A Francisella mutant in lipid A carbohydrate modification elicits protective immunity.

Francisella tularensis (Ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow-derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88(-/-) mice infected with either strain. MyD88(-/-) mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.

Pharmacokinetics of valerenic acid after single and multiple doses of valerian in older women.

Insomnia is a commonly reported clinical problem with as many as 50% of older adults reporting difficulty in falling and/or remaining asleep. Valerian (Valeriana officinalis) is a commonly used herb that has been advocated for promoting sleep. Valerenic acid is used as a marker for quantitative analysis of valerian products with evidence of pharmacological activity relevant to the hypnotic effects of valerian. The objective of this study was to determine the pharmacokinetics of valerenic acid in a group of elderly women after receiving a single nightly valerian dose and after 2 weeks of valerian dosing. There was not a statistically significant difference in the average peak concentration (C(max)), time to maximum concentration (T(max)) area under the time curve (AUC), elimination half-life (T(1/2)) and oral clearance after a single dose compared with multiple dosing. There was considerable inter- and intra-subject variability in the pharmacokinetic parameters. C(max) and AUC deceased and T(1/2) increased with increased body weight. The variability between the capsules was extremely low: 2.2%, 1.4% and 1.4%, for hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic acid, respectively. In conclusion, large variability in the pharmacokinetics of valerenic acid may contribute to the inconsistencies in the effect of valerian as a sleep aid.

Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium.

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate-type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography-mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe-enterobactin or Fe-salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.

Estrogen nitration kinetics and implications for wastewater treatment.

Understanding estrogen-removal mechanisms in wastewater treatment is imperative, as estrogens have environmental effects at trace concentrations. Previous research investigating co-metabolic degradation of 17alpha-ethinylestradiol (EE2) by ammonia-oxidizing bacteria (AOB) revealed that, in batch tests where high nitrite-nitrogen (NO2-N) concentrations occurred as a result of ammonia-nitrogen (NH4-N) oxidation by AOB, an abiotic estrogen nitration reaction actually was occurring--not co-metabolic degradation. This paper addresses nitration kinetics. A first-order abiotic nitration model was developed that predicts nitration of EE2, 17beta-estradiol (E2), and estrone (El) as a function of temperature, pH, estrogen (EE2, E2, and E1), and NO2-N concentration. A contact time of 3.6 to 4.1 days is required for 90% estrogen nitration at 500 mg/L NO2-N and pH 6.4. At 20 degrees C and pH 6.4, the threshold NO2-N concentration for nitration to occur is 9 mg/L; therefore, estrogen nitration is not likely in activated sludge treatment of domestic wastewater, but has potential for high-NH4-N-strength wastewaters.

Effects of testosterone undecanoate administered alone or in combination with letrozole or dutasteride in female to male transsexuals.

INTRODUCTION: Testosterone undecanoate (TU) has potential as androgen therapy for ovariectomized female to male (FtM) transsexual subjects; however, the long-term physiologic effects of TU treatment, the significance of testosterone (T), and the T metabolites dihydrotestosterone (DHT) and estradiol (E) on specific outcome parameters are currently unknown. AIM: The aim of this study was to investigate the long-term treatment of TU with regard to bone metabolism, body composition, and lipid profile in FtM subjects, and to evaluate the relationship between observed effects and circulating levels of T, E, and DHT. MAIN OUTCOME MEASURES: Circulating follicle-stimulating hormone, luteinizing hormone, T, E, DHT, and lipid concentrations were measured, as well as bone metabolism, body composition, and insulin resistance. METHODS: This was a 1-year, randomized treatment, open-label, uncontrolled safety study. Fifteen ovariectomized FtM subjects from an outpatient clinic were divided into three groups to receive TU 1,000 mg alone or in combination with oral administration of letrozole (L) 2.5 mg/die or dutasteride (D) 0.5 mg/die for a period of 54 weeks. RESULTS: TU alone and TU + D treatments were successful in terms of hormone adjustment, did not result in any adverse effects, and were well-tolerated. Bone mineral density decreased by an average of 0.9 g/cm(2) in the TU + L group, and the addition of D resulted in a failure to gain lean mass. CONCLUSIONS: This study confirmed that TU is a successful and safe treatment for FtM subjects. These data indicate that E has an important role in bone metabolism and that DHT may play a role in muscle metabolism.

Intratesticular androgens and spermatogenesis during severe gonadotropin suppression induced by male hormonal contraceptive treatment.

Male hormonal contraceptive regimens function by suppressing gonadotropin secretion, resulting in a dramatic decrease in testicular androgen biosynthesis and spermatogenesis. Animal studies suggest that persistent intratesticular (iT)-androgen production has a stimulatory effect on spermatogenesis in the setting of gonadotropin suppression. We hypothesized that men with incompletely suppressed spermatogenesis (>1,000,000 sperm/mL) during male hormonal contraceptive treatment would have higher iT-androgen concentrations than men who achieved severe oligospermia (

Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography-mass spectrometry.

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is approximately 20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.

Metabolism-based cyclophosphamide dosing for hematopoietic cell transplant.

When cyclophosphamide (120 mg/kg) is used for hematopoietic cell transplant, the increased area under the curve of carboxyethylphosphoramide mustard (AUC(CEPM)) is related to liver toxicity and death. We determined the feasibility of dose-adjusting cyclophosphamide to a preset metabolic endpoint (AUC(CEPM), 325 +/- 25 micromol/L.h). In 20 patients blood sampling was done over a 16-hour period after administration of 45 mg/kg cyclophosphamide; AUC(CEPM) from 0 to 16 hours was calculated by noncompartmental analysis. The expected AUC(CEPM) for 0 to 48 hours was estimated, and the second cyclophosphamide dose was determined. The mean second cyclophosphamide dose was 42 mg/kg, and the mean total cyclophosphamide dose was 86 mg/kg (range, 54-120 mg/kg). The mean AUC(CEPM) for the time from 0 to 48 hours was 296 micromol/L.h (95% confidence interval, 275-317 micromol/L.h). A retrospective analysis indicated that AUC(CEPM) could be more accurately predicted by use of a population pharmacokinetic model. We conclude that metabolism-based dosing of cyclophosphamide is feasible and that a lower cyclophosphamide dose does not affect engraftment.

A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells.

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of approximately 50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.

Transiently altered acetaminophen metabolism after liver transplantation.

BACKGROUND AND OBJECTIVES: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in drug metabolism occur after organ transplantation. This study was designed to characterize acetaminophen disposition during the first 6 months after liver transplantation. METHODS: Thirteen liver transplant patients received an oral dose of acetaminophen (500 mg) on days 2, 10, 90, and 180 after transplantation. Serial blood samples were collected for 8 hours, and urine was collected for 24 hours. Liver biopsy specimens were obtained from the donor liver during transplantation (day 0) and on days 10, 90, and 180 after transplantation. RESULTS: There were significant time-dependent changes in acetaminophen metabolism after liver transplantation. When day 2 and day 10 were compared with day 180, the respective mean urinary recovery was 137% and 81% higher for thioether conjugates derived from NAPQI (P =.0002 and P =.01, respectively); 31% and 22% lower for acetaminophen sulfate (P =.0006 and P =.008, respectively); and 22% and 27% lower for acetaminophen glucuronide (P =.05 and P =.004, respectively). Metabolite formation clearances changed in concordance with the fractional urinary recovery. It was surprising that hepatic CYP2E1 content on day 10 after transplantation was only 20% higher, on average, than that found on day 180 (not significant). In contrast, hepatic CYP3A4 content was 984% higher, on average, when tissue from days 10 and 180 was compared after transplantation (P =.007). CONCLUSIONS: Increased recovery of acetaminophen thioether conjugates during the first 10 days after liver transplantation was a result of impaired glucuronidation and sulfation and enhanced NAPQI formation.

Validation of a soy food-frequency questionnaire and evaluation of correlates of plasma isoflavone concentrations in postmenopausal women.

BACKGROUND: Soy foods may have various health benefits, but little is known about the patterns and correlates of soy consumption among postmenopausal women in the United States. OBJECTIVE: We assessed the reliability and validity of a soy food-frequency questionnaire (FFQ) and examined demographic, lifestyle, and dietary correlates of plasma isoflavone concentrations in postmenopausal women. DESIGN: In this cross-sectional study, soy isoflavone intake and plasma isoflavone concentration were analyzed in 96 postmenopausal women aged 50-79 y; the data were obtained at 2 visits that were 1 wk apart. Intake was determined with a 20-item soy FFQ and a comprehensive FFQ that included questions about tofu and soymilk. Fasting plasma daidzein and genistein concentrations were determined with liquid chromatography-mass spectrometry. RESULTS: Intraclass correlations between week 1 and week 2 values were >0.98 for both the soy and comprehensive FFQs. Median reported isoflavone intake was <2 mg/d. Pearson's product-moment correlation coefficients relating isoflavone intakes with plasma isoflavone concentrations ranged from 0.35 to 0.43. Plasma isoflavone concentrations were positively associated with age, fiber consumption, servings of fruit and vegetables, and dietary supplement use and were inversely associated with caffeine consumption; no associations with body mass index, education, dietary beliefs, activity level, alcohol intake, or fat intake were observed. CONCLUSIONS: Within a population with low soy consumption, the soy FFQ and comprehensive FFQ showed good reliability and moderate validity. Associations of plasma isoflavone concentrations with other dietary behaviors suggest that these compounds may serve as biomarkers of health behaviors in populations with low soy consumption.

Validation of a soy food frequency questionnaire with plasma concentrations of isoflavones in US adults.

OBJECTIVE: To validate assessment of soy intake using food frequency questionnaires (FFQs) compared with plasma isoflavone (genistein and daidzein) concentrations. DESIGN: Cross-sectional analysis of soy isoflavone intake and plasma analysis of isoflavones. SUBJECTS: 77 men and women, age range 20 to 40 years, recruited from the Seattle metropolitan area. MAIN OUTCOME MEASURES: Isoflavone intake was determined from responses to a 40-item soy FFQ and from tofu and soymilk intake assessed as part of a comprehensive FFQ used for the Women's Health Initiative (WHI FFQ). Isoflavone concentrations in fasting blood samples were determined by liquid chromatography-mass spectrometry. STATISTICAL ANALYSES: Correlation coefficients were calculated for: a) isoflavone intake assessed by the soy FFQ and the WHI FFQ, b) intake assessed by the soy FFQ and plasma isoflavone concentrations, and c) intake assessed by the WHI FFQ and plasma isoflavone concentrations. RESULTS: Isoflavone intake was highly correlated between the soy FFQ and the WHI FFQ (r = 0.84). Genistein and daidzein intakes determined by the soy FFQ were significantly correlated with plasma concentrations (r = 0.53 and 0.45, respectively). Isoflavone intake assessed from the WHI FFQ was also correlated with plasma concentration (r = 0.46 and 0.45). Soymilk and tofu were the two major contributors to isoflavone intake (38.6%). CONCLUSIONS: A soy-specific, 40-item FFQ assessed isoflavone intake with good validity. Isoflavone intake assessed by the WHI FFQ (tofu and soymilk) had lower correlations with plasma concentrations compared with the soy FFQ. Nonetheless, assessment of the two foods is a reasonably good marker for soy food consumption in this sample.

Estrogen biodegradation kinetics and estrogenic activity reduction for two biological wastewater treatment methods.

Estrogens from anthropogenic and livestock sources are a serious concern for aquatic ecosystems at concentrations less than 1 ng/L Fundamental process parameters to reduce estrogenic activity were investigated for two biotreatment methods: heterotrophic bacterial degradation in municipal activated sludge (AS) and a nitration process that is applicable to high NH4-N wastewaters. Batch tests with estrogen and nitro-estrogen compounds were conducted at nanogram per liter concentrations with mixed liquor from an AS wastewater treatment facility (WWTF) operating at a 3 day solids retention time (SRT) and a membrane bioreactor (MBR) WWTF operating at a 30-40 day SRT. The estrogenic activities of estrone (E1), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) were reduced 80-97% following nitration. First-order biological degradation rate coefficients (kb) of the nitrated estrogens were 10-50% lower than the parent estrogen compounds. The kb values for EE2 in MBR and AS mixed liquors were similar, 1.67 and 1.63 L/gVSS-day respectively, indicating that the bacteria responsible for EE2 degradation were present at long and short SRTs. The kb values for E1 and E2 were 2 orders of magnitude greater than for EE2. EE2 degradation was 7.5 times faster in the presence of E1 and E2, and no effect was observed with other estrogen mixtures.

A sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid A.

A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources.

Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity.

Plasma membrane monoamine transporter (PMAT) is a novel polyspecific organic cation transporter that transports organic cations and the purine nucleoside, adenosine. PMAT is expressed in the kidney, but the specific localization and function of this transporter in renal cells are unclear. In this study, we developed a polyclonal antibody toward a 14-amino acid sequence in the last intracellular loop of PMAT and determined the precise cellular localization of PMAT in human and rat kidneys. Surprisingly, we found that the PMAT protein was predominantly expressed in the glomerulus with minimal expression in tubular cells. Within the glomerulus, dual-color immunofluorescence labeling showed that the PMAT protein was specifically localized to the visceral glomerular epithelial cells, i.e., podocytes. There was no significant PMAT immunoreactivity in mesangial or glomerular endothelial cells. We further showed that puromycin aminonucleoside (PAN), a classic podocyte toxin that induces massive proteinuria and severe glomerulopathy, is transported by PMAT. Expression of PMAT in Madin-Darby canine kidney cells significantly increased cell sensitivity to PAN. Decynium 22, a potent PMAT inhibitor, abolished PAN toxicity in PMAT-expressing cells. Together, our data suggest that PMAT is specifically expressed in podocytes and may play an important role in PAN-induced kidney injury.

Pharmacokinetics and pharmacodynamics of oral testosterone enanthate plus dutasteride for 4 weeks in normal men: implications for male hormonal contraception.

Oral administration of testosterone enanthate (TE) and dutasteride increases serum testosterone and might be useful for male hormonal contraception. To ascertain the contraceptive potential of oral TE and dutasteride by determining the degree of gonadotropin suppression mediated by 4 weeks of oral TE plus dutasteride, 20 healthy young men were randomly assigned to 4 weeks of either 400 mg oral TE twice daily or 800 mg oral TE once daily in a double-blinded, controlled fashion at a single site. All men received 0.5 mg dutasteride daily. Blood for measurement of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, dihydrotesterone (DHT), and estradiol was obtained prior to treatment, weekly during treatment, and 1, 2, 4, 8, 12, 13, 14, 16, 20, and 24 hours after the morning dose on the last day of treatment. FSH was significantly suppressed throughout treatment with 800 mg TE once daily and after 4 weeks of treatment with 400 mg TE twice daily. LH was significantly suppressed after 2 weeks of treatment with 800 mg TE, but not with 400 mg TE. Serum DHT was suppressed and serum estradiol increased during treatment in both groups. High-density lipoprotein cholesterol was suppresed during treatment, but liver function tests, hematocrit, creatinine, mood, and sexual function were unaffected. The administration of 800 mg oral TE daily combined with dutasteride for 28 days significantly suppresses gonadotropins without untoward side effects and might have utility as part of a male hormonal contraceptive regimen.

17alpha-ethinylestradiol transformation via abiotic nitration in the presence of ammonia oxidizing bacteria.

Impacts of trace concentrations of estrogens on aquatic ecosystems are a serious environmental concern, with their primary source being wastewater treatment facility effluents. Increased removal of 17alpha-ethinylestradiol (EE2) has been reported for activated sludge treatment with long enough solids retention time for nitrification. Previous work based on batch tests with Nitrosomonas europaea and nitrifying activated sludge at high EE2 concentrations (>300 000 ng/L) and high NH4-N concentrations (>200 mg/L) has led to the hypothesis that ammonia oxidizing bacteria cometabolically degrade EE2. This work investigated EE2 transformation with N. europaea and Nitrosospira multiformis at environmentally relevant EE2 concentrations and LC-MS-MS to observe transformation products. Degradation of EE2 was not observed in batch tests with no NH4-N addition or with 10 mg/L NH4-N fed daily. At increased NH4-N concentrations (200-500 mg/L) EE2 transformation was observed, but the only detected products were nitrated EE2. Abiotic assays with growth medium confirmed EE2 removal by nitration, which is enhanced at low pH and high NO2-N concentrations. These results suggest that EE2 removal at low concentrations found in municipal treatment activated sludge systems is not due to cometabolic degradation by ammonia oxidizing bacteria, or to abiotic nitration, but most likely due to heterotrophic bacteria.

Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth.

Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,25-dihydroxyvitamin D3 conjugation.

The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 >> 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 microM and 33.7 +/- 1.4 and 32.9 +/- 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.

Role of cytochrome P450 2C8 and 2J2 genotypes in calcineurin inhibitor-induced chronic kidney disease.

OBJECTIVES: The calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus (Tac) help prevent allograft rejection but are associated with nephrotoxicity. Cytochrome P450 2C8 (CYP2C8) and CYP2J2 are polymorphic enzymes expressed in the kidney that metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids, promoting kidney homeostasis. This study examined the association between CNI-induced nephrotoxicity in liver transplant patients and CYP2C8 and CYP2J2 polymorphisms. METHODS: Liver transplantation patients receiving CNIs for at least 3 years were genotyped for CYP2C8*3, CYP2C8*4, CYP2C8 Haplotypes B and C, and CYP2J2*7 and evaluated for nephrotoxicity (serum creatinine > or = 1.6 mg/dl) 3-year post-transplantation. CYP2C8 proteins were also engineered in E. coli and their activity towards AA and inhibition by CNIs was investigated in vitro. RESULTS: The risk of kidney disease post-transplantation was positively associated with CYP2C8*3 genotype. Odds ratios for all participants carrying at least one CYP2C8*3 allele were significant [odds ratio=2.38 (1.19-4.78)]. Stratification by CNI indicated a significant association between CYP2C8*3 and nephrotoxicity among patients receiving Tac but not CsA. The risk of renal dysfunction was not significantly influenced by CYP2C8*4, CYP2J2*7, or CYP2C8 haplotype B genotypes although inheritance of haplotype C seems to be protective. In vitro, the gene products of CYP2C8*3 and CYP2C8*4 were deficient in AA epoxidation, retaining 26 and 18% of wild-type activity, respectively. Circulating plasma concentrations of CsA and Tac inhibited CYP2C8 wild-type in vitro epoxidation of AA by 17 and 35%, respectively. CONCLUSION: Inheritance of CYP2C8*3 is associated with a higher risk of developing renal toxicity in patients treated chronically with CNIs, and especially Tac, possibly by reducing formation of kidney protecting vasodilatory epoxyeicosatrienoic acids.