Use cases, best practice and reporting standards for metabolomics in regulatory toxicology

Metabolomics is a widely used technology in academic research, yet its application to regulatory science has been limited. The most commonly cited barrier to its translation is lack of performance and reporting standards. The MEtabolomics standaRds Initiative in Toxicology (MERIT) project brings together international experts from multiple sectors to address this need. Here, we identify the most relevant applications for metabolomics in regulatory toxicology and develop best practice guidelines, performance and reporting standards for acquiring and analysing untargeted metabolomics and targeted metabolite data. We recommend that these guidelines are evaluated and implemented for several regulatory use cases

Development of data processing methods for high resolution mass spectrometry-based metabolomics with an application to human liver transplantation

Direct Infusion (DI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) is becoming a popular measurement platform in metabolomics. This thesis aims to advance the data processing and analysis pipeline of the DI FT-ICR based metabolomics, and broaden its applicability to a clinical research. To meet the first objective, the issue of missing data that occur in a final data matrix containing metabolite relative abundances measured for each sample analysed, is addressed. The nature of these data and their effect on the subsequent data analyses are investigated. Eight common and/or easily accessible missing data estimation algorithms are examined and a three stage approach is proposed to aid the identification of the optimal one. Finally, a novel survival analysis approach is introduced and assessed as an alternative way of missing data treatment prior univariate analysis. To address the second objective, DI FT-ICR MS based metabolomics is assessed in terms of its applicability to research investigating metabolomic changes occurring in liver grafts throughout the human orthotopic liver transplantation (OLT). The feasibility of this approach to a clinical setting is validated and its potential to provide a wealth of novel metabolic information associated with OLT is demonstrated

Using community metabolomics as a new approach to discriminate marine microbial particulate organic matter in the western English Channel

Metabolomics provides an unbiased assessment of a wide range of metabolites and is an emerging ‘omics technique in the marine sciences. Here we use ‘non-targeted’ community metabolomics to determine patterns in metabolite profiles associated with particulate organic matter (POM) from two long-term monitoring stations in the western English Channel (station L4; 50° 15′N, 4° 13′W and E1; 50° 02′N, 4° 22′W). Particulates, composed of mainly phytoplankton, were sampled from the two stations during May 2009 by filtering onto glass fibre filters (> 0.7 μm to < 200 μm) from surface waters and from below thermocline. The polar metabolite fraction was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), and the lipid fraction by direct infusion mass spectrometry (DIMS), these were then analysed to statistically compare their distributions. Results show significantly different profiles of metabolites across the four locations with the largest differences for both the polar and lipid fractions found between the two stations relative to the smaller differences associated with depth. We putatively annotate the most discriminant metabolites revealing a range of amino-acid derivatives, diacylglyceryltrimethylhomoserine (DGTS) lipids, oxidised fatty acids (oxylipins), glycosylated compounds, oligohexoses, phospholipids, triacylglycerides (TAGs) and oxidised TAGs. The majority of the polar metabolites were most abundant in the surface waters at L4 and least abundant in the deep waters at E1 (E1-70m). In contrast, the oxidised TAGS were most abundant at E1, particularly at E1-70m. Differentiated metabolites are discussed in relation to supporting data on nutrients, carbon and chlorophyll, and to metatranscriptome-derived phytoplankton taxonomy. Our results show for the first time the power of community metabolomics in discriminating metabolite patterns associated with marine POM

Metabolomics and lipidomics: Expanding the molecular landscape of exercise biology

Dynamic changes in circulating and tissue metabolites and lipids occur in response to exercise-induced cellular and whole-body energy demands to maintain metabolic homeostasis. The metabolome and lipidome in a given biological system provides a molecular snapshot of these rapid and complex metabolic perturbations. The application of metabolomics and lipidomics to map the metabolic responses to an acute bout of aerobic/endurance or resistance exercise has dramatically expanded over the past decade thanks to major analytical advancements, with most exercise-related studies to date focused on analyzing human biofluids and tissues. Experimental and analytical con-siderations, as well as complementary studies using animal model systems, are warranted to help overcome challenges associated with large human interindividual variability and decipher the breadth of molecular mechanisms underlying the metabolic health-promoting effects of exercise. In this review, we provide a guide for exercise researchers regarding analytical techniques and experimental workflows commonly used in metabolomics and lipidomics. Furthermore, we discuss advancements in human and mammalian exercise research utilizing metabolomic and lipidomic approaches in the last decade, as well as highlight key technical considerations and remaining knowledge gaps to continue expanding the molecular landscape of exercise biology

The discovery and validation of metabolites as candidate biomarkers for the diagnosis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) constitutes a major disease burden worldwide. Much of its high mortality-to-incidence ratio can be attributed to late diagnosis, resulting in poor survival. Motivated by the need to develop a novel non-invasive diagnostic test to improve the chances of early diagnosis, this thesis makes use of metabonomic technologies to discover and validate metabolites as potential novel diagnostic markers for HCC. First, a systematic review of the literature on the topic was conducted to collate all published evidence of metabolites that were reported to be discriminatory for HCC. A bespoke risk of bias assessment tool was developed for metabonomic studies and a weighted score system was implemented to rank metabolites based on the strength of evidence. This resulted in a ranked list of metabolites with the greatest potential to be followed up for validation for each of the sample types (tissue, blood and urine). Then, validation of urinary metabolites with the greatest potential concluded from the systematic review was performed using data acquired from a UK cohort. None of the previously reported difference between HCC and cirrhosis groups could be reproduced, indicating the current lack of candidate markers specific for HCC detectable in urine. Finally, an exploratory analysis of serum 1H-NMR data from a UK and a Nigerian cohort was performed. Common and different alterations in metabolite levels between the two cohorts were compared. Glutamine-to-glutamate ratio was identified as a potential marker with the best discriminatory power between HCC and cirrhosis patients and this was validated using an independent cohort from the Gambia. This work adds to the ongoing effort to elucidate metabolites with the best potential to be further validated with the goal of developing a novel diagnostic test for HCC.Open Acces

Single sample pathway analysis in metabolomics: performance evaluation and application

Background Single sample pathway analysis (ssPA) transforms molecular level omics data to the pathway level, enabling the discovery of patient-specific pathway signatures. Compared to conventional pathway analysis, ssPA overcomes the limitations by enabling multi-group comparisons, alongside facilitating numerous downstream analyses such as pathway-based machine learning. While in transcriptomics ssPA is a widely used technique, there is little literature evaluating its suitability for metabolomics. Here we provide a benchmark of established ssPA methods (ssGSEA, GSVA, SVD (PLAGE), and z-score) alongside the evaluation of two novel methods we propose: ssClustPA and kPCA, using semi-synthetic metabolomics data. We then demonstrate how ssPA can facilitate pathway-based interpretation of metabolomics data by performing a case-study on inflammatory bowel disease mass spectrometry data, using clustering to determine subtype-specific pathway signatures. Results While GSEA-based and z-score methods outperformed the others in terms of recall, clustering/dimensionality reduction-based methods provided higher precision at moderate-to-high effect sizes. A case study applying ssPA to inflammatory bowel disease data demonstrates how these methods yield a much richer depth of interpretation than conventional approaches, for example by clustering pathway scores to visualise a pathway-based patient subtype-specific correlation network. We also developed the sspa python package (freely available at https://pypi.org/project/sspa/), providing implementations of all the methods benchmarked in this study. Conclusion This work underscores the value ssPA methods can add to metabolomic studies and provides a useful reference for those wishing to apply ssPA methods to metabolomics data

Novel statistical approaches for missing values in truncated high-dimensional metabolomics data with a detection threshold.

Despite considerable advances in high throughput technology over the last decade, new challenges have emerged related to the analysis, interpretation, and integration of high-dimensional data. The arrival of omics datasets has contributed to the rapid improvement of systems biology, which seeks the understanding of complex biological systems. Metabolomics is an emerging omics field, where mass spectrometry technologies generate high dimensional datasets. As advances in this area are progressing, the need for better analysis methods to provide correct and adequate results are required. While in other omics sectors such as genomics or proteomics there has and continues to be critical understanding and concern in developing appropriate methods to handle missing values, handling of missing values in metabolomics has been an undervalued step. Missing data are a common issue in all types of medical research and handling missing data has always been a challenge. Since many downstream analyses such as classification methods, clustering methods, and dimension reduction methods require complete datasets, imputation of missing data is a critical and crucial step. The standard approach used is to remove features with one or more missing values or to substitute them with a value such as mean or half minimum substitution. One of the major issues from the missing data in metabolomics is due to a limit of detection, and thus sophisticated methods are needed to incorporate different origins of missingness. This dissertation contributes to the knowledge of missing value imputation methods with three separate but related research projects. The first project consists of a novel missing value imputation method based on a modification of the k nearest neighbor method which accounts for truncation at the minimum value/limit of detection. The approach assumes that the data follows a truncated normal distribution with the truncation point at the detection limit. The aim of the second project arises from the limitation in the first project. While the novel approach is useful, estimation of the truncated mean and standard deviation is problematic in small sample sizes (N \u3c 10). In this project, we develop a Bayesian model for imputing missing values with small sample sizes. The Bayesian paradigm has generally been utilized in the omics field as it exploits the data accessible from related components to acquire data to stabilize parameter estimation. The third project is based on the motivation to determine the impact of missing value imputation on down-stream analyses and whether ranking of imputation methods correlates well with the biological implications of the imputation

Metabolomics enables precision medicine: “A White Paper, Community Perspective”

Introduction: Background to metabolomics: Metabolomics is the comprehensive study of the metabolome, the repertoire of biochemicals (or small molecules) present in cells, tissues, and body fluids. The study of metabolism at the global or “-omics” level is a rapidly growing field that has the potential to have a profound impact upon medical practice. At the center of metabolomics, is the concept that a person’s metabolic state provides a close representation of that individual’s overall health status. This metabolic state reflects what has been encoded by the genome, and modified by diet, environmental factors, and the gut microbiome. The metabolic profile provides a quantifiable readout of biochemical state from normal physiology to diverse pathophysiologies in a manner that is often not obvious from gene expression analyses. Today, clinicians capture only a very small part of the information contained in the metabolome, as they routinely measure only a narrow set of blood chemistry analytes to assess health and disease states. Examples include measuring glucose to monitor diabetes, measuring cholesterol and high density lipoprotein/low density lipoprotein ratio to assess cardiovascular health, BUN and creatinine for renal disorders, and measuring a panel of metabolites to diagnose potential inborn errors of metabolism in neonates. Objectives of White Paper—expected treatment outcomes and metabolomics enabling tool for precision medicine: We anticipate that the narrow range of chemical analyses in current use by the medical community today will be replaced in the future by analyses that reveal a far more comprehensive metabolic signature. This signature is expected to describe global biochemical aberrations that reflect patterns of variance in states of wellness, more accurately describe specific diseases and their progression, and greatly aid in differential diagnosis. Such future metabolic signatures will: (1) provide predictive, prognostic, diagnostic, and surrogate markers of diverse disease states; (2) inform on underlying molecular mechanisms of diseases; (3) allow for sub-classification of diseases, and stratification of patients based on metabolic pathways impacted; (4) reveal biomarkers for drug response phenotypes, providing an effective means to predict variation in a subject’s response to treatment (pharmacometabolomics); (5) define a metabotype for each specific genotype, offering a functional read-out for genetic variants: (6) provide a means to monitor response and recurrence of diseases, such as cancers: (7) describe the molecular landscape in human performance applications and extreme environments. Importantly, sophisticated metabolomic analytical platforms and informatics tools have recently been developed that make it possible to measure thousands of metabolites in blood, other body fluids, and tissues. Such tools also enable more robust analysis of response to treatment. New insights have been gained about mechanisms of diseases, including neuropsychiatric disorders, cardiovascular disease, cancers, diabetes and a range of pathologies. A series of ground breaking studies supported by National Institute of Health (NIH) through the Pharmacometabolomics Research Network and its partnership with the Pharmacogenomics Research Network illustrate how a patient’s metabotype at baseline, prior to treatment, during treatment, and post-treatment, can inform about treatment outcomes and variations in responsiveness to drugs (e.g., statins, antidepressants, antihypertensives and antiplatelet therapies). These studies along with several others also exemplify how metabolomics data can complement and inform genetic data in defining ethnic, sex, and gender basis for variation in responses to treatment, which illustrates how pharmacometabolomics and pharmacogenomics are complementary and powerful tools for precision medicine. Conclusions: Key scientific concepts and recommendations for precision medicine: Our metabolomics community believes that inclusion of metabolomics data in precision medicine initiatives is timely and will provide an extremely valuable layer of data that compliments and informs other data obtained by these important initiatives. Our Metabolomics Society, through its “Precision Medicine and Pharmacometabolomics Task Group”, with input from our metabolomics community at large, has developed this White Paper where we discuss the value and approaches for including metabolomics data in large precision medicine initiatives. This White Paper offers recommendations for the selection of state of-the-art metabolomics platforms and approaches that offer the widest biochemical coverage, considers critical sample collection and preservation, as well as standardization of measurements, among other important topics. We anticipate that our metabolomics community will have representation in large precision medicine initiatives to provide input with regard to sample acquisition/preservation, selection of optimal omics technologies, and key issues regarding data collection, interpretation, and dissemination. We strongly recommend the collection and biobanking of samples for precision medicine initiatives that will take into consideration needs for large-scale metabolic phenotyping studie