Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry.

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that permits the quantification of major circulating vitamin D(3) metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction, and Diels-Alder derivatization procedure prior to LC-MS/MS analysis.

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.

Estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer.

Background: Estrogens suppress tumor growth in prostate cancer which progresses despite anorchid serum androgen levels, termed castration resistant prostate cancers (CRPC), although the mechanisms are unclear. We hypothesize that estrogen inhibits CRPC in anorchid animals by suppressing tumoral androgens, an effect independent of the estrogen receptor.

Methods: The human CRPC xenograft LuCaP 35V was implanted into orchiectomized male SCID mice and established tumors were treated with placebo, 17beta-estradiol or 17beta-estradiol and estrogen receptor antagonist ICI 182,780.

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.

Serum LH correlates highly with intratesticular steroid levels in normal men.

Sex steroids are essential for spermatogenesis; however, normal intratesticular concentrations of these hormones in man have not been extensively studied. To improve our understanding of intratesticular hormone concentrations, we performed bilateral testicular aspirations in a group of normal men, determined sex steroid concentrations within each testis, and compared these levels to serum hormone concentrations. Ten healthy human subjects aged 20-49 underwent bilateral testicular aspirations.

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.

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.

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.

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.

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.

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.

Sensitive and specific LC-MS assay for quantification of digoxin in human plasma and urine.

Digoxin, a commonly prescribed cardiac glycoside with a narrow therapeutic window, is routinely used in pharmacokinetic studies to assess the in vivo activity of the drug efflux pump P-glycoprotein. To minimize adverse events, a sub-therapeutic dose of digoxin is usually administered, producing low plasma concentrations requiring a sensitive detection technique. Commonly available immunoassay techniques do not provide the required sensitivity to measure these low plasma concentrations and are potentially non-specific in certain subject populations.

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.

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.

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.

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.

Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry.

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives.

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 ( View Article and Find Full Text PDF

Determination of free and total cortisol in plasma and urine by liquid chromatography-tandem mass spectrometry.

Cortisol is an important adrenal steroid hormone involved in the regulation of metabolic homeostasis. A new liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) multiple reactant monitoring (MRM) procedure for the measurement of cortisol concentration in plasma ultrafiltrate, whole plasma, and urine was developed and validated. Plasma, plasma ultrafiltrate, or urine was extracted by ethyl acetate.

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.

Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3).

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).

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.

Frequency of soy food consumption and serum isoflavone concentrations among Chinese women in Shanghai.

Objective: The food-frequency questionnaire (FFQ) can be an efficient tool to evaluate dietary intake in large, population-based studies, especially for specific foods. The objective of this study was to validate the assessment of soy and isoflavone (daidzein and genistein) intakes, measured by an FFQ, by comparing intakes with serum isoflavone concentrations.

Design And Setting: Soy and isoflavone intakes and serum isoflavone concentrations were determined as part of a case-control study of dietary factors and risks of benign breast disease and breast cancer.