Glenville Jones

Queen's University, Kingston, Ontario, Canada

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Publications (35)203.69 Total impact

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    ABSTRACT: Context: The discovery of hypercalcemic diseases due to loss-of-function mutations in CYP24A1 has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D. Objective: We describe a novel LC-MS/MS-based method involving derivatization with DMEQ-TAD to simultaneously assay multiple vitamin D metabolites including 25-OH-D and 24,25-(OH)2Dusing 100 μ l of serum with a 5-minute run-time. Design: The assay uses a newly-synthesized internal standard d6-24,25-(OH)2D3 enabling quantitation of 24,25-(OH)2D3, as well as the determination of the ratio of 25-OH-D3:24,25-(OH)2D3, a physiologically useful parameter. Setting: We report data on >1000 normal and disease samples involving D deficiency or hypercalcemia, as well as studies involving knockout mouse models. Results: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2 to -5% bias), exhibited low inter- and intra-assay coefficients of variation (4-7%), and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2)=0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3:24,25-(OH)2D3 which remained <25 in vitamin D sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia (IIH). Conclusions: The new method also showed good utility in clinical settings involving vitamin D deficiency, supplementation with vitamin D and IIH, as well as in animal models with ablation of selected CYPs involved in vitamin D.
    The Journal of Clinical Endocrinology and Metabolism 03/2014; · 6.31 Impact Factor
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    ABSTRACT: Context: Hypercalcemia, hypercalciuria, and recurrent nephrolithiasis are all common clinical problems. This case report illustrates a newly-described but possibly not uncommon cause of this presenting complex. Objective: Report a patient studied for over 30 years ,with the diagnosis finally made with modern biochemical and genetic tools. Design: Case report and review of literature Setting: University Referral Center Patient: Single patient with hypercalcemia, hypercalciuria, and recurrent nephrolithiasis Intervention: Treatment with low calcium diet, low vitamin D intake, prednisone, and ketoconazole. Main Outcome Measure: Clinical and biochemical response to interventions above. Results: Calcium absorption by dual isotope absorptiometry was elevated at 37.4%. Serum levels of 24,25-dihydroxyvitamin D were very low as measured in two laboratories (0.62ng/mL, nl 3.49+/-1.57, and 0.18mg/mL). Genetic analysis of CYP24A1 revealed homozygous mutation E143del previously described. The patient's serum calcium and renal function improved markedly on treatment with ketoconazole but not with prednisone. Conclusions: Chronic hypercalcemia, hypercalciuria, and/or nephrolithiasis may be caused by mutations in CYP24A1 causing failure to metabolize 1,25-dihydroxyvitamin D.
    The Journal of Clinical Endocrinology and Metabolism 01/2014; · 6.31 Impact Factor
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    ABSTRACT: Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α,25-dihydroxyvitamin D3. It is clear that the second or 1α-hydroxylation step is carried out by a single enzyme, 25-hydroxyvitamin D 1α-hydroxylase CYP27B1. However, it is not certain what enzyme or enzymes are responsible for the initial 25-hydroxylation. An excellent case has been made for vitamin D 25-hydroxylase CYP2R1, but this hypothesis has not yet been tested. We have now produced Cyp2r1(-/-) mice. These mice had greater than 50% reduction in serum 25-hydroxyvitamin D3. Curiously, the 1α,25-dihydroxyvitamin D3 level in the serum remained unchanged. These mice presented no health issues. A double knockout of Cyp2r1 and Cyp27a1 maintained a similar circulating level of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Our results support the idea that the CYP2R1 is the major enzyme responsible for 25-hydroxylation of vitamin D, but clearly a second, as-yet unknown, enzyme is another contributor to this important step in vitamin D activation.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
  • Glenville Jones, David E Prosser, Martin Kaufmann
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    ABSTRACT: The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3 which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression, described in companion chapters in this review series. This review will update our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We will focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We will highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease and hyperproliferative diseases such as psoriasis and cancer; as well as to explore potential future developments in the field.
    The Journal of Lipid Research 04/2013; · 4.39 Impact Factor
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    ABSTRACT: In early 2011, a committee convened by the Institute of Medicine issued a report on the Dietary Reference Intakes for calcium and vitamin D. The Endocrine Society Task Force in July 2011 published a guideline for the evaluation, treatment, and prevention of vitamin D deficiency. Although these reports are intended for different purposes, the disagreements concerning the nature of the available data and the resulting conclusions have caused confusion for clinicians, researchers, and the public. In this commentary, members of the Institute of Medicine committee respond to aspects of The Endocrine Society guideline that are not well supported and in need of reconsideration. These concerns focus on target serum 25-hydroxyvitamin D levels, the definition of vitamin D deficiency, and the question of who constitutes a population at risk vs. the general population.
    The Journal of Clinical Endocrinology and Metabolism 03/2012; 97(4):1146-52. · 6.31 Impact Factor
  • Glenville Jones
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    ABSTRACT: Vitamin D has gone through a renaissance with the association of vitamin D deficiency with a wide array of common diseases including breast, colorectal and prostate cancers, cardio-vascular disease, autoimmune conditions and infections. Vitamin D analogs constitute a valuable group of compounds which can be used to regulate gene expression in functions as varied as calcium and phosphate homeostasis, as well as cell growth regulation and cell differentiation of a wide spectrum of cell types. This review will discuss the full range of vitamin D compounds currently available, some of their possible uses, and potential mechanisms of action.
    Rheumatic diseases clinics of North America 02/2012; 38(1):207-32, xi. · 2.59 Impact Factor
  • Glenville Jones, David E Prosser, Martin Kaufmann
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    ABSTRACT: CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D(3)-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D(3) (25-OH-D(3)) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) into 24-hydroxylated products, which constitute the degradation of the vitamin D molecule. This review focuses on recent data in the CYP24A1 field, including biochemical, physiological and clinical developments. Notable among these are: the first crystal structure for rat CYP24A1; mutagenesis studies which change the regioselectivity of the enzyme; and the finding that natural inactivating mutations of CYP24A1 cause the genetic disease idiopathic infantile hypercalcemia (IIH). The review also discusses the emerging correlation between rising serum phosphate/FGF-23 levels and increased CYP24A1 expression in chronic kidney disease, which in turn underlies accelerated degradation of both serum 25-OH-D(3) and 1,25-(OH)(2)D(3) in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states.
    Archives of Biochemistry and Biophysics 11/2011; 523(1):9-18. · 3.37 Impact Factor
  • Journal of the American Dietetic Association 10/2011; 111(10):1467. · 3.80 Impact Factor
  • Martin Kaufmann, David E Prosser, Glenville Jones
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    ABSTRACT: CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.
    Journal of Biological Chemistry 06/2011; 286(33):28729-37. · 4.65 Impact Factor
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    ABSTRACT: Vitamin D supplementation for the prevention of rickets is one of the oldest and most effective prophylactic measures in medicine, having virtually eradicated rickets in North America. Given the potentially toxic effects of vitamin D, the recommendations for the optimal dose are still debated, in part owing to the increased incidence of idiopathic infantile hypercalcemia in Britain in the 1950s during a period of high vitamin D supplementation in fortified milk products. We investigated the molecular basis of idiopathic infantile hypercalcemia, which is characterized by severe hypercalcemia, failure to thrive, vomiting, dehydration, and nephrocalcinosis. We used a candidate-gene approach in a cohort of familial cases of typical idiopathic infantile hypercalcemia with suspected autosomal recessive inheritance. Identified mutations in the vitamin D-metabolizing enzyme CYP24A1 were evaluated with the use of a mammalian expression system. Sequence analysis of CYP24A1, which encodes 25-hydroxyvitamin D 24-hydroxylase, the key enzyme of 1,25-dihydroxyvitamin D(3) degradation, revealed recessive mutations in six affected children. In addition, CYP24A1 mutations were identified in a second cohort of infants in whom severe hypercalcemia had developed after bolus prophylaxis with vitamin D. Functional characterization revealed a complete loss of function in all CYP24A1 mutations. The presence of CYP24A1 mutations explains the increased sensitivity to vitamin D in patients with idiopathic infantile hypercalcemia and is a genetic risk factor for the development of symptomatic hypercalcemia that may be triggered by vitamin D prophylaxis in otherwise apparently healthy infants.
    New England Journal of Medicine 06/2011; 365(5):410-21. · 54.42 Impact Factor
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    ABSTRACT: The vitamin D metabolite, 1,25-(OH)₂D₃, binds the vitamin D receptor (VDR) to exert its regulatory effects at the transcription level. VDR is expressed in professional antigen-presenting cells (pAPCs), such as macrophages (Mø) and dendritic cells (DCs). We show for the first time that the 24-hydroxylase enzyme is activated in bone marrow-derived dendritic cell (BMDC), due to 1,25(OH)₂D₃ stimulation which resulted in the induction of its gene, CYP24A1. Furthermore, we provide evidence that the influence of 1,25-(OH)₂D₃ on TLR-4-L-induced activation of pAPC is dependent on the order of VDR and TLR-4 engagement. Thus, pre-treatment of pAPC with 1,25-(OH)₂D₃ partially inhibited LPS-induced nitric oxide (NO) production. However, these inhibitory effects were not observed when LPS and 1,25-(OH)₂D₃ were added simultaneously or when LPS preceded 1,25-(OH)₂D₃. Moreover, we found that 1,25-(OH)₂D₃ pre-treatment of pAPCs did not cause general suppression since it interfered with NO levels but not with the cytokines IL-6 or TNF-α. Consequently, engagement of VDR by 1,25-(OH)₂D₃ can partially interfere with TLR-4-L-induced activation of pAPCs only when it occurs before TLR-4 stimulation.
    Immunobiology 04/2011; 216(9):988-96. · 2.81 Impact Factor
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    ABSTRACT: The Institute of Medicine Committee to Review Dietary Reference Intakes for Calcium and Vitamin D comprehensively reviewed the evidence for both skeletal and nonskeletal health outcomes and concluded that a causal role of calcium and vitamin D in skeletal health provided the necessary basis for the 2011 Estimated Average Requirement (EAR) and Recommended Dietary Allowance (RDA) for ages older than 1 year. For nonskeletal outcomes, including cancer, cardiovascular disease, diabetes, infections, and autoimmune disorders, randomized clinical trials were sparse, and evidence was inconsistent, inconclusive as to causality, and insufficient for Dietary Reference Intake (DRI) development. The EAR and RDA for calcium range from 500 to 1,100 and 700 to 1,300 mg daily, respectively, for ages 1 year and older. For vitamin D (assuming minimal sun exposure), the EAR is 400 IU/day for ages older than 1 year and the RDA is 600 IU/day for ages 1 to 70 years and 800 IU/day for 71 years and older, corresponding to serum 25-hydroxyvitamin D (25OHD) levels of 16 ng/mL (40 nmol/L) for EARs and 20 ng/mL (50 nmol/L) or more for RDAs. Prevalence of vitamin D inadequacy in North America has been overestimated based on serum 25OHD levels corresponding to the EAR and RDA. Higher serum 25OHD levels were not consistently associated with greater benefit, and for some outcomes U-shaped associations with risks at both low and high levels were observed. The Tolerable Upper Intake Level for calcium ranges from 1,000 to 3,000 mg daily, based on calcium excretion or kidney stone formation, and from 1,000 to 4,000 IU daily for vitamin D, based on hypercalcemia adjusted for uncertainty resulting from emerging risk relationships. Urgently needed are evidence-based guidelines to interpret serum 25OHD levels relative to vitamin D status and intervention.
    Journal of the American Dietetic Association 04/2011; 111(4):524-7. · 3.80 Impact Factor
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    ABSTRACT: This article summarizes the new 2011 report on dietary requirements for calcium and vitamin D from the Institute of Medicine (IOM). An IOM Committee charged with determining the population needs for these nutrients in North America conducted a comprehensive review of the evidence for both skeletal and extraskeletal outcomes. The Committee concluded that available scientific evidence supports a key role of calcium and vitamin D in skeletal health, consistent with a cause-and-effect relationship and providing a sound basis for determination of intake requirements. For extraskeletal outcomes, including cancer, cardiovascular disease, diabetes, and autoimmune disorders, the evidence was inconsistent, inconclusive as to causality, and insufficient to inform nutritional requirements. Randomized clinical trial evidence for extraskeletal outcomes was limited and generally uninformative. Based on bone health, Recommended Dietary Allowances (RDAs; covering requirements of ≥97.5% of the population) for calcium range from 700 to 1300 mg/d for life-stage groups at least 1 yr of age. For vitamin D, RDAs of 600 IU/d for ages 1-70 yr and 800 IU/d for ages 71 yr and older, corresponding to a serum 25-hydroxyvitamin D level of at least 20 ng/ml (50 nmol/liter), meet the requirements of at least 97.5% of the population. RDAs for vitamin D were derived based on conditions of minimal sun exposure due to wide variability in vitamin D synthesis from ultraviolet light and the risks of skin cancer. Higher values were not consistently associated with greater benefit, and for some outcomes U-shaped associations were observed, with risks at both low and high levels. The Committee concluded that the prevalence of vitamin D inadequacy in North America has been overestimated. Urgent research and clinical priorities were identified, including reassessment of laboratory ranges for 25-hydroxyvitamin D, to avoid problems of both undertreatment and overtreatment.
    The Journal of Clinical Endocrinology and Metabolism 01/2011; 96(1):53-8. · 6.31 Impact Factor
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    ABSTRACT: The analysis of vitamins D and their metabolites and analogues has been reviewed by us on two occasions (Makin et al., 1995; Jones and Makin, 2000) over the last 10-15 years. In this chapter, we have drawn heavily on the 2000 review, up-dating it to take account of the developments in methodology that have occurred in the intervening years, but including elements of our 1995 review so that the reader can get a picture of the historical context as well as the modern developments.
    06/2010: pages 967-1096;
  • Glenville Jones
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    ABSTRACT: Vitamin D has gone through a renaissance with the association of vitamin D deficiency with a wide array of common diseases including breast, colorectal and prostate cancers, cardio-vascular disease, autoimmune conditions and infections. Vitamin D analogs constitute a valuable group of compounds which can be used to regulate gene expression in functions as varied as calcium and phosphate homeostasis, as well as cell growth regulation and cell differentiation of a wide spectrum of cell types. This review will discuss the full range of vitamin D compounds currently available, some of their possible uses, and potential mechanisms of action.
    Endocrinology and metabolism clinics of North America 06/2010; 39(2):447-72, table of contents. · 3.56 Impact Factor
  • Glenville Jones
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    ABSTRACT: The roles of vitamin D, mediated through its conversion to 1,25-dihydroxyvitamin D(3) (calcitriol), have been expanded recently through new knowledge about the range of tissues capable of activating it and the breadth of the genes under its regulatory control. This basic science together with the fact that numerous studies across North America are revealing that vitamin D insufficiency/deficiency (as defined by 25-OH-D levels <30 ng/ml) is extremely common (>80%) in dialysis patients are indicative that these patients have two vitamin D-related problems that require different treatment regimens. Combinations of vitamin D(3) (cholecalciferol) or vitamin D(2) (ergocalciferol) and an active calcitriol analog should be used to treat their vitamin D deficiency and their calcitriol hormone insufficiency, respectively. This mini-review provides the case for combination therapy.
    Seminars in Dialysis 01/2010; 23(3):239-43. · 2.25 Impact Factor
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    ABSTRACT: Plasma 25(OH)D has emerged as a valuable biomarker for the many varied health-related effects of vitamin D in the clinic mainly because of the recognition of the importance of the enzyme, CYP27B1, or the 25(OH)D-alpha-hydroxylase in the extrarenal, target cell production of calcitriol. This review briefly assesses current methodology for plasma 25(OH)D assay focusing mainly on currrent controversies surrounding the definition of the normal range and performance characteristics of the assay, separate measurement of both 25(OH)D(2) and 25(OH)D(3), and quality assurance tesing of laboratories offering the test. Clinicians have two main types of 25(OH)D assay based on either high-performance liquid chromatography with UV or mass detection or higher throughput kits based on protein (competitive protein binding assay or radioimmunoassay) binding. Based on 30 yr of experience with measuring 25(OH)D levels, it is concluded that, in the hands of appropriately trained experts, both types of assay provide reliable and accurate results, but all laboratories providing 25(OH)D data need frequent external quality assurance service to ensure that this performance is maintained.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 01/2008; 22 Suppl 2:V11-5. · 6.04 Impact Factor
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    ABSTRACT: Studies of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1alpha,25-(OH)(2)D(3), leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1alpha,25-(OH)(2)D(3)-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design.
    Proceedings of the National Academy of Sciences 08/2007; 104(31):12673-8. · 9.81 Impact Factor
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    ABSTRACT: Vinyl carbamate (VC) is derived from ethyl carbamate, a carcinogen formed in fermentation of food and alcoholic products. We have undertaken studies to test the hypothesis that an epoxide generated from VC oxidation leads to formation of 1,N6-ethenodeoxyadenosine (epsilon dAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry for identification and quantitation of epsilon dAS. Scanning and fragment ion analyses confirmed the identity of epsilon dAS based on the molecular ion [M + H]+ m/z 276 and the specific fragment ion m/z 160. Chemical oxidation of VC in reactions containing 2'-deoxyadenosine produced epsilon dAS with 1H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic epsilon dAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of epsilon dAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of epsilon dAS in incubations of lung microsomes or recombinant CYP2E1 with VC was dependent on protein concentrations, CYP2E1 enzyme levels, and incubation time. The rates of epsilon dAS formation were highly correlated with VC concentrations. Peak rates were produced by lung microsomes and recombinant CYP2E1 at 3.0 and 2.5 mM VC, respectively. In inhibitory studies, incubations of VC were performed using lung microsomes from mice treated with the CYP2E1 inhibitor diallyl sulfone (100 mg/kg, p.o.). Results from these studies showed significantly decreased epsilon dAS formation in microsomes incubated with VC, with an inhibition of 70% at 3.0 mM. These findings suggested that CYP2E1 is a major enzyme mediating VC oxidation, leading to the formation of a metabolite that alkylates DNA to form the epsilon dAS adduct.
    Drug Metabolism and Disposition 06/2007; 35(5):713-20. · 3.36 Impact Factor
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    ABSTRACT: A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1alpha,25-dihydroxyvitamin D3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design.
    Archives of Biochemistry and Biophysics 05/2007; 460(2):177-91. · 3.37 Impact Factor