)D(3), no significant difference was observed on the suppressive effect of 4 microg i.v. of either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) on the plasma-PTH levels in the uraemic patients. This might suggest the existence of an effect of 1alpha(OH)D(3) on the parathyroid glands which is independent of the plasma-1,25(OH)(2)D(3) levels, that are achieved after oral or i.v. administration of 1alpha(OH)D(3).

Quantitative evaluation of 1-alpha-hydroxycholecalciferol as a cholecalciferol substitute for broilers.

Two experiments were conducted using a corn-soybean meal diet that meets or exceeds the NRC (1984) requirements for all nutrients except cholecalciferol (D3) to determine the effectiveness of 1-alpha-hydroxycholecalciferol (1alpha-OHD3) as a substitute for D3 in the diet of young broilers. Ross x Ross mixed-sex, 1-d-old chicks were reared in Petersime battery brooders not exposed to ultraviolet light with feed and water supplied ad libitum for 16 d. In Experiment 1, D3 was fed at 0, 2.5, 5, 10, 20, and 40 microg/kg and one source of 1alpha-OHD3-(Hoffmann-LaRoche, Inc.; HLR) was fed at 0.625, 1.25, 2.5, 5, and 10 microg/kg of diet. In Experiment 2, the D3 was fed at 0, 2.5, 5, and 10 microg and two sources of 1alpha-OHD3-[HLR and Majestic Research Inc. (MRI)] were fed at 0, 0.625, 1.25, and 5 microg/kg of diet. Slope ratio analysis of data from the measurement of 16-d body weight, plasma Ca, rickets, and bone ash indicated bioavailability of the 1alpha-OHD3 as compared to D3 from 1.88 to 21.2. Percentage bone ash gave the most precise values in both experiments. Considering all the data from both experiments, the 1alpha-OHD3 appears to be approximately eight times as effective as D3 for satisfying the requirements of several criteria in two experiments with broiler chickens.

Synthesis and pharmacokinetics of 1 alpha-hydroxyvitamin D3 tritiated at 22 and 23 positions showing high specific radioactivity.

A novel synthesis of a radioactive compound of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-(3)H4]1 alpha OHD3) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22,23-(3)H4]1 alpha OHD3 (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studies with rats. In the pharmacokinetics studies, the plasma concentration level of the active form of vitamin D3, 1 alpha,25-dihydroxy-vitamin D3 [1 alpha,25(OH)2D3], after oral or intravenous administration of [22,23-(3)H4]1 alpha OHD3 (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1 alpha,25(OH)2D3 tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1 alpha OHD3 (1) over the treatment of 1 alpha,25(OH)2D3 (2).

Sustained osteoblast nuclear receptor binding of converted 1alpha, 25-dihydroxyvitamin D3 after administration of 3H-1alpha-hydroxyvitamin D3: a combined receptor autoradiography and radioassay time course study with comparison to 3H-1alpha, 25-dihydroxyvitamin D3.

The present study was undertaken to clarify the receptor distribution and the pharmacokinetics of 3H-1alpha(OH)D3, and 3H-1alpha,25(OH)2D3 for comparison. Receptor autoradiography was used after intravenous injection to 3-day-old neonatal rats and radioassay-HPLC after oral application to young adult rats. Corresponding results were obtained from both receptor autoradiography and radioassay. After 3H-1alpha(OH)D3 administration, uptake was delayed but sustained over a long period of time and the concentration of silver grains (autoradiography) or recovered 3H-1alpha,25(OH)2D3 (radioassay) peaked at a lower level. After 3H-1alpha,25(OH)2D3 administration, osteoblast nuclear, whole bone uptake and retention of radiolabeled compound were relatively rapid and short in duration. Nuclear uptake in osteoblasts after administration of 3H-1alpha(OH)D3 was abolished in competition studies with 10-fold unlabeled 1alpha,25(OH)2D3. These results indicate that 1alpha(OH)D3 continuously supplies osteoblasts with converted 1alpha,25(OH)2D3 and would not spread to the cells because of the low binding affinity of the receptor. Accordingly, 1alpha(OH)D3 appears to have some therapeutic properties different from 1alpha,25(OH)2D3 because of a relatively slow and sustained accumulation of the receptor and less Cmax (pharmacokinetics) compared with 1alpha,25(OH)2D3.

Pharmacokinetics of 1 alpha-hydroxycholecalciferol after intraperitoneal, intravenous and oral administration in patients undergoing peritoneal dialysis.

Intravenous (i.v.) injection of vitamin D3 has a well known suppressive effect on the release of parathyroid hormone. However, the i.v. route is inconvenient in patients undergoing peritoneal dialysis. Moreover, no study has been published on the pharmacokinetics of 1 alpha-hydroxycholecalciferol (1 alpha-OHD3) after intraperitoneal (IP), i.v. and oral administration. Therefore, the appearance of 1,25-(OH)2D3 after administration of 1 alpha-OHD3 was studied in 8 peritoneal dialysis patients. Open, prospective, randomized cross-over design with single doses of 1 alpha-OHD3 (80 ng/kg BW) given on 3 separate occasions either IP, i.v. or oral was applied. After the administration of 1 alpha-OHD3, blood was collected at baseline and 0.5, 1, 2, 3, 4, 6, 12 and 24 h for measurement of circulating 1,25-(OH)2D3. A one compartment model with first order absorption and elimination (Cpl = Be-ke*t-Ae-ka*t) was fitted to the concentrations following i.v. administration. Following IP and oral administration the concentrations did not reach maximum levels within the time of blood sampling. In all cases, the 24 h area under the time/concentration curve for 1,25-(OH)2D3 (AUC24) was calculated using the trapezoidal method. Residual areas were calculated using the terminal slope from i.v. administration, and added to AUC24 giving AUC0-->infinity. After i.v. administration A, ka, B, ke, t1/2, AUC24 and AUC0-->infinity were (mean +/- SD) 62.9 +/- 16.4 pg/ml, 0.76 +/- 0.30 h-1, 71.6 +/- 14.7 pg/ml, 0.017 +/- 0.015 h-1, 109.4 +/- 129.5 h, 1315.8 +/- 236.9 pg/ml x h and 10322.2 +/- 11473.7 pg/ml x h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

1 alpha-Hydroxycholecalciferol adsorption to peritoneal dialysis bags: influence of time, glucose concentration, temperature, and albumin.

The present study examines the adsorptive phenomenon of 1 alpha-hydroxycholecalciferol (1 alpha-OHD3) to peritoneal dialysis bags. One minute after injection of 1 alpha-OHD3 into the dialysis bags, a significant adsorption to the bags was observed in experiments where albumin was not added. A higher temperature (37 degrees versus 23 degrees C) in bags containing 13.6 mg glucose/ml resulted in less adsorption (P < 0.05). Adding albumin to bags at 37 degrees C with a glucose concentration of 13.6 mg/ml, reduced the adsorptive phenomenon significantly (P < 0.01). Bags tested at 37 degrees C with a glucose concentration of 38.6 mg/ml resulted in a significantly higher 1 alpha-OHD3 adsorption than bags containing 13.6 mg glucose/ml (P < 0.01). It is concluded that adsorption of 1 alpha-OHD3 to dialysis bags is affected by time, albumin, temperature and glucose concentrations. Substantial amounts of 1 alpha-OHD3 is retained by the peritoneal dialysis system when albumin is not added.

Pharmacokinetics of active vitamins D3, 1 alpha-hydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3 in patients on chronic hemodialysis.

Two active forms of vitamin D3, 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25 [OH] 2D3) and 1 alpha-hydroxyvitamin D3 (1 alpha, [OH] D3) are widely used for treating osteodystrophy in patients with chronic renal failure. Since the pharmacokinetics of these agents during long-term oral administration are unclear, we measured the circulating concentrations of 1 alpha, 25 (OH) 2D before and after their long-term oral administration in patients receiving maintenance hemodialysis. After 12 weeks of treatment with a daily dose of 1 alpha, (OH) D3 (0.5 micrograms), the administration of a single dose (2 micrograms) of 1 alpha, (OH) D3 showed that the area under the curve over 24 h (AUC) was increased significantly compared with day 1 of therapy. In contrast, after the treatment with a daily dose of 1 alpha, 25 (OH) 2D3 (0.25 micrograms), a single dose of 1 alpha, 25 (OH) 2D3 (1 microgram) did not exhibit this effect on the AUC. A single dose of each agent produced no significant change in either the peak increment above basal values or the elimination half-time (T 1/2) of circulating plasma 1 alpha, 25 (OH) 2D. The overall basal concentration of 1 alpha, 25 (OH) 2D achieved by 1 alpha, (OH) D3 after 12 weeks of administration was cumulative, but this effect was not observed in patients on 1 alpha, 25 (OH) 2D3. These data indicate that the pharmacokinetics of the two forms of active vitamin D3 did not differ as to peak increment and T1/2 except for the AUC, even after long-term dosage.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of pulse therapy using 1 alpha(OH)D on secondary hyperparathyroidism in patients on maintenance hemodialysis].

The effect of pulse therapy on severe secondary hyperparathyroidism related to chronic renal failure has been examined in 11 patients on maintenance hemodialysis by using an oral administration of 8 micrograms of 1 alpha(OH)D maximum per week. Throughout the 10 months of this treatment, the serum levels of intact-PTH, HS-PTH, and C-PTH were followed up. Additionally, to estimate the peak level of 1,25(OH)2D, its serum concentration at 10 hours after the 1 alpha (OH)D ingestion was measured. Results have shown that the serum levels of the intact-PTH, HS-PTH, and C-PTH were lowered in 9 of these 11 patients, two of this number especially showing a marked suppression of the serum intact-PTH to a low that was near to the normal upper limit level. In the two other cases of these 11 patients, no suppression was seen in any of serum PTH levels throughout the 10 months. In the cases that showed a good response, the 1,25(OH)2D serum concentration elevated significantly, to more than 100 pg/ml at 10 hours after the 1 alpha (OH)D intake. In contrast, in the 2 cases that showed no response, no appreciable elevation in the serum concentrations was noted. Thus, since the pulse therapy using 1 alpha (OH)D decreased the serum levels of the intact-PTH, HS-PTH, and C-PTH in 9 out of the 11 cases, we have concluded that pulse therapy using 1 alpha(OH)D is a valid therapy for secondary hyperparathyroidism in patients on maintenance hemodialysis.

Metabolism of 25-hydroxyvitamin D in copper-laden rat: a model of Wilson's disease.

Wilson's disease results in excess tissue accumulation of copper and is often complicated by skeletal and mineral abnormalities. We investigated vitamin D metabolism in rats fed a copper-laden diet rendering hepatic copper content comparable with that found in Wilson's disease. Injection of 25-hydroxyvitamin D3 [25(OH)D3] resulted in reduced 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in copper-intoxicated rats. In vitro 25(OH)D-1 alpha-hydroxylase activity was impaired in renal mitochondria from copper-intoxicated animals. Activity was also inhibited in mitochondria from controls when copper was added to incubation media. Impaired conversion of 25(OH)D to 1,25(OH)2D occurs in copper intoxication and suggests that altered vitamin D metabolism is a potential factor in the development of bone and mineral abnormalities in Wilson's disease.

Circulating 1 alpha,25-dihydroxyvitamin D levels after a single dose of 1 alpha,25-dihydroxyvitamin D3 or 1 alpha-hydroxyvitamin D3 in normal men.

In order to clarify the differences between 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 1 alpha-hydroxyvitamin D3 (1(OH)D3) as a drug, we determined the serum levels of 1,25(OH)2D at different times after oral administration of each drug to normal men. In the paired test which was carried out on the same people, the integrated change of serum 1,25(OH)2D levels, defined as the mean area under the concentration curve over 24 h, was significantly higher in the 1,25(OH)2D3 group than in the 1(OH)D3 group. The mean of the peak serum level (Cmax) was also significantly higher in the former group. On the other hand, the mean time to reach the peak level (Tmax) was significantly shorter in the 1,25(OH)2D3 group than in the 1(OH)D3 group. The same results were obtained even when we performed the non-paired administration tests. In the present study, the peak serum level of 1,25(OH)2D after ingestion of a single dose of 1,25(OH)2D3 was reached rapidly, i.e., between 4 and 6 h, and the value had usually returned to the baseline by 24 h. On the contrary, 1(OH)D3 administration resulted in a slow rise in the serum level of 1,25(OH)2D with a relatively low peak level. Therefore, clinical use of these two drugs demands careful consideration of their differences.