Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic ...changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.
Metabolic profiling (metabolomics/metabonomics) is the measurement in biological systems of the complement of low-molecular-weight metabolites and their intermediates that reflects the dynamic ...response to genetic modification and physiological, pathophysiological, and/or developmental stimuli. The measurement and interpretation of the endogenous metabolite profile from a biological sample (typically urine, serum, or biological tissue extract) have provided many opportunities to investigate the changes induced by external stimuli (e.g., drug treatment) or enhance our knowledge of inherent biological variation within subpopulations. This article will focus on the basic principles of metabolic profiling and how the tools (nuclear magnetic resonance NMR, liquid chromatography–mass spectrometry LC-MS) can be applied in toxicology and pathology. Metabolic profiling can complement conventional methodologies and other “omics” technologies in investigating preclinical drug development issues. Case studies will illustrate the value of metabolic profiling in improving our understanding of phospholipidosis and peroxisome proliferation. A key message will be that metabolic profiling offers huge potential to highlight biomarkers and mechanisms in support of toxicology and pathology investigations in preclinical drug development.
Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in ...combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.
Phenotyping of 1,200 ‘healthy’ adults from the UK has been performed through the investigation of diverse classes of hydrophilic and lipophilic metabolites present in serum by applying a series of ...chromatography–mass spectrometry platforms. These data were made robust to instrumental drift by numerical correction; this was prerequisite to allow detection of subtle metabolic differences. The variation in observed metabolite relative concentrations between the 1,200 subjects ranged from less than 5 % to more than 200 %. Variations in metabolites could be related to differences in gender, age, BMI, blood pressure, and smoking. Investigations suggest that a sample size of 600 subjects is both necessary and sufficient for robust analysis of these data. Overall, this is a large scale and non-targeted chromatographic MS-based metabolomics study, using samples from over 1,000 individuals, to provide a comprehensive measurement of their serum metabolomes. This work provides an important baseline or reference dataset for understanding the ‘normal’ relative concentrations and variation in the human serum metabolome. These may be related to our increasing knowledge of the human metabolic network map. Information on the Husermet study is available at
http://www.husermet.org/
. Importantly, all of the data are made freely available at MetaboLights (
http://www.ebi.ac.uk/metabolights/
).
1H NMR spectroscopic and pattern recognition (PR)-based methods were used to investigate the biochemical variability in urine obtained from control rats and from rats treated with a hydrazine (a ...model hepatotoxin) or HgCl2 (a model renal cortical toxin). The 600 MHz 1H NMR spectra of urine samples obtained from vehicle- or toxin-treated Han-Wistar (HW) and Sprague-Dawley (SD) rats were acquired, and principal components analysis (PCA) and soft independent modeling of class analogy (SIMCA) analysis were used to investigate the 1H NMR spectral data. Variation and strain differences in the biochemical composition of control urine samples were assessed. Control urine 1H NMR spectra obtained from the two rat strains appeared visually similar. However, chemometric analysis of the control urine spectra indicated that HW rat urine contained relatively higher concentrations of lactate, acetate, and taurine and lower concentrations of hippurate than SD rat urine. Having established the extent of biochemical variation in the two populations of control rats, PCA was used to evaluate the metabolic effects of hydrazine and HgCl2 toxicity. Urinary biomarkers of each class of toxicity were elucidated from the PC loadings and included organic acids, amino acids, and sugars in the case of mercury, while levels of taurine, β-alanine, creatine, and 2-aminoadipate were elevated after hydrazine treatment. SIMCA analysis of the data was used to build predictive models (from a training set of 416 samples) for the classification of toxicity type and strain of rat, and the models were tested using an independent set of urine samples (n = 124). Using models constructed from the first three PCs, 98% of the test samples were correctly classified as originating from control, hydrazine-treated, or HgCl2-treated rats. Furthermore, this method was sensitive enough to predict the correct strain of the control samples for 79% of the data, based upon the class of best fit. Incorporation of these chemometric methods into automated NMR-based metabonomics analysis will enable on-line toxicological assessment of biofluids and will provide a tool for probing the mechanistic basis of organ toxicity.
The systemic biochemical effects of oral hydrazine administration (dosed at 75, 90, and 120 mg/kg) have been investigated in male Han Wistar rats using metabonomic analysis of 1H NMR spectra of urine ...and plasma, conventional clinical chemistry, and liver histopathology. Plasma samples were collected both pre- and 24 h postdose, while urine was collected predose and daily over a 7 day postdose period. 1H NMR spectra of the biofluids were analyzed visually and via pattern recognition using principal component analysis. The latter showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular weight compounds in urine and plasma, which was correlated with the severity of the hydrazine induced liver lesions. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-d-hydroxybutyrate, creatine, histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, β-alanine, citrulline, Nα-acetylcitrulline and argininosuccinate excretion was increased. Moreover, the most notable effect was the appearance in urine and plasma of 2-aminoadipate, which has previously been shown to lead to neurological effects in rats. High urinary levels of 2-aminoadipate may explain the hitherto poorly understood neurological effects of hydrazine. Metabonomic analysis of high-resolution 1H NMR spectra of biofluids has provided a means of monitoring the progression of toxicity and recovery, while also allowing the identification of novel biomarkers of development and regression of the lesion.
The use of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC–MS) as complementary analytical techniques for open metabolic profiling is illustrated in the ...context of defining urinary biochemical discriminators between male and female Sprague–Dawley rats. Subsequent to the discovery of a female-specific urinary discriminator by LC–MS, further LC, MS, and NMR methods have been applied in a coordinated effort to identify this urinary component. Thereafter, the biological relevance and context of the identified component, in this case a steroid metabolite, has been achieved. This approach will be deployed in future studies of disease, drug efficacy, and toxicity to discover and identify biologically relevant markers.
Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools
are needed for assessing the phenotypes of the many mouse models that ...are now being generated, where genes have been âknocked
out,â âknocked in,â or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of
cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify
mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy,
two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background
is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may
result in very different responses in diverse populations. Despite these differences associated with strain, multivariate
statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could
be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.
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