Stimulation of human T cells with sulfonamides and sulfonamide metabolites

Exposure to sulfonamides is associated with a high incidence of hypersensitivity reactions. Antigen-specific T cells are involved in the pathogenesis; however, the nature of the antigen interacting with specific T-cell receptors is not fully defined.

Attention-deficit hyperactivity disorder (ADHD) and glial integrity: an exploration of associations of cytokines and kynurenine metabolites with symptoms and attention

In contrast to studies of depression and psychosis, the first part of this study showed no major differences in serum levels of cytokines and tryptophan metabolites between healthy children and those with attention-deficit/hyperactivity disorder of the combined type (ADHD). Yet, small decreases of potentially toxic kynurenine metabolites and increases of cytokines were evident in subgroups. Therefore we examined predictions of biochemical associations with the major symptom clusters, measures of attention and response variability.

Optimizing heroin-assisted treatment (HAT): Assessment of the contribution of direct ethanol metabolites in identifying hazardous and harmful alcohol use

Heavy alcohol consumption may accelerate the progression of hepatitis C-related liver disease and/or limit efforts at antiviral treatment in opioid-dependent patients receiving heroin-assisted treatment (HAT). Our study aims to assess alcohol intake among HAT patients by self-reports compared to direct ethanol metabolites.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.

Stereoselective determination of drugs and metabolites in body fluids, tissues and microsomal preparations by capillary electrophoresis (2000-2010)

During the past two decades, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs and their metabolites in body fluids, tissues and in vitro preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed during the past 10 years and presents an overview of the key achievements encountered in that time period. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and bioanalysis of drug enantiomers of toxicological, forensic and doping interest. Chiral CE was extensively employed for research purposes to investigate the stereoselectivity associated with hydroxylation, dealkylation, carboxylation, sulfoxidation, N-oxidation and ketoreduction of drugs and metabolites. Enantioselective CE played a pivotal role in many biomedical studies, thereby providing new insights into the stereoselective metabolism of drugs in different species which might eventually lead to new strategies for optimization of pharmacotherapy in clinical practice.

Tradeoffs associated with constitutive and induced plant resistance against herbivory

Several prominent hypotheses have been posed to explain the immense variability among plant species in defense against herbivores. A major concept in the evolutionary ecology of plant defenses is that tradeoffs of defense strategies are likely to generate and maintain species diversity. In particular, tradeoffs between constitutive and induced resistance and tradeoffs relating these strategies to growth and competitive ability have been predicted. We performed three independent experiments on 58 plant species from 15 different plant families to address these hypotheses in a phylogenetic framework. Because evolutionary tradeoffs may be altered by human-imposed artificial selection, we used 18 wild plant species and 40 cultivated garden-plant species. Across all 58 plant species, we demonstrate a tradeoff between constitutive and induced resistance, which was robust to accounting for phylogenetic history of the species. Moreover, the tradeoff was driven by wild species and was not evident for cultivated species. In addition, we demonstrate that more competitive species—but not fast growing ones—had lower constitutive but higher induced resistance. Thus, our multispecies experiments indicate that the competition–defense tradeoff holds for constitutive resistance and is complemented by a positive relationship of competitive ability with induced resistance. We conclude that the studied genetically determined tradeoffs are indeed likely to play an important role in shaping the high diversity observed among plant species in resistance against herbivores and in life history traits.

Novel metabolites and roles for α-tocopherol in humans and mice discovered by mass spectrometry-based metabolomics

Contradictory results from clinical trials that examined the role of vitamin E in chronic disease could be a consequence of interindividual variation, caused by factors such as xenobiotic use. Cometabolism of vitamin E with other pharmaceutical products could affect the bioavailability of the drug. Thus, it is necessary to understand fully the metabolic routes and biological endpoints of vitamin E.

Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.

Human urine certified reference material CZ 6010: creatinine and toluene metabolites (hippuric acid and o-cresol) and a benzene metabolite (phenol)

A reference material for the biological monitoring of occupational exposure to toluene, benzene and phenol was prepared. O-cresol and hippuric acid (metabolites of toluene) are used for the biological monitoring of occupational exposure to toluene. Phenol, a metabolite of benzene, is used for the biological monitoring of exposure to benzene, but phenol can of course also be used as an indicator of exposure to phenol as well. The reference material (RM) used for the determination of these metabolites was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to toluene and those taking part in an inhalation experiment. Tests for homogeneity and stability were performed by determining urine concentrations of o-cresol, hippuric acid, creatinine and phenol. To investigate the stability of the RM, the urinary concentrations of o-cresol and phenol were monitored for eighteen months using GC and HPLC, while those of hippuric acid and creatinine were followed for five and six years, respectively, using HPLC. Analysis of variance showed that the concentrations did not change. The certified concentration values (and their uncertainties) of the substances in this reference material (phenol concentration c=6.46+/-0.58 mg l(-1); o-cresol concentration c=1.17+/-0.15 mg l(-1); hippuric acid concentration c=1328+/-30 mg l(-1); creatinine concentration c=0.82+/-0.10 g l(-1)) were evaluated via the interactive statistical programme IPECA.

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice

ABSTRACT: BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Urinary metabolites and antioxidant products of exogenous melatonin in the mouse

Exogenous melatonin is widely used for sleep disorders and has potential value in neuroprotection, cardioprotection and as an antioxidant. Here, a novel method is described for the determination of melatonin and six metabolites in mouse urine by use of LC-MS/MS and GC-MS. LC-MS/MS is used for the measurement of melatonin, N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin (6-HMEL), while GC/MS is used for the determination of N-[2-(5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (2-OMEL) and cyclic 3-hydroxymelatonin (3-HMEL) with detection limits on column of 0.02-0.5 pmol, depending on the metabolite. Following oral administration of melatonin to mice, a 0-24 hr urine collection revealed the presence of melatonin (0.2% dose), 6-HMEL (37.1%) and NAS (3.1%) comprising >90% of the total metabolites; AMK and AFMK were also detected at 0.01% each; 2-OMEL was found at 2.2% of the dose, which is >100 times more than the AMK/AFMK pathway, and comprises >5% of the melatonin-related material detected in mouse urine. 3-HMEL was largely found as a sulfate conjugate. These studies establish sensitive assays for determination of six melatonin metabolites in mouse urine and confirm the potential for antioxidant activity of melatonin through the identification in vivo of AMK and AFMK, ring-opened metabolites with a high capacity for scavenging reactive oxygen species.

Muscle metabolites: functional MR spectroscopy during exercise imposed by tetanic electrical nerve stimulation

Permission from the ethics committee and informed consent were obtained. The purpose of this study was to prospectively evaluate a method developed for the noninvasive assessment of muscle metabolites during exercise. Hydrogen 1 magnetic resonance (MR) spectroscopy peaks were measured during tetanic isometric muscle contraction imposed by supramaximal repetitive nerve stimulation. The kinetics of creatine-phosphocreatine and acetylcarnitine signal changes (P < .001) could be assessed continuously before, during, and after exercise. The control peak (trimethylammonium compounds), which served as an internal reference, did not change. This technique-that is, functional MR spectroscopy-opens the possibility for noninvasive diagnostic muscle metabolite testing in a clinical setting.