Gene expression of vitamin D hydroxylase and megalin in the remnant kidney of nephrectomized rats

BACKGROUND: Regulation of vitamin D hydroxylase genes in the early stage of chronic renal failure is not fully understood. Using nephrectomized rats, we examined changes in mRNA levels of CYP27B1 (25-hydroxyvitamin D3-1 alpha-hydroxylase), CYP24 (25-hydroxyvitamin D3-24-hydroxylase), and vitamin D receptor in relation to megalin, recently found to participate in renal vitamin D metabolism. METHODS: A rat model of moderate renal failure was induced by 3/4 nephrectomy. Plasma parameters, including vitamin D metabolite concentrations, were measured at weeks 2, 4 and 8, and poly(A)+ RNA extracted from the remnant kidneys was subjected to Northern blot hybridization. RESULTS: Plasma creatinine concentration at week 2 was 0.40 +/- 0.02 mg/dL in the sham-operated and 0.93 +/- 0.15 mg/dL in the nephrectomized rats, and both values remained constant up to week 8. Plasma concentrations of 25(OH)D3, 1 alpha,25(OH)2D3, and 24,25(OH)2D3 were unchanged between nephrectomized and sham-operated rats at week 8. Intact parathyroid hormone (PTH) increased at week 8 in nephrectomized rats. CYP27B1 mRNA in nephrectomized rats did not vary at week 2, but increased approximately two- and four-fold at weeks 4 and 8, respectively, compared to the sham-operated rats. CYP24 and megalin mRNAs, on the other hand, began to decline as early as at week 2 in nephrectomized rats and kept decreasing throughout the experiment. The expression of vitamin D receptor was modestly but significantly decreased only at week 8. CONCLUSION: Coordinated and reciprocal alterations of the increase in CYP27B1 mRNA and the decrease in CYP24 mRNA may play a pivotal role in maintaining the plasma level of 1 alpha,25(OH)2D3 in the face of reduced nephron mass and/or megalin expression.     

5alpha-dihydrotestosterone inhibits 1alpha,25-dihydroxyvitamin D3-induced expression of CYP24 in human prostate cancer cells

BACKGROUND: A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. METHODS: The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay. RESULTS: Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. CONCLUSIONS: We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact.     

Metabolism of a New Local Anesthetic, Ropivacaine, by Human Hepatic Cytochrome P450

Background: Ropivacaine is a local anesthetic with a long duration of action. Although it is less toxic than bupivacaine, local anesthetic toxicity is possible when the plasma concentration is increased. Because ropivacaine is an amide-type local anesthetic, it is metabolized by cytochrome P450 (P450) in the liver, and its elimination and plasma concentration can be dependent on the level of P450. The purpose of this investigation was to elucidate the metabolism of ropivacaine by human hepatic P450.

Methods: The metabolism of ropivacaine was compared using recombinant human and purified rat hepatic P450 isozymes. An inhibition study using antibodies against rat P450 was performed using hepatic microsomes from human and rat to identify which P450s are involved in ropivacaine metabolism.

Results: Ropivacaine was metabolized to 2',6'-pipecoloxylidide (PPX), 3'-hydroxyropivacaine (3'-OH Rop), and 4'-hydroxyropivacaine (4'-OH Rop) by hepatic microsomes from human and rat. PPX was a major metabolite of both human and rat hepatic microsomes. In a reconstituted system with rat P450, PPX was produced by CYP2C11 and 3A2, 4'-OH Rop by CYP1A2, and 3'-OH Rop by CYP1A2 and 2D1. Formation of PPX in rat hepatic microsomes was inhibited by anti CYP3A2, but not by CYP2C11 antibody, and formation of 3'-OH Rop was inhibited by CYP1A2 and 2D1 antibodies. Anti CYP3A2 and 1A2 antibodies inhibited the formation of PPX and 3'-OH Rop in human hepatic microsomes, respectively. Recombinant human P450s expressed in lymphoblast cells were used for further study. CYP3A4 and 1A2 formed the most PPX and 3'-OH Rop, respectively. Ropivacaine N-dealkylation and 3'-hydroxylation activities correlated well with the level of CYP3A4 and 1A2 in human hepatic microsomes, respectively.     

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)     

Sequential changes in mineral metabolism and serum vitamin D metabolite concentrations produced by phenobarbital administration in the rat

Summary We examined the effect of daily phenobarbital administration on serum vitamin D metabolite levels and indices of vitamin D biologic activity in 7-week-old male rats maintained on parenteral vitamin D supplementation (125 ng/day). Treatment with phenobarbital (75 mg/kg/day) produced a biphasic response in parameters of vitamin D biologic effect, including serum calcium concentration, serum inorganic phosphate concentration, and intestinal⁴⁵Ca calcium absorption. An initial increase in these values, maximal after 3–5 days of treatment, was followed by a subsequent decline to subnormal levels by day 21. A parallel biphasic pattern was observed for serum 25-hydroxyvitamin D (25OHD) concentration. Serum 25OHD reached a peak increase of 87% above control levels (P<0.01), observed after 5 days of treatment, and subsequently declined to 62% of control animal values (P<0.01) by 21 days. Serum 24,25(OH)₂D concentration followed a similar course and exhibited a strong positive correlation with serum 25OHD concentrations (r=0.74,P<0.01). In contrast, serum 1,25(OH)₂D concentration was not significantly different from control values after 5 days but was increased 80% over control values (P<0.05) by day 21. Serum vitamin D concentration declined progressively in treated animals, falling to 50% of control levels (P<0.05) by day 5 and to 27% of control levels (P<0.001) by day 21. At the point of maximal increase in serum 25OHD concentration, hepatic microsomal vitamin D₃-25-hydroxylase activity was not increased in the treated animals whereas hepatic mitochondrial vitamin D₃-25-hydroxylase activity was increased by 2.4-fold. Increased hepatic mitochondrial vitamin D₃-25-hydroxylase activity persisted through 21 days of phenobarbital treatment. It is concluded that phenobarbital administration in the rat produces an initial increase in vitamin D biologic effect which correlates temporally with increased circulating levels of 25OHD, the latter possibly resulting from increased hepatic mitochondrial vitamin D-25-hydroxylase activity. A subsequent decline in serum 250HD concentration may be the result of decreased availability of vitamin D as substrate. This sequence of alterations in vitamin D metabolism bears potentially important implications for the timing of prophylactic vitamin D supplementation in patients treated with anticonvulsant drugs.     

Newly established assay method for 25-hydroxyvitamin D3 24-hydroxylase revealed much lower Km for 25-hydroxyvitamin D3 than for 1alpha,25-dihydroxyvitamin D3.

An accurate assay method of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was established. Kidney mitochondria prepared from vitamin D-replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial-type electron transferring proteins, adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by high-performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D-replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24-hydroxylase for 25-hydroxyvitamin D3 [25(OH)D3] was 0.6 microM, which was 35-fold lower than that for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] which was 20.9 microM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35-fold higher than that to 1alpha,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1alpha,25(OH)2D3.     

1 Alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3 have anabolic effects on cortical bone, but induce intracortical remodeling at toxic doses in ovariectomized rats

It is well established that vitamin D metabolites have anabolic properties on cancellous bone in rats. However, few data are available on cortical bone effects of vitamin D metabolites. In this study, we examined the effects of the synthetic vitamin D analogs 1alpha-hydroxyvitamin D2 (1alpha(OH)D2) and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) on cortical bone of the tibial shaft in ovariectomized (OVX) rats using bone histomorphometry. Six-month-old Fischer 344 rats were either OVX or sham-operated (SHAM). OVX rats received vehicle, 1alpha(OH)D2 or 1alpha(OH)D3 orally via the diet in a dose range from 0.025 to 0.2 microg/kg/day. All animals were killed 3 months postsurgery after in vivo fluorochrome labeling. Relative to SHAM rats, vehicle-treated OVX rats showed a reduction in cortical bone area (%) due to expansion of the marrow cavity. Treatment of OVX rats with either 1alpha(OH)D2 or 1alpha(OH)D3 dose-dependently decreased marrow area, and increased cortical area, periosteal perimeter, and periosteal and endocortical bone formation rate compared with OVX vehicle controls. Interestingly, OVX animals receiving the highest doses showed intracortical resorption cavities, a phenomenon only exceptionally observed in rats. The intracortical hole area was significantly lower in 1alpha(OH)D2-treated compared with 1alpha(OH)D3-treated rats. We conclude that 1alpha(OH)D2 and 1alpha(OH)D3 prevent cortical bone loss in OVX rats and have anabolic effects on cortical bone at higher doses. However, very high, toxic doses of both vitamin D analogs induce intracortical remodeling as an untoward side effect.     

Identification of the pharmacogenetic determinants of alfentanil metabolism: cytochrome P-450 3A4. An explanation of the variable elimination clearance.

There is considerable variability in the elimination clearance of the opioid analgesic alfentanil. It has been shown previously that alfentanil clearance is independent of the polymorphic debrisoquine hydroxylase (P-450 2D6), and it is therefore of interest to identify the human cytochrome P-450 enzymes involved in noralfentanil formation, the primary reaction involved in the oxidative N-dealkylation at the piperidine nitrogen. Purified human P-450 3A4 showed appreciable catalytic activity, and yeast recombinant P-450 3A4 also showed alfentanil oxidation activity. When microsomes prepared from different human liver samples were compared, noralfentanil formation activity was well correlated (r = 0.95,P less than 0.005) with nifedipine oxidation (a P-450 3A4 marker) but not with markers of other P-450s, including phenacetin O-deethylation (P-450 1A2), chlorzoxazone 6-hydroxylation (P-450 2E1), and (S)-mephenytoin 4'-hydroxylation (a P-450 2C enzyme). Using antibodies that recognize specific human P-450 enzymes (immunoinhibition techniques), it was possible to demonstrate that anti-P-450 3A4 nearly completely inhibited alfentanil oxidation activity in the human liver microsomes, but no other antibodies showed a measurable inhibitory effect. Selective chemical inhibitors of P-450 3A4, gestodene and troleandomycin, inhibited as much as 90% of the microsomal noralfentanil formation activity, but other chemical inhibitors did not show a detectable inhibitory effect. 7,8-Benzoflavone inhibited as much as 90% of the alfentanil oxidation activity of the microsomal or reconstituted P-450 3A4 system. This work indicates that P-450 3A4 contributes significantly to human liver microsomal alfentanil oxidation, whereas P-450 2D6 does not contribute.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D–Dependent Rickets Type 1B (25‐Hydroxylase Deficiency): A Rare Condition or a Misdiagnosed Condition?

Vitamin D requires a two‐step activation by hydroxylation: The first step is catalyzed by hepatic 25‐hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α‐hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25‐(OH)₂D. Mutations of CYP2R1 have been associated with vitamin D–dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss‐of‐function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss‐of‐function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25‐OH‐D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25‐(OH)₂D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa‐hydroxyvitamin D₃ or alfacalcidol (1α‐OH‐D₃) treatment, and we observed a dramatic increase in the 1,25‐(OH)₂D serum concentration, which indicated the role of accessory 25‐hydroxylase enzymes. Lastly, in patients who received calcifediol (25‐OH‐D₃), we documented normal 24‐hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25‐OH‐D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research.     

Halothane-dependent lipid peroxidation in human liver microsomes is catalyzed by cytochrome P4502A6 (CYP2A6)

BACKGROUND: Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction. METHODS: Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry. RESULTS: Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody. CONCLUSIONS: Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction prevents halothane-dependent lipid peroxidation in vitro.     

Halothane-dependent Lipid Peroxidation in Human Liver Microsomes Is Catalyzed by Cytochrome P4502A6 (CYP2A6)

Background: Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction.

Methods: Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry.

Results: Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody.     

Suppression of rat hepatic vitamin D-25-hydroxylase by cholecalciferol,but not by 25-hydroxy- or 1,25-dihydroxymetabolites

Summary Hepatic vitamin D-25-hydroxylase activity is greater in vitamin D-depleted than replete animals. We investigated whether vitamin D itself or a metabolite of vitamin D was responsible for modulating the activity of vitamin D-25-hydroxylase. Accordingly, we repleted vitamin D-depleted rats with subcutaneous injections of 2600, 520, and 130 pmoles of cholecalciferol (D₃), 25-hydroxycholecalciferol (25(OH)₂D₃), and 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃), respectively, for up to 3 weeks. Repletion resulted in accelerated weight gain and in increased activity of gut mucosal alkaline phosphatase. Using an improved assay to measure vitamin D-25-hydroxylase activity in liver homogenates, we found 78% reduction (P<0.001) in the D₃-repleted group, maximal by 1 week, in contrast to no change in those groups treated with D₃ metabolites. D₃, 25(OH)D₃, and D₃-esters remaining in livers at the time of assay were estimated in a parallel experiment using [³H]D₃-repleted rats. Residual D₃ accounted for only a 9% dilution of substrate in the assay. 25(OH)D₃ was present in the liver at concentrations two orders of magnitude lower than the amount required to inhibit vitamin D-25-hydroxylase activityin vitro. D₃ esters had no inhibitory effectin vitro at 250-fold excess of that found in the repleted rat liver. Vitamin D appears to modulate its D-25-hydroxylase activity in biological systems by a mechanism other than feedback inhibition by 25(OH)D₃, 1,25(OH)₂D₃, or D₃-esters. Partial presentation of this work occurred in poster form at Digestive Disease Week, New Orleans, Louisiana, May 1984 (Gastroenterology (1984), 86: 1931) and the 6th Workshop on Vitamin D, Merano, Italy, March 1985.     

Enhancement of hepatic 4‐hydroxylation of 25‐hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug‐induced osteomalacia

Long‐term therapy with certain drugs, especially cytochrome P450 (P450; CYP)‐inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25‐hydroxyvitamin D₃ (25OHD₃) were evaluated after exposure to P450‐inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8‐fold increase in formation of the major metabolite of 25OHD₃, 4β,25(OH)₂D₃. This inductive effect was blocked by the addition of 6′,7′‐dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK‐2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)₂D₃ was the predominant monohydroxy metabolite produced from 25OHD₃, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)₂D₃ was increased 60% (p < 0.01) after short‐term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)₂D₃ (?10%; p = 0.03), and a nonsignificant change in 24R,25(OH)₂D₃ (?8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)₂D₃ and decrease in 1α,25(OH)₂D₃ levels. Examination of the plasma monohydroxy metabolite/25OHD₃ ratios indicated selective induction of the CYP3A4‐dependent 4β‐hydroxylation pathway of 25OHD₃ elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug‐induced osteomalacia. © 2013 American Society for Bone and Mineral Research.     

Fentanyl inhibits metabolism of midazolam: competitive inhibition of CYP3A4 in vitro

Fentanyl decreases clearance of midazolam administered i.v., but the mechanism remains unclear. To elucidate this mechanism, we have investigated the effect of fentanyl on metabolism of midazolam using human hepatic microsomes and recombinant cytochrome P450 isoforms (n = 6). Midazolam was metabolized to l'-hydroxymidazolam (l'-OH MDZ) by human hepatic microsomes, with a Michaelis-Menten constant (K(m)) of 5.0 (SD 2.7) microgramsmol litre-1. Fentanyl competitively inhibited metabolism of midazolam in human hepatic microsomes, with an inhibition constant (Ki) of 26.8 (12.4) microgramsmol litre-1. Of the seven representative human hepatic P450 isoforms, CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4, only CYP3A4 catalysed hydroxylation of midazolam, with a K(m) of 3.6 (0.8) microgramsmol liter-1. Fentanyl competitively inhibited metabolism of midazolam to l'-OH MDZ by CYP3A4, with a Ki of 24.2 (6.8) microgramsmol litre-1, comparable with the Ki obtained in human hepatic microsomes. These findings indicate that fentanyl competitively inhibits metabolism of midazolam by CYP3A4.      

Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.     

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.     

1a,25-(OH)2D3 Regulates 25-Hydroxyvitamin D3 24R-Hydroxylase Activity in Growth Zone Costochondral Growth Plate Chondrocytes via Protein Kinase C

Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1alpha- and 24R-hydroxylase activities and metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1alpha,25-(OH)2D3 exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10(-9) - 10(-7) M 1alpha,25-(OH)2D3 or 24R,25-(OH)2D, for 24 hours, and 1alpha- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10(-10) - l0(-8) M 1alpha,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1alpha,25-(OH)2D3 (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)2D3-dependent effect on 24,25-(OH)2D3 production was determined by comparing the response to 1alpha,25-(OH)2D3 to the response to 1beta,25-(OH)2D3. 1alpha,25-(OH)2D3 increased 24R-hydroxylase specific activity in a dose-dependent manner, 24,25-(OH)2D3 production by intact cells was also increased. The effect of 1alpha,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1alpha,25-(OH)2D3 caused an increase; 1beta,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours. 1alpha-hydroxylase activity was unaffected by either metabolite at 24 hours. 1alpha,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1alpha,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1alpha,25(OH)2D3-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.