482 results found
Gray N, Gethings LA, Plumb RS, et al., 2020, UHPLC-MS-Based lipidomic and metabonomic investigation of the metabolic phenotypes of wild type and hepatic CYP reductase null (HRN) mice, Journal of Pharmaceutical and Biomedical Analysis, Vol: 186, Pages: 1-9, ISSN: 0731-7085
Hepatic cytochrome P450 reductase (EC 22.214.171.124, POR) deficient mice provide a useful means of investigating liver-related CYP450 drug metabolism. However, the organ-wide inactivation of CYP450s has wide ranging effects on liver physiology. Untargeted UHPLC-MS metabolic and lipid profiling of aqueous and organic solvent extracts has been employed to compare the metabolic phenotypes of livers obtained from either wild type (C57Bl6) or hepatic P450 reductase null (HRNTM) mice. The metabolic phenotyping of polar aqueous extracts revealed differences between wild type and HRNTM mice for bile acids with taurochendeoxycholic acid, and tauroursodeoxycholic acid increased in proportion in the latter and taurocholic acid reduced. Lipidomic profiling demonstrated that there were numerous differences in the lipidome, particularly relating to phospholipid synthesis with significant changes in the relative amounts of phosphatidylcholines (PC) and phosphatidylethanolamines (PE). These results illustrate the wide ranging disruptive effects on the normal hepatic phenotype that result from POR-deficiency in the the HRNTM animals.
Wilson I, Athersuch T, Bradshaw P, et al., 2020, Acyl Glucuronide Reactivity in Perspective, Drug Discovery Today, ISSN: 1359-6446
Letertre M, Munjoma N, Wolfer K, et al., 2020, A two-way interaction between methotrexate and the gut microbiota of male Sprague Dawley rats, Journal of Proteome Research, ISSN: 1535-3893
Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague–Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.
Letertre M, Munjoma NC, Slade SE, et al., 2020, Metabolic Phenotyping Using UPLC–MS and Rapid Microbore UPLC–IM–MS: Determination of the Effect of Different Dietary Regimes on the Urinary Metabolome of the Rat, Chromatographia, ISSN: 0009-5893
Wilson I, Dargue R, Zia R, et al., 2020, Metabolism and effects on endogenous metabolism of paracetamol (acetaminophen) in a porcine model of liver failure, Toxicological Sciences, Vol: 175, Pages: 87-97, ISSN: 1096-0929
The metabolic fate, toxicity and effects on endogenous metabolism of paracetamol (acetaminophen, APAP) in 22 female Landrace cross large white pigs were evaluated in a model of acute liver failure (ALF). Anaesthetized pigs were initially dosed at 250 mg/kg via an oroduodenal tube with APAP serum concentrations maintained above 300 mg/L using maintenance doses of 0.5-4g/h until ALF. Studies were undertaken to determine both the metabolic fate of APAP and its effects on the endogenous metabolic phenotype of ALF in using 1H NMR spectroscopy. Increased concentrations of citrate combined with pre-ALF increases in circulating lactate, pyruvate and alanine in plasma suggest mitochondrial dysfunction and a switch in hepatic energy metabolism to glycolysis in response to APAP treatment. A specific liquid chromatography-tandem mass spectrometry assay was used to quantify APAP and metabolites. The major circulating and urinary metabolite of APAP was the phenolic glucuronide (APAP-G), followed by p-aminophenol glucuronide (PAP-G) formed from N-deacetylated APAP. The PAP produced by N-deacetylation was the likely cause of the methaemoglobinemia and kidney toxicity observed in this, and previous, studies in the pig. The phenolic sulfate of APAP, and the glutathione-derived metabolites of the drug were only found as minor components (with the cysteinyl conjugate detected but not the mercapturate). Given its low sulfation, combined with significant capacity for N-deacetylation the pig may represent a poor translational model for toxicology studies for compounds undergoing significant metabolism by sulfation, or which contain amide bonds which when hydrolysed to unmask an aniline lead to toxicity. However, the pig may provide a useful model where extensive amide hydrolysis is seen for drugs or environmental chemicals in humans, but not in e.g., the rat and dog which are the pre-clinical species normally employed for safety assessment.
Dargue R, Grant I, Nye L, et al., 2020, The analysis of acetaminophen (paracetamol) and 7 metabolites in rat, pig and human plasma by U(H)PLC-MS, Bioanalysis, Vol: 12, Pages: 485-500, ISSN: 1757-6180
A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.
Bradshaw P, Richards S, Wilson I, et al., 2020, Kinetic modelling of acyl glucuronide and glucoside reactivity and development of structure-property relationships, Organic and Biomolecular Chemistry, Vol: 18, Pages: 1389-1401, ISSN: 1477-0520
Acyl glucuronide metabolites have been implicated in the toxicity of several carboxylic acid-containing drugs, and the rate of their degradation via intramolecular transacylation and hydrolysis has been associated with the degree of protein adduct formation. Although not yet proven, the formation of protein adducts in vivo-and subsequent downstream effects -has been proposed as amechanism of toxicity for carboxylic acid-containing xenobiotics capable of forming acyl glucuronides. A structurally-related series of metabolites, the acyl glucosides, have also been shown to undergo similar degradation reactions and consequently the potential todisplay a similar mode of toxicity.Here we reportdetailedkinetic modelsof each transacylation and hydrolysis reaction for a series of phenylacetic acid acyl glucuronides and their analogous acyl glucosides. Differences in reactivity were observed for the individual transacylation steps between the compound series; our findings suggest that the chargedcarboxylateion and neutral hydroxyl group in the glucuronideand glucoside conjugates, respectively, are responsible for these differences. The transacylation reaction was modelled using density functional theory and the calculated activation energy for this reaction showed a close correlation with thedegradation rateof the 1-banomer.Comparison of optimised geometries between the two series of conjugates revealed differences in hydrogen bondingwhich may further explain the differencesin reactivity observed. Together, these models may find application in drug discovery for prediction ofacyl glucuronide and glucoside metabolitebehaviour.
King AM, Trengove RD, Mullin LG, et al., 2020, Rapid profiling method for the analysis of lipids in human plasma using ion mobility enabled-reversed phase-ultra high performance liquid chromatography/mass spectrometry, Journal of Chromatography A, Vol: 1611, Pages: 1-10, ISSN: 0021-9673
The incorporation of ion mobility (IM) into LC–MS analysis has been demonstrated to result in the generation of superior quality MS and MS/MS spectral data as well as providing enhanced resolution in the IM dimension based on lipid class. Here a sub 4 min microbore LC-ion mobility-accurate mass MS (LC-IM-MS) method has been developed for the rapid, profiling of lipids in biological fluids. The method was scaled directly from a conventional, 12 min, LC-MS analysis maintaining the chromatographic performance and lipid separation observed in the longer methodology giving a 75% saving in mobile phase consumption and analysis time. Because of the additional dimension of separation provided by IM, improvements in mass spectral quality from the increased resolution of co-eluting species were also seen when compared to the same separation without IM, thus aiding the identification of target lipids. When applied to human plasma samples some 5037 (positive ESI) and 2020 (negative ESI) mass/retention time features were detected following adduct deconvolution and, of these, 3727 and 800 of those present in the pooled plasma QC samples had a CV of below 30% for positive and negative ESI modes respectively. The method was applied to the analysis of a pilot set of commercially sourced breast cancer plasma samples enabling the differentiation of samples from healthy controls and patients based on their lipid phenotypes. Analysis of the resulting data showed that phosphatidylcholines, triglycerides and diglycerides exhibited lower expression and phosphatidylserine showed increased expression in the breast cancer samples compared to those of healthy subjects. The coefficients of variation, determined by reference to the QC data, for all of the features identified as potential markers of disease, were 6% or less.
King A, Baginski M, Morikawa Y, et al., 2019, Application of a novel mass spectral data acquisition approach to lipidomic analysis of liver extracts from sitaxentan-treated liver-humanized PXB mice., Journal of Proteome Research, Vol: 18, Pages: 4055-4064, ISSN: 1535-3893
The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their entering the collision cell. By rapidly scanning the resolving quadrupole window over a specified mass range, co-eluting precursor ions are transmitted sequentially into the collision cell, where collision energies are cycled between low and elevated levels to induce fragmentation. This method of data generation provides both precursor and fragment ion information at high specificity, allowing for greater accuracy of compound identification, whether using a database, spectral libraries, or comparison to authentic standards. The value of the approach in simplifying and "de-cluttering" the spectra of co-eluting lipids is shown with examples from lipidomic profiles obtained in investigations of the composition of organic extracts of livers obtained from SCID and chimeric liver-humanized mice administered under various experimental conditions.
Nye LC, Williams JP, Munjoma NC, et al., 2019, A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry: Application to the characterisation of metabolites in rat urine, Journal of Chromatography A, Vol: 1602, Pages: 386-396, ISSN: 0021-9673
A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1–2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.
Gika H, Virgiliou C, Theodoridis G, et al., 2019, Untargeted LC/MS-based metabolic phenotyping (metabonomics/metabolomics): The state of the art, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol: 1117, Pages: 136-147, ISSN: 1570-0232
Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these “routine” approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or “new” approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.
Lees HJ, Swann JR, Poucher S, et al., 2019, Obesity and cage environment modulate metabolism in the Zucker rat: a multiple biological matrix approach to characterizing metabolic phenomena, Journal of Proteome Research, Vol: 18, Pages: 2160-2174, ISSN: 1535-3893
Obesity and its comorbidities are increasing worldwide imposing a heavy socioeconomic burden. The effects of obesity on the metabolic profiles of tissues (liver, kidney, pancreas), urine, and the systemic circulation were investigated in the Zucker rat model using 1H NMR spectroscopy coupled to multivariate statistical analysis. The metabolic profiles of the obese ( fa/ fa) animals were clearly differentiated from the two phenotypically lean phenotypes, ((+/+) and ( fa/+)) within each biological compartment studied, and across all matrices combined. No significant differences were observed between the metabolic profiles of the genotypically distinct lean strains. Obese Zucker rats were characterized by higher relative concentrations of blood lipid species, cross-compartmental amino acids (particularly BCAAs), urinary and liver metabolites relating to the TCA cycle and glucose metabolism; and lower amounts of urinary gut microbial-host cometabolites, and intermatrix metabolites associated with creatine metabolism. Further to this, the obese Zucker rat metabotype was defined by significant metabolic alterations relating to disruptions in the metabolism of choline across all compartments analyzed. The cage environment was found to have a significant effect on urinary metabolites related to gut-microbial metabolism, with additional cage-microenvironment trends also observed in liver, kidney, and pancreas. This study emphasizes the value in metabotyping multiple biological matrices simultaneously to gain a better understanding of systemic perturbations in metabolism, and also underscores the need for control or evaluation of cage environment when designing and interpreting data from metabonomic studies in animal models.
Whiley LW, Nye L, Grant I, et al., 2019, Ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization quantification of tryptophan metabolites and markers of gut health in serum and plasmaapplication to clinical and epidemiology cohorts, Analytical Chemistry, Vol: 91, Pages: 5207-5216, ISSN: 0003-2700
A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1–12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.
King AM, Mullin LG, Wilson ID, et al., 2019, Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC-MS and ion mobility enabled HILIC-MS, Metabolomics, Vol: 15, ISSN: 1573-3882
IntroductionAs large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening.ObjectivesTo develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system.MethodsA rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation.ResultsThe rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high coll
Gray N, Plumb RS, Wilson ID, et al., 2019, A validated UPLC-MS/MS assay for the quantification of amino acids and biogenic amines in rat urine, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol: 1106, Pages: 50-57, ISSN: 1570-0232
A UPLC-MS/MS assay, employing a reversed-phase separation, has been applied to the analysis of a number of common amino acids and biogenic amines in rat urine. Analytes were derivatised, using 6‑aminoquinolyl‑N‑hydroxysuccinimidyl carbamate (AccQTag Ultra™). Derivatisation with this reagent, by increasing the hydrophobicity of the analytes, enables better retention by improving reversed-phase chromatographic properties and also improves ionisation efficiency to enhance MS-detection. The method allows for the determination of 38 amino compounds in 7.5 min, including baseline resolution of critical isomers. The assay has been validated for the absolute quantification of 29 amino compounds in rat urine, over a concentration range of 0.6–200 μM, for the purpose of exploratory metabolite phenotyping. Acceptable linearity (R2 > 0.995) and intra- and inter-day accuracy (<20.7%) and precision (<20.1%) for these analytes was achieved. The limits of detection ranged from 1.2–12 fmol on column with 20 μL of sample. The remaining nine amines examined were not accurately quantified by this method but can be monitored for relative/fold change in biological samples. The use of the method is exemplified by the monitoring of changes in healthy male Sprague-Dawley rat urinary amino acid concentrations over a 7-day period.
Beger RD, Dunn WB, Bandukwala A, et al., 2019, Towards quality assurance and quality control in untargeted metabolomics studies, Metabolomics, Vol: 15, Pages: 1-5, ISSN: 1573-3882
We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day ‘Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies’ held at the National Cancer Institute’s Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.
Adesina-Georgiadis KN, Gray N, Plumb RS, et al., 2018, The metabolic fate and effects of 2-bromophenol in male Sprague-Dawley rats, Xenobiotica, ISSN: 0049-8254
1. The metabolic fate and urinary excretion of 2-bromophenol, a phenolic metabolite of bromobenzene, was investigated in male Sprague Dawley rats following single intraperitoneal doses at either 0, 100 or 200 mg/kg. 2. Urine was collected for seven days and samples analysed using 1H NMR spectroscopy, inductively coupled plasma (ICP)MS, and UPLC-MS. 3. 1H NMR spectroscopy of the urine samples showed that, at these doses, 2-bromophenol had little effect on endogenous metabolite profiles, supporting histopathology and clinical chemistry data which showed no changes associated with the administration of 2-bromophenol at these doses. 4. The use of ICP-MS Provided a means for the selective detection and quantification of bromine-containing species and showed that between 15 and 30% of the dose was excreted via the urine over the 7 days of the study for both the 100 and 200 mg doses respectively. 6. The bulk of the excretion of Br-containing material had occurred by 8 hr post administration. UPLC-MS of urine revealed a number of metabolites of 2-bromophenol, with 2-bromophenol glucuronide and 2-bromophenol sulphate identified as the major species. A number of minor hydroxylated metabolites were also detected as their glucuronide, sulphate or O-methyl conjugates. There was no evidence for the production of reactive metabolites.
Ekdahl A, Weidolf L, Baginski M, et al., 2018, The metabolic fate of fenclozic acid in chimeric mice with a humanized liver, Archives of Toxicology, Vol: 92, Pages: 2819-2828, ISSN: 0340-5761
The metabolic fate of the human hepatotoxin fenclozic acid ([2-(4-chlorophenyl)-1,3-thiazol-4-yl]acetic acid) (Myalex) was studied in normal and bile-cannulated chimeric mice with a humanized liver, following oral administration of 10 mg/kg. This in vivo animal model was investigated to assess its utility to study “human” metabolism of fenclozic acid, and in particular to explore the formation of electrophilic reactive metabolites (RMs), potentially unique to humans. Metabolism was extensive, particularly involving the carboxylic acid-containing side chain. Metabolism resulted in the formation of a large number of metabolites and involved biotransformation via both oxidative and conjugative routes. The oxidative metabolites detected included a variety of hydroxylations as well as cysteinyl-, N-acetylcysteinyl-, and cysteinylglycine metabolites. The latter resulted from the formation of glutathione adducts/conjugates providing evidence for the production of RMs. The production of other classes of RMs included acyl-glucuronides, and the biosynthesis of acyl carnitine, taurine, glutamine, and glycine conjugates via potentially reactive acyl-CoA intermediates was also demonstrated. A number of unique “human” metabolites, e.g., those providing evidence for side-chain extension, were detected in the plasma and excreta of the chimeric liver-humanized mice that were not previously characterised in, e.g., the excreta of rat and C57BL/6 mice. The different pattern of metabolism seen in these chimeric mice with a humanized liver compared to the conventional rodents may offer clues to the factors that contributed to the drug-induced liver injury seen in humans.
King AM, Grant I, Rainville PD, et al., 2018, Capillary ultra performance liquid chromatography-tandem mass spectrometry analysis of tienilic acid metabolites in urine following intravenous administration to the rat, Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol: 1087-1088, Pages: 142-148, ISSN: 1570-0232
Capillary scale (100 mm × 150 μm id) UPLC/MS/MS, performed using reversed-phase gradient chromatography on sub 2 μm particles, has been successfully employed for the characterization of the metabolites of the drug tienilic acid (TA) excreted via the urine following oral administration to the rat. The capillary LC system provided a significant increase (range ca. 11-33-fold) in sensitivity compared with a conventional 150 mm × 2.1 mm id UPLC system. An investigation of the effect of the injection volume and sample mass loading on the capillary column on the results obtained for both endogenous metabolites and TA was performed. This demonstrated that the injection of up to 2 μL of rat urine onto the system was permitted whilst still providing excellent chromatographic results and robustness. Qualitative analysis of the urine revealed the presence of TA itself and a total of 15 metabolites of the drug, including those resulting from biotransformations such as hydroxylation or conjugation. The capillary chromatography system was shown to be robust, and capable of providing comprehensive drug metabolite profiles from small format urine samples such as those obtained from preclinical studies in rodents.
Wilson CE, Dickie AP, Schreiter K, et al., 2018, The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice, Archives of Toxicology, Vol: 92, Pages: 1953-1967, ISSN: 0340-5761
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 r
Broadhurst D, Goodacre R, Reinke SN, et al., 2018, Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies, Metabolomics, Vol: 14, ISSN: 1573-3882
BackgroundQuality assurance (QA) and quality control (QC) are two quality management processes that are integral to the success of metabolomics including their application for the acquisition of high quality data in any high-throughput analytical chemistry laboratory. QA defines all the planned and systematic activities implemented before samples are collected, to provide confidence that a subsequent analytical process will fulfil predetermined requirements for quality. QC can be defined as the operational techniques and activities used to measure and report these quality requirements after data acquisition.Aim of reviewThis tutorial review will guide the reader through the use of system suitability and QC samples, why these samples should be applied and how the quality of data can be reported.Key scientific concepts of reviewSystem suitability samples are applied to assess the operation and lack of contamination of the analytical platform prior to sample analysis. Isotopically-labelled internal standards are applied to assess system stability for each sample analysed. Pooled QC samples are applied to condition the analytical platform, perform intra-study reproducibility measurements (QC) and to correct mathematically for systematic errors. Standard reference materials and long-term reference QC samples are applied for inter-study and inter-laboratory assessment of data.
Athersuch TJ, Antoine DJ, Boobis AR, et al., 2018, Paracetamol metabolism, hepatotoxicity, biomarkers and therapeutic interventions: a perspective, Toxicology Research, Vol: 7, Pages: 347-357, ISSN: 2045-452X
After over 60 years of therapeutic use in the UK, paracetamol (acetaminophen, N-acetyl-p-aminophenol, APAP) remains the subject of considerable research into both its mode of action and toxicity. The pharmacological properties of APAP are the focus of some activity, with the role of the metabolite N-arachidonoylaminophenol (AM404) still a topic of debate. However, that the hepatotoxicity of APAP results from the production of the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI/NABQI) that can deplete glutathione, react with cellular macromolecules, and initiate cell death, is now beyond dispute. The disruption of cellular pathways that results from the production of NAPQI provides a source of potential biomarkers of the severity of the damage. Research in this area has provided new diagnostic markers such as the microRNA miR-122 as well as mechanistic biomarkers associated with apoptosis, mitochondrial dysfunction, inflammation and tissue regeneration. Additionally, biomarkers of, and systems biology models for, glutathione depletion have been developed. Furthermore, there have been significant advances in determining the role of both the innate immune system and genetic factors that might predispose individuals to APAP-mediated toxicity. This perspective highlights some of the progress in current APAP-related research.
Bradshaw P, Wilson I, Upcott Gill R, et al., 2018, Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems, Scientific Reports, Vol: 8, ISSN: 2045-2322
The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5–10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.
Theodoridis GA, Gika HG, Wilson ID, 2018, Preface
Gika HG, Theodoridis GA, Wilson ID, 2018, Metabolic Profiling: Status, Challenges, and Perspective., Pages: 3-13
Metabolic profiling has advanced greatly in the past decade and evolved from the status of a research topic of a small number of highly specialized laboratories to the status of a major field applied by several hundreds of laboratories, numerous national centers, and core facilities. The present chapter provides our view on the status of the remaining challenges and a perspective of this fascinating research area.
Begou O, Gika HG, Theodoridis GA, et al., 2018, Quality Control and Validation Issues in LC-MS Metabolomics., Pages: 15-26
Global metabolic profiling (untargeted metabolomics) of different and complex biological matrices aims to implement an holistic, hypothesis-free analysis of (potentially) all the metabolites present in the analyzed sample. However, such an approach, although it has been the focus of great interest over the past few years, still faces many limitations and challenges, particularly with regard to the validation and the quality of the obtained results. The present protocol describes a quality control (QC) procedure for monitoring the precision of the analytical process involving untargeted metabolic phenotyping of urine and plasma/serum. The described/suggested methodology can be applied to different biological matrices, such as biological biofluids, cell, and tissue extracts.
Pickup K, Martin S, Partridge EA, et al., 2017, Acute liver effects, disposition and metabolic fate of [14C]-fenclozic acid following oral administration to normal and bile-cannulated male C57BL/6J Mice, Archives of Toxicology, Vol: 91, Pages: 2643-2653, ISSN: 1432-0738
The distribution, metabolism, excretion and hepatic effects of the human hepatotoxin fenclozic acid were investigated following single oral doses of 10 mg/kg to normal and bile-duct cannulated male C57BL/6J mice. Whole body autoradiography showed distribution into all tissues except the brain, with radioactivity still detectable in blood, kidney and liver at 72 h post dose. Mice dosed with [14C]-fenclozic acid showed acute centrilobular hepatocellular necrosis but no other regions of the liver were affected. The majority of the [14C]-fenclozic acid-related material recovered was found in the urine/aqueous cage wash, (49%) whilst a smaller portion (13%) was eliminated via the faeces. Metabolic profiles for urine, bile and faecal extracts, obtained using liquid chromatography and a combination of mass spectrometric and radioactivity detection, revealed extensive metabolism of fenclozic acid in mice that involved biotransformations via both oxidation and conjugation. These profiling studies also revealed the presence of glutathione-derived metabolites providing evidence for the production of reactive species by mice administered fenclozic acid. Covalent binding to proteins from liver, kidney and plasma was also demonstrated, although this binding was relatively low (less than 50 pmol eq./mg protein).
Glymenaki M, Barnes A, Hagan SO, et al., 2017, Stability in metabolic phenotypes and inferred metagenome profiles before the onset of colitis-induced inflammation., Scientific Reports, Vol: 7, ISSN: 2045-2322
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.
Wilson ID, Begou O, Gika HG, et al., 2017, Hyphenated MS–Based Targeted approaches in metabolomics, Analyst, Vol: 142, Pages: 3079-3100, ISSN: 1364-5528
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.
Dickie AP, Wilson CE, Schreiter K, et al., 2017, The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice, Biochemical Pharmacology, Vol: 135, Pages: 139-150, ISSN: 0006-2952
The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10 mg/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 ± 0.08 μg/mL at 0.25–0.5 h post-dose with an AUCinf of 1.74 ± 0.52 μg h/mL and an effective half-life for the drug of 1.42 ± 0.72 h (n = 3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15 ± 0.08 μg/mL at 0.25 h post-dose with an AUCinf of 1.94 ± 0.22 μg h/mL and an effective half-life of 1.28 ± 0.02 h (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.
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