The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice.

The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUCinf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.

Hyphenated MS-based targeted approaches in metabolomics.

While global metabolic profiling (untargeted metabolomics) has been the center of much interest and research activity in the past few decades, more recently targeted metabolomics approaches have begun to gain ground. These analyses are, to an extent, more hypothesis-driven, as they focus on a set of pre-defined metabolites and aim towards their determination, often to the point of absolute quantification. The continuous development of the technological platforms used in these studies facilitates the analysis of large numbers of well-characterized metabolites present in complex matrices. The present review describes recent developments in the hyphenated chromatographic methods most often applied in targeted metabolomic/lipidomic studies (LC-MS/MS, CE-MS/MS, and GC-MS/MS), highlighting applications in the life and food/plant sciences. The review also underlines practical challenges-limitations that appear in such approaches.

Stability in metabolic phenotypes and inferred metagenome profiles before the onset of colitis-induced inflammation.

Inflammatory bowel disease (IBD) is associated with altered microbiota composition and metabolism, but it is unclear whether these changes precede inflammation or are the result of it since current studies have mainly focused on changes after the onset of disease. We previously showed differences in mucus gut microbiota composition preceded colitis-induced inflammation and stool microbial differences only became apparent at colitis onset. In the present study, we aimed to investigate whether microbial dysbiosis was associated with differences in both predicted microbial gene content and endogenous metabolite profiles. We examined the functional potential of mucus and stool microbial communities in the mdr1a -/- mouse model of colitis and littermate controls using PICRUSt on 16S rRNA sequencing data. Our findings indicate that despite changes in microbial composition, microbial functional pathways were stable before and during the development of mucosal inflammation. LC-MS-based metabolic phenotyping (metabotyping) in urine samples confirmed that metabolite profiles in mdr1a -/- mice were remarkably unaffected by development of intestinal inflammation and there were no differences in previously published metabolic markers of IBD. Metabolic profiles did, however, discriminate the colitis-prone mdr1a -/- genotype from controls. Our results indicate resilience of the metabolic network irrespective of inflammation. Importantly as metabolites differentiated genotype, genotype-differentiating metabolites could potentially predict IBD risk.

Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A metabonomic study of gut microbial influence on host brain metabolism.

1H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results revealed a number of differences between germ free (GF) and conventional (CV) rats or specific pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species- and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine, NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF hippocampus was characterized by higher taurine and phosphocholine concentrations and lower quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be greater in the absence of an established microbiome. The results provide further evidence that brain biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy metabolism demonstrating biochemical dialogue between the microbiome and brain.

The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08µg/mL at 0.25-0.5h post-dose with an AUCinf of 1.74±0.52µgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08µg/mL at 0.25h post-dose with an AUCinf of 1.94±0.22µgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans.

Detection of thiol modifications by hydrogen sulfide.

Hydrogen sulfide (H2S) is an important gasotransmitter in both animals and plants. Many physiological events, including responses to stress, have been suggested to involve H2S, at least in part. On the other hand, numerous responses have been reported following treatment with H2S, including changes in the levels of antioxidants and the activities of transcription factors. Therefore, it is important to understand and unravel the events that are taking place downstream of H2S in signaling pathways. H2S is known to interact with other reactive signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO). One of the mechanisms by which ROS and NO have effects in a cell is the modification of thiol groups on proteins, by oxidation or S-nitrosylation, respectively. Recently, it has been reported that H2S can also modify thiols. Here we report a method for the determination of thiol modifications on proteins following the treatment with biological samples with H2S donors. Here, the nematode Caenorhabditis elegans is used as a model system but this method can be used for samples from other animals or plants.

The effect of a paced auditory serial addition test (PASAT) intervention on the profile of volatile organic compounds in human breath: a pilot study.

This study sought to identify if detectable changes in human breath profiles may be observed following a psychological intervention designed to induce stress, a paced auditory serial addition test (PASAT). Breath samples were collected from 22 participants (10 male and 12 female) following a double cross-over randomized design with two experimental interventions. One intervention required participants to listen to classical music chosen to be neutral. The other intervention required participants to undertake a PASAT that induced cardiovascular responses consistent with acute stress. Both interventions also involved two sequences of cognitive function tests. Blood-pressure and heart-rate were recorded throughout each intervention and distal breath samples were collected onto Tenax® TA/Carbograph 1 thermal desorption tubes, using an adaptive breath sampler. Samples were collected before and after the PASAT. Breath samples were analysed by thermal desorption gas chromatography-mass spectrometry. Data registration using retention indexing and peak deconvolution followed by partial least-squares discriminant analysis identified six stress sensitive compounds. A principal components analysis model based on these components generated a model that predicted post-PASAT versus post-neutral intervention samples with a sensitivity of 83.3% and a selectivity of 91.6% for females, compared to 100% sensitivity and 90% selectivity for males. Of the six compounds indole, 2-hydroxy-1-phenylethanone, benzaldehyde, and 2-ethylhexan-1-ol were identified on the basis of mass spectral, retention indexing and confirmation against pure standards. 2-methylpentadecane was tentatively identified from mass spectral and retention indexing, whilst one component has yet to be assigned, although the mass spectrum is indicative of a terpene. Indole and 2-methylpentadecane concentrations increased in response to the PASAT intervention, while the other compounds reduced in their abundance in human breath, possibly as a result of ventilation effects.

Comparison of metabolomic profiles obtained using chemical ionization and electron ionization MS in exhaled breath.

The exhaled breath is rich in a wide range of volatile organic compounds with the potential to provide readily accessible biomarkers for metabolic activity in the body as a result of normal or abnormal/disease processes. Exhaled breath samples from five healthy volunteers have been analyzed by thermal desorption GC-MS using electron impact and chemical ionization. A total of eight compounds: 2-propenoic acid; 2-methyl, methyl ester; toluene; hexanal; 1,4-cyclohexadiene, 1-methyl-4-(1methlethyl); phenol; nonanal; dodecane and indole, have been evaluated to establish differences in selectivity and sensitivity using these two mechanisms of ionization. The combination of both electron impact and chemical ionization profiles could prove valuable when prospecting for breath-derived biomarkers as they result in complementary information that aids the identification of unknown components.

Extraction methods for the removal of phospholipids and other endogenous material from a biological fluid.

BACKGROUND: A comparison of three different sample preparation techniques for the analysis of plasma samples has been investigated to highlight the effect that these approaches have on the removal of endogenous material. The three techniques under investigation are: SPE, support assisted liquid-liquid extraction and nonspecific solvent-based protein precipitation. RESULTS: Comparisons are made on the practicalities of each approach and to allow a semiquantitative assessment between the effectiveness of these different techniques the relative amounts of phospholipids present within the sample are analyzed. Total ion chromatograms are also obtained to further study the effects of different extraction techniques in the removal of endogenous components from a biological matrix. Both of these approaches provide a very coarse measure of the cleanliness of the extracts and demonstrate that support assisted liquid-liquid extraction and an optimized SPE approach remove a greater amount of endogenous material. CONCLUSION: This study highlights the importance of sample preparation in removing endogenous material, which may have a detrimental effect on the performance of a bioanalytical assay.

Nitric oxide and ABA in the control of plant function.

Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to control many aspects of physiology. ABA has been extensively studied in the mechanisms which control stomatal movement as well as in seed dormancy and germination and plant development. The addition of either ABA or NO to plant cells is known to instigate the actions of many signal transduction components. Both may have an influence on the phosphorylation of proteins in cells mediated by effects on protein kinases and phosphatases, as well as recruiting a wide range of other signal transduction molecules to mediate the final effects. Both ABA and NO may also lead to the regulation of gene expression. However, it is becoming more apparent that NO may be acting downstream of ABA, with such action being mediated by reactive oxygen species such as hydrogen peroxide in some cases. However not all ABA responses require the action of NO. Here, examples of where ABA and NO have been put together into the same signal transduction pathways are discussed.

Integrated HPLC-MS and (1)H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides.

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-beta-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an alpha,alpha-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37 degrees C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and (1)H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, (1)H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of (1)H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than (1)H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-beta-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

The role of gut microbiota in drug response.

Higher organisms such as mammals exist in a symbiotic relationship with their gut microbiota, formed from a diverse and highly metabolically active consortium of species. The gut microbiota, in addition to their ability to process dietary derived material, are also capable of performing a range of biotransformations on xenobiotics, such as drugs and their metabolites, in ways that can affect absorption and bioavailability. The potential for the gut microflora to influence drug metabolism and toxicity in unexpected ways is discussed.

Evaluation of the use of UPLC-TOFMS with simultaneous [14C]-radioflow detection for drug metabolite profiling: application to propranolol metabolites in rat urine.

The non-selective beta-adrenergic receptor antagonist propranolol [1-(isopropylamino)-3-(1-naphthoxy)-2-propanol] is metabolised extensively in vivo. Enumerating and identifying the many metabolites that result from multiple biotransformations provides a considerable analytical challenge, greatly aided by efficient chromatography coupled to sensitive mass spectrometric detection. Here the use of the newly introduced high-resolution technique of "ultra performance liquid chromatography" (UPLC) linked to quadrupole time-of-flight mass spectrometry (TOFMS) with simultaneous [(14)C]-radioflow detection was applied to rapid metabolite profiling. [14C]-propranolol, dosed intraperitoneally to rat at 25 mg kg(-1) and 200 microCi kg(-1) was used as a model compound for this evaluation. Some 14 metabolites were detected in the urine by this technique including a number of conjugated metabolites such as sulphates, several isobaric glucuronides and two novel di-glucuronides.

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using (19)F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS ((127)I).

A combination of (19)F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by (19)F-NMR spectroscopy and HPLC-(127)I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.

The application of microbore UPLC/oa-TOF-MS and 1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin.

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.

Metabolism of [14C]-5-chloro-1,3-benzodioxol-4-amine in male Wistar-derived rats following intraperitoneal administration.

[14C]-5-chloro-1,3-benzodioxol-4-amine was administered intraperitoneally (i.p.) to bile duct-cannulated rats (Alpk:ApfSD, Wistar derived) at 25 mg kg-1 to determine the rates and routes of excretion of the compound and to investigate its metabolic fate. A total of 89.1% of the dose was excreted in the 48 h following administration, the majority being recovered in the urine during the first 12 h. The main metabolite in both urine and bile, detected by high-performance liquid chromatography (HPLC) with radioprofiling and mass spectrometry, was identified as a demethylenated monosulfate conjugate. Unchanged parent compound formed a major component of the radiolabel excreted in urine and, in addition to unchanged parent and demethylenated sulphate conjugate, a large number of minor metabolites were detected in urine and bile. The overall metabolic fate of 5-chloro-1,3-benzodioxol-4-amine in the rat was complex, with some similarities to previously studied methylenedioxyphenyl compounds.

Statistically integrated metabonomic-proteomic studies on a human prostate cancer xenograft model in mice.

A novel statistically integrated proteometabonomic method has been developed and applied to a human tumor xenograft mouse model of prostate cancer. Parallel 2D-DIGE proteomic and 1H NMR metabolic profile data were collected on blood plasma from mice implanted with a prostate cancer (PC-3) xenograft and from matched control animals. To interpret the xenograft-induced differences in plasma profiles, multivariate statistical algorithms including orthogonal projection to latent structure (OPLS) were applied to generate models characterizing the disease profile. Two approaches to integrating metabonomic data matrices are presented based on OPLS algorithms to provide a framework for generating models relating to the specific and common sources of variation in the metabolite concentrations and protein abundances that can be directly related to the disease model. Multiple correlations between metabolites and proteins were found, including associations between serotransferrin precursor and both tyrosine and 3-D-hydroxybutyrate. Additionally, a correlation between decreased concentration of tyrosine and increased presence of gelsolin was also observed. This approach can provide enhanced recovery of combination candidate biomarkers across multi-omic platforms, thus, enhancing understanding of in vivo model systems studied by multiple omic technologies.

A multi-analytical platform approach to the metabonomic analysis of plasma from normal and Zucker (fa/fa) obese rats.

Plasma obtained from 20 week old normal Wistar-derived and Zucker (fa/fa) rats was analysed using a number of different analytical methodologies to obtain global metabolite profiles as part of metabonomic investigations of animal models of diabetes. Samples were analysed without sample pre-treatment using 1H NMR spectroscopy, after acetonitrile solvent protein precipitation by ultra-performance liquid chromatography-MS (UPLC-MS) and after acetonitrile protein precipitation and derivatisation for capillary gas chromatography-MS (GC-MS). Subsequent data analysis using principal components analysis revealed that all three analytical platforms readily detected differences between the plasma metabolite profiles of the two strains of rat. There was only limited overlap between the metabolites detected by the different methodologies and the combination of all three methods of metabolite profiling therefore provided a much more comprehensive profile than would have been provided by their use individually.

The comparative metabonomics of age-related changes in the urinary composition of male Wistar-derived and Zucker (fa/fa) obese rats.

The global metabolite profiles of endogenous compounds excreted in urine by male Wistar-derived and Zucker (fa/fa) obese rats were investigated from 4 to 20 weeks of age using both 1H NMR spectroscopy and HPLC-TOF/MS with electrospray ionisation (ESI). Multivariate data analysis was then performed on the resulting data which showed that the composition of the samples changed with age, enabling age-related metabolic trajectories to be constructed. At 4 weeks it was possible to observe differences between the urinary metabolite profiles from the two strains, with the difference becoming more pronounced over time resulting in a marked divergence in their metabolic trajectories at 8-10 weeks. The changes in metabolite profiles detected using 1H NMR spectroscopy included increased protein and glucose combined with reduced taurine concentrations in the urine of the Zucker animals compared to the Wistar-derived strain. In the case of HPLC-MS a number of ions were found to be present at increased levels in the urine of 20 week old Zucker rats compared to Wistar-derived rats including m/z 71.0204, 111.0054, 115.0019, 133.0167 and 149.0454 (negative ion ESI) and m/z 97.0764 and 162.1147 (positive ion ESI). Conversely, ions m/z 101.026 and 173.085 (negative ion ESI) and m/z 187.144 and 215.103 (positive ion ESI) were present in decreased amounts in urine from Zucker compared to Wistar-derived rats. Metabolite identities proposed for these ions include fumarate, maleate, furoic acid, ribose, suberic acid, carnitine and pyrimidine nucleoside. The utility of applying metabonomics to understanding disease processes and the biological relevance of some of the findings are discussed.