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Background. Rheumatoid arthritis (RA) is a complex, chronic autoimmune disease characterized by various inflammatory symptoms, including joint swelling, joint pain, and both structural and functional joint damage. The most commonly used animal model for studying RA is mice with collagen‐induced arthritis (CIA); the wide use of this model is due primarily to many similarities with RA in human patients. Metabolomics is used increasingly in biological studies for diagnosing disease and for predicting and evaluating drug interventions, as a large number of disease‐associated metabolites can be analyzed and interpreted from a biological perspective. Aim. To profile free amino acids and their biogenic metabolites in CIA mice plasma. Method. Ultra‐high‐performance liquid chromatography/tandem mass spectrometry coupled with multiple reaction monitoring (MRM) was used for metabolomics study. Results. Profile of 45 amine metabolites, including free amino acids and their biogenic metabolites in plasma was obtained from CIA mice. We found that the plasma levels of 20 amine metabolites were significantly decreased in the CIA group. Conclusion. The results suggest that a disordered amine response is linked to RA‐associated muscle wasting and energy expenditure.

Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1×106 cells. The platform consists of two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC-MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC-MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%. © 2011 Elsevier B.V.

A validated in vitro model of the large intestine (TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides (GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 (Bb-12) (iii) on GOS degradation, microbial growth (L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S rRNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S rRNA gene-targeted qPCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher (P < 0.05) for pig than for human faeces. However, lactate accumulation was higher (P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The widespread use of organophosphate (OP) pesticides has resulted in ubiquitous exposure in humans, primarily through their diet. Exposure to OP pesticides may have adverse health effects, including neurobehavioral deficits in children. The optimal design of new studies requires data on the reliability of urinary measures of exposure. In the present study, urinary concentrations of six dialkyl phosphate (DAP) metabolites, the main urinary metabolites of OP pesticides, were determined in 120 pregnant women participating in the Generation R Study in Rotterdam. Intra-class correlation coefficients (ICCs) across serial urine specimens taken at o18, 18–25, and 425 weeks of pregnancy were determined to assess reliability. Geometric mean total DAP metabolite concentrations were 229 (GSD 2.2), 240 (GSD 2.1), and 224 (GSD 2.2) nmol/g creatinine across the three periods of gestation. Metabolite concentrations from the serial urine specimens in general correlated moderately. The ICCs for the six DAP metabolites ranged from 0.14 to 0.38 (0.30 for total DAPs), indicating weak to moderate reliability. Although the DAP metabolite levels observed in this study are slightly higher and slightly more correlated than in previous studies, the low to moderate reliability indicates a high degree of within-person variability, which presents challenges for designing well-powered epidemiological studies.

We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R2 > 0.995), sensitivity (limit of detection 0.1-1 ng), repeatability, and reproducibility (relative standard deviation <10%). The IP-LC-ESI-MS method was shown to be a useful tool for microbial metabolomics, i.e., the comprehensive quantitative analysis of metabolites in extracts of microorganisms, and for the determination of the energy charge, i.e., the cellular energy status, as an overall quality measure for the sample workup and analytical protocols. © 2006 American Chemical Society.

In the field of metabolomics, hundreds of metabolites are measured simultaneously by analytical platforms such as gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and NMR to obtain their concentration levels in a reliable way. Analytical repeatability (intrabatch precision) is a common figure of merit for the measurement error of metabolites repeatedly measured in one batch on one platform. This measurement error, however, is not constant as its value may depend on the concentration level of the metabolite. Moreover, measurement errors may be correlated between metabolites. In this work, we introduce new figures of merit for comprehensive measurements that can detect these nonconstant correlated errors. Furthermore, for the metabolomics case we identified that these nonconstant correlated errors can result from sample instability between repeated analyses, instrumental noise generated by the analytical platform, or bias that results from data pretreatment. © 2011 American Chemical Society.

In sports doping, as well in man as in horseracing, stanozolol (Stan) was abused and became the subject of metabolism research. Also in veterinary practice, stanozolol became an important misused anabolic steroid. Like most other anabolic steroids, stanozolol has poor gas chromatographic behavior. It is difficult to detect in urine, because of low urinary excretion and renal clearance. This is due to the rapid metabolization, leading to low concentration levels of the parent compound found in urine. Therefore, most research studies have focused on the detection of its urinary metabolites. For the identification of the metabolites, different methods of extraction and detection are described in the literature. These are reviewed in this article. Most authors use a hydrolysis to free the phase II metabolites. Extraction procedures vary from solid-phase extraction (SPE), liquid-liquid (L-L) extraction to immunoaffinity chromatography (IAC). For the final detection, the use of gas chromatography (GC)-mass spectrometry (MS) can be compared with liquid chromatography (LC)-MSn. Different metabolites are identified depending on the administration of stanozolol in the animal experiment (oral or intramuscular). Analyses for these analytes in other matrices are also briefly discussed. © 2002 Elsevier Science B.V. All rights reserved.

Objective: The methods and results are described of a study on the dermal absorption of chlorpyrifos (CPF) in humans established via urinary excretion of the metabolite 3,5,6-trichloro-2-pyridinol (TCP). Methods: Two dermal, single, doses of CPF were applied in two study groups (A and B) each comprising three apparently healthy male volunteers who gave their written informed consent. The clinical part of the study was conducted in compliance with the ICH Guideline and the EC principles of good clinical practice (GCP). An approximately 0.5 ml dilution of CPF in ethanol was applied to an area of ∼100 cm2 of the volar aspect of the forearm, resulting in doses of either 5 mg (A) or 15 mg (B) of CPF per study subject. Duration of dermal exposure was 4 h, after which the non-absorbed fraction was washed off. The following samples were collected at pre-determined intervals for the determination of either CPF or its metabolite TCP: dosing solutions, wash-off fractions and urine samples collected up to 120 h after dosing. Results: A relatively large fraction of CPF (42%-67% of the applied dose) was washed off from the exposed skin area. Application of either 5 mg (A) or 15 mg CPF (B) resulted in the total urinary excretion of 131.8 μg (A) or 115.6 μg (B) of TCP 120 h after dosing. This indicated that 4.3% of the applied dose has been absorbed (A), while in group (B) no significant increase in urinary TCP (115.6 μg) was established. The latter indicates that an increase in the dermal dose at a fixed area does not increase absorption, which suggests that the percutaneous penetration rate was constant. Further, it was observed that the clearance of CPF by the body was not completed within 120 h, suggesting that CPF or TCP was retained by the skin and/or accumulated in the body. A mean elimination half-life of 41 h was established. Conc lusion: The results show that daily occupational exposure to CPF may result in accumulation of CPF and/or its metabolites, possibly resulting in adverse effects. © Springer-Verlag 2004.

Metabolic flows, especially those of amino acids, were determined and analyzed at different stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense. The method is mainly based on mass balances and measurements of amino acids and other metabolites. The measurements included the consumption rate of glucose, intake rates of all amino acids and production rates of carbon dioxide, cell mass, and transglutaminase. Three groups of metabolic flows were determined by three different methods. Those in the first group are determined by solely using measurement results. The second group deals with the synthesis of most amino acids. The metabolic flows were determined by using a mass-balancing method considering the contribution of these amino acids to the synthesis of cells and product, i.e., transglutaminase. The third group includes the reactions covering all other important intermediates. The metabolic flows in this group were calculated by a metabolite-balancing method. Metabolic flows during different fermentation phases were thus determined. The distribution of metabolic flows of amino acids implies that growth and transglutaminase production are active as long as there are free amino acids available in the medium. An important factor which limits further growth and production is probably the cross-linking action of transglutaminase on the nitrogen source in the medium. The results suggest that a nitrogen source other than peptone and/or amino acids might improve growth and production.

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-staterelated physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilmreactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilmreactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e.with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniformcolonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

Microbial metabolites may influence the metabolic integrity of intestinal epithelial cells and induce mucosal immune responses. Therefore, we investigated the effects of the microbial metabolites butyrate, iso-valerate, and ammonium on Caco-2 cells and macrophages. Barrier functioning was determined by measuring transepithelial electrical resistance and basolateral recoveries of metabolites. The barrier function of Caco-2 cells remained intact after exposures. Basolateral recoveries ranged from 6.2% to 15.2%. Tumour necrosis factor-α and interleukin-10 were measured to determine immune reactions. The Caco-2 cells did not secrete both cytokines. Physiological concentrations of butyrate and iso-valerate stimulated the secretion of tumour necrosis factor-α and suppressed the secretion of interleukin-10 by macrophages that are not protected by an epithelial barrier. In contrast, ammonium concentrations as high as those produced by microbiotas of IBD patients suppressed the release of both cytokines when the barrier function is impaired. © 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The hypothesis was studied that intestinal microbial metabolites play a role in the pathogenesis of inflammatory bowel disease. For that purpose, an in vitro model of the colon was inoculated with fresh feces of six healthy individuals and eight inflammatory bowel disease patients. Samples were taken from the model over time to analyze metabolites from both saccharolytic and proteolytic fermentation. Microbiotas from inflammatory bowel disease patients produced significantly more short-chain fatty acids and ammonia than microbiotas from healthy individuals. Furthermore, the branched-chain fatty acid production was 25% higher after inoculation with microbiotas from patients than after inoculation with microbiotas from healthy individuals. Phenolic compounds were produced by all microbiotas, with large interindividual variation. The production of (potentially toxic) metabolites may play a role in the onset or chronicity of inflammatory bowel disease, because they were produced in higher amounts by microbiotas from these patients than by microbiotas from healthy individuals.

A high-performance liquid chromatography-high resolution Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR-MS) method was developed to investigate the metabolism of ginsenosides in in vitro models of the gastro-intestinal tract. The metabolites were identified by high-resolution tandem mass spectrometry. Degradation and bioconversion routes of the different ginsenosides at acidic (gastric) conditions and in the presence of intestinal microbiota were elaborated. Besides hydrolysis (deglycosylation) also hydration reactions occurred at acidic conditions. The results illustrate the value of metabolite profiling by HPLC-FTICR-MS for understanding of the mechanisms in bioavailability of herbal drugs and their metabolites. © 2008 Elsevier B.V. All rights reserved.

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion of l-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-mol C-mol-1) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9 mM (1.8 g l-1) was obtained, at a yield of 8.5% (C-mol C-mol-1). A 2-fold increase of the specific p-hydroxybenzoate production rate (qp) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that l-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, qp increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient l-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified. © 2007 Elsevier B.V. All rights reserved.

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed. © 2006 Elsevier B.V. All rights reserved.

We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI-). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01-1 μM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n > 250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations. © 2008 Elsevier B.V. All rights reserved.