Metabolomics as a Tool in Disease Diagnosis in Animals

Metabolomics as a Tool in Disease Diagnosis in Animals

The metabolome of the organism is closely linked to its phenotype and thus forms an important tool in studying disease biology. We currently use Mass Spectrometry based analysis of malaria-infected erythrocytes from in vitro cultures as well as from patients’ serum to identify unique malaria-specific biomarkers. This would enable accurate detection of sub-microscopic infections overcoming the limitations of traditional malaria-detection techniques. A similar approach is also used for studying other human and animal diseases in our laboratory. A quantitative, targeted as well as un-targeted, global metabolomics approach is being used to decipher the metabolic pathways in human diseases.

Metabolomics is the emerging field of Omics Science. It refers to technique in which various low/ small molecular weight molecule or metabolites of biological sample like urine, blood, saliva, milk, breath exhalate etc of cell, tissue ,organ are taken for study and metabolomics profiling is performed involving either mass –spectrometry or nuclear magnetic resonance for comparison. Example antibiotics, pigments (resins, terpenes). Metabolome refers to complete set of metabolites in biological cell, organ, tissue or organism, which are usually cellular processes end products, example fructose, sucrose. This level of metabolites keeps on changing with stimuli (external/internal), stress or diseased condition of animal. Metabolomics term was first coined in 1990 and used for studying metabolome in field of nutrition, inborn metabolic errors and drug application effect, like in detection of cancer. Metabolomics is only one part of different large scale analyses of omics’ world like genomics, proteomics, spliceomics, epigenomics, transcriptomics and pharmacogenomics.While genomics deals with DNA,transcriptomics with RNA,proteomics with proteins,metabolomics is related with study of sugars,nucleotide,aminoacids, lipids(lipidome) metabolites which are responsible for phenotypic character and functioning in living being. This is very much related with bioinformatics . Metabolomics study is important because the metabolome is closely knit with genotype of an organism, physiology and the environment that is what type of food organism is consuming or air is being inhaled. This technique helps in having closer look at genotype-phenotype and genotype-envirotype relationships. It has been reported that all abnormalities/changes which are detected in genome or transcriptome are not causative agent of abnormality/disease that is there may be silent mutations. Likewise it is not so that all enzymes or protein products detected through proteomics may be functional. It may happen that all environmental influences occurring at different stages are not taken into consideration. But this technique may be used for monitoring changes occurring in genome or for measuring effects of up/down regulation of specific gene transcript.Usually; metabolites are result of cellular pathway which takes into account the variations taking place at genome, transcriptome, proteome including metabolic influences. Proteomics study may involve two dimensional gel electrophoresis.matrix-assisted laser desorption/ionization or time-of-flight mass spectrometry. Metabolic fingerprinting involves measurement of subset of whole profile with little quantitation or differentiation of metabolites. The target isotope-based analysis mainly focuses on metabolome particular segment by analyzing few selected metabolites which comprise specific biochemical pathway. We also use the mass spectrometry approach to identify in-born errors of metabolism in neonates. These disorders constitute a diverse heterogenous group of disorders and a significant percentage of neonates suffer from these disorders. Early detection by using the highly sensitive method of mass spectrometry could potentially help provide early diagnosis and appropriate disease management strategy for children suffering from these diseases.

Procedure:

In first step sample collection is done followed by treatment and processing.Metabolomic assessment can be pursed either in vitro or in vivo using tissue, fluids or cells. Mostly biofluids are preferred as it is easy to collect like serum, urine, plasma, saliva, bronchial washes, pleural fluids or prostatic secretions. But mostly use of serum and urine are in practice. Try to maintain low temperature and consistent samples extraction as it is essential. For biofluids 0.1-0.5 mL is the standard sample volume. Nowadays some scientists have also shown interest in using tissues directly. For NMR there is requirement of minimal sample preparation. Seperation techniques used mainly include methods like gas chromatography, high performance liquid chromatography (HPLC), Ultra performance Liquid chromatography and capillary electrophoresis. The detection technique for qualitative and quantitative assessment involves use of nuclear magnetic resonance spectroscopy (NMR) or mass spectrometry.NMR uses isotope possessing property of magnetic spin. Isotopes mainly used are 1H and 13 C .NMR spectroscopy are used to measure phosphorylated lipid metabolites and high energy phosphate metabolites. The acquisition time is about 10 minutes. As it preserves tissue architecture pathological evaluation is not compromised. The metabolites detected in cancer includes aminoacids (leucine, Isoleucine, valine, alanine, glutamine, tyrosine, asparagine, lysine, free choline, phenylalanine, glycine, taurine, glycine),beta hydroxybutyrate, alpha ketoisovalerate, beta glucose, alpha glucose, formate, UTP and UDP, phosphatidylcholine, plasmalogen, acetate, glutathione,succinate,fumarate.Dimethylalanine, OInorganic phosphate, triacylglycerol, creatine, phosphocreatine, betaine,ADP and ATP,sugar phosphates, phosphatidyl-glycerol, myo-inositol, cholesterol and esters. Both NMR and MS involve intial chromatography stage followed by separation according to their mass to charge ratio. All metabolites cannot be ionized to an equal extent.MS is more sensitive for metabolite detection and requires more tissue destruction and there is difficulty in quantification while NMR spectroscopy is less sensitive for metabolite detection, having easy quantification, is nondestructive and requires little sample handling and preparation. Although other techniques are also available like ion –mobility spectrometry, electrochemical detection (coupled to HPLC), radiolabelling techniques (when combined with thin layer chromatography), MRSI (Magnetic resonance spectroscopic imaging) and PET scan. Data Analysis is done using multivariate analysis, like Partial least square method (PLS), Principle component Analysis or orthogonal PLS (OPLS). In final step validation is done followed by clinical application.

Applications:

Metabolomics is used in variety of health applications including pharmacology and preclinical drug trials, transplant monitoring, oncology, toxicology, new-born screening, clinical biochemistry and as a tool for functional genomics. In poultry there is effect of breed and feed on egg composition which can be well judged using albumin metabolites ( erythritol, threitol, ribitol, linoleic acid, isoleucine, dihydrouracil, 4- hydroxyphenyllactic acid, alanine, glycine, N-butyrylglycine, pyruvic acid, valine, sugar alcohols and yolk metabolites(erythritol, threitol,urea,sugar alcohol). Acyglycine is diagnostic marker of inborn errors of metabolism. In ruminants Non-esterified fatty acids, creatinine, albumin, BHBA, growth hormones, enzymes, cholesterol, urea, Inulin, triiodothyronine, lactose can be used as indicators.

Metabolomics as Part of Multi-omics Approaches

Because the small molecules detected via metabolomics studies can be impacted by the genome, transcriptome, proteome, and environment, metabolomics is the only true reflection of phenotype at any given point in time. Any changes in or interactions between gene expression, protein expression, and the environment can be directly observed through the metabolome, making metabolomics the most complex and informative of all the omics techniques.

While each omic approach on its own provides useful information, combining different approaches can reveal important biological and physiological signatures that can help elucidate the mechanisms behind specific disease etiologies, drug responses, and more.

Applications of Metabolomics

Metabolomics has several applications across the academic, clinical, and industrial sectors, from identifying disease biomarkers to accelerating pharmaceutical drug discovery to aiding farmers in selecting the most promising cultivars. Below we discuss some of the exciting capabilities realized through metabolomics in each of these sectors.

Academic Applications of Metabolomics

Academic research, which can also be called “bench science” or “basic science research” lays the foundational knowledge required to better understand biological systems and to make progress outside of the laboratory. The early stages of drug discovery, biomarker identification, microbial-host interactions in health and disease (for humans, plants, and animals), and more, typically start in the academic laboratory.

One of the hottest areas of research today is the human microbiome and its role in human health and disease. While the potential for leveraging the human microbiome to optimize human health has been recognized for several years, its therapeutic potential has been limited by knowledge gaps in precisely how the human microbiome interacts with its host. By adding metabolomics components to their (traditionally DNA-based) studies, researchers are beginning to understand how the microbiome impacts our immune systems,¹ digestive system,² metabolism,³ skin, and brain function. These studies are laying the foundation for pre-clinical and, eventually, clinical studies that will make testing and addressing the microbiome in the clinic as common as measuring blood pressure.

Histone modification⁴ is also a growing area of academic research, because it plays such an integral role in life—from basic biological processes like DNA repair to complex physiologies like disease etiology. Several metabolomics studies have reported crucial relationships between metabolites and histone modifications, and how fluctuations in these relationships can impact diseases such as cancer.⁵ Basic research like this that provides mechanistic understanding often drives drug discovery work, to eventually result in effective therapeutics used in clinical practice.

Clinical Applications of Metabolomics

Metabolic signatures have been used clinically to some capacity for over 30 years (and in some cases, even longer). For example: the heel-prick test done on babies after their birth detects certain inborn errors in metabolism; cholesterol tests measure lipid metabolites; and the Warburg effect⁶ is a well-documented metabolic hallmark of cancer. But these are all tests or signatures applied to a global population—there is nothing individualized about them. They are performed and read in the exact same way for everyone.

Metabolomics, by definition, is the high-throughput measurement of hundreds to thousands of metabolites at a time from multiple samples at once. This makes it the perfect tool for advancing precision medicine, which accounts for patient-specific variables that impact health and disease. Metabolomics provides a deep look into the metabolites present in any given sample at any given time, so it can be used to characterize metabolic anomalies associated with disease, discover and validate new therapeutic targets and biomarkers, and dictate personalized therapeutic approaches for patients based on their own unique metabolic profiles.

Metabolomics can also help direct dosing strategies for clinical trials and help troubleshoot failed interventions. In one case study, investigators managed to turn a failed phase II trial into a successful phase III trial using metabolomics to re-design the trial parameters. In another case study, metabolomics was integrated into a clinical trial design so investigators could gain insight on a precision treatment for pulmonary arterial hypertension. These are just two examples of many.

Commercial Applications of Metabolomics

There are several commercial applications for metabolomics, including pharmaceutical, cosmetic, nutritional science, and agriculture. Pharmaceuticals are a significant opportunity area for metabolomics, which can provide early information on drug toxicity⁷ and pharmacokinetics to increase the chances of a drug proceeding through clinical trials and gaining regulatory approval through the FDA and EMA. Cosmetics also stand to benefit from metabolomics studies, which are being used to drive research into personalized skin care and, similar to drug toxicity studies, can characterize cosmetic product toxicity and activity⁸—a particularly important factor for companies that do not engage in animal testing.

Metabolomics can also be used to assist food breeders in optimizing for traits such as flavor⁹ and selecting the cultivars most likely to succeed commercially—as well as prevent large-scale losses by detecting food adulteration and contamination.¹⁰ Other research groups and companies are leveraging microbial metabolomics to improve fertilizers, while still more are using metabolomics to better understand nutrition and address malnutrition on a global scale. It is also being used to drive innovation in animal nutrition, which can impact not only the health of our beloved pets but also the health of animals intended for food (and, therefore, our own health).

Problems and challenges:

1. Metabolites have variations in molecular weight and concentration

2.Metabolites are more dynamic and so make metabolome more time sensitive.

3. Loss of metabolites like glutathione may take place during tissue extraction.

4. All metabolites cannot be detected. It is concluded that Metabolomics will be solution for animal problems in future.

Compiled  & Shared by- Team, LITD (Livestock Institute of Training & Development)

 Image-Courtesy-Google

 Reference-On Request.

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