WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Rao et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 7.632

Volume 9, Issue 4, 868-886 Case Study ISSN 2278 – 4357

METABOLOMICS: THE STUDY OF METABOLITES

Nutan Rao*, Akshata Desai, Shraddha Gaur and Madhusudan Bhoir

Department of Quality Assurance, Oriental College of Pharmacy, Sanpada (E), Navi

Mumbai-400705, India.

ABSTRACT
Article Received on
31 Jan. 2020, Metabolomics is the newest ―omics‖ approach for analysis of
Revised on 20 Feb. 2020, metabolites. Metabolomics measure the metabolites which are present
Accepted on 11 March 2020
DOI: 10.20959/wjpps20204-15875 in the biological samples to obtain ‗metabolomes‘. One of the most
popular aims of metabolomic study is biomarker discovery. This
biomarker helps us in identifying the disease state. Various analytical
*Corresponding Author
techniques are used for metabolite measurements. Most commonly
Nutan Rao
Department of Quality used technique is mass spectrometry due to its high specificity and
Assurance, Oriental College excellent limit of detection. Metabolomics has several applications.
of Pharmacy, Sanpada (E), Typical data handling practices, data outputs and instrumentation for
Navi Mumbai-400705,
these experiments are introduced in this article. Description of
India.
pharmacogenomics and pharmacoproteomics is also provided. A case
study involving the use of metabolomics approach is also described in this article.

KEYWORDS: Metabolomics, Metabolite, Metabolomes, Omics System.

INTRODUCTION
Current clinical practice depends upon on the prognosis, diagnosis, and treatment of diseases
using methods determined and averaged for the specific diseased cohort/population. The
control and eradication of a number of diseases are subsequently hampered in many
individuals due to misdiagnosis, treatment failure, relapse, and adverse drug reactions. These
occurrences can be explained by individual variation in the genome, transcriptome, proteome,
and metabolome of a patient. With the intention of developing personalized approaches to
diagnose diseases and for treatment, various ―omics‖ approaches have analysed the influence
of these factors on a molecular level. Metabolomics is the newest addition to the ―omics‖
system which represents the complete set of metabolites in a biological cell, tissue, organ or
organism, which are the end products of cellular processes. The metabolites profile of two or

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more disease phenotypes are compared which helps to identify biomarkers. These biomarkers
then are used to develop personalized medicines. Biomarkers are also used for diagnostic and
treatment of diseases. These biomarkers serves as a functional end-point of physiological and
pathophysiological processes illustrating both environmental influences like nutrition,
exercise or medication and genetic predisposition.[1,2]

The systematic identification and quantification of the small molecule, metabolic products
(the metabolome) of a biological system (cell, tissue, organ, biological fluid, or organism) at
a specific point in time is known as metabolomics.[3,4]

The quantitative measurement of the dynamic multiparametric metabolic response of living

systems to genetic modification or pathophysiological stimuli is known as metabonomics.

The difference between the two terms i.e. between metabolomics and metabonomics is that
metabolomics is more associated with mass spectrometry-based techniques and
metabonomics is associated with NMR spectroscopy, this is simply because of usages
amongst different groups that have popularized the different terms.

Although in different fields metabolomics study has achieved great successes, measuring
thousand of biological samples in one study is still a challenge to ensure high quality of data
due to analytical variances, environmental fluctuation and instrument drift.[5,6] When large-
scale metabolomics is performed following issues should be covered.[7] Fig.1
1. To increase throughput sample pretreatment method should be highly efficient.
2. For ensuring rich metabolic profiling information and high analytical throughput, data
collection method is critical.
3. Among different batches and different instruments data repeatability and integration are
the key factors in large-scale metabolomics study.

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Fig.1: large scale metabolomics study.

There are three types of experiments mainly Targeted metabolomics, untargeted

metabolomics and Semi-targeted metabolomics.

Targeted metabolomics experiment is to quantify specific metabolites within a sample. Here

they target a limited number of analytes. For example: to observe what happens to the
concentration of a drug in a patient‘s body over time, therefore detection of only that drug is
required.[8-14]

Untargeted metabolomics experiments are to measure as many metabolites as possible

within a sample. Samples are most frequently analysed using LC–MS (although GC–MS can
be used) with a high-resolution mass analyser scanning a wide mass range, e.g., 50–2000
Threshold. Resulted components are then compared with other sample sets to find out
differences and biologically-relevant information.[8,9]

Semi-targeted metabolomics experiments are for examination of specific portion of the

metabolome or a subclass of metabolites, such as only the organic acids or lipids.[10,15,16]
Four main points in Analysis of metabolomics data are:
 Efficient and unbiased
 Separation of analytes
 Detection
 Identification and quantification

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Fig. 2: Difference between metabolomics, proteomics, genomics.

PHARMACOGENOMICS
Pharmacogenomics is a platform that combines pharmacology and genomics. Genetic
polymorphisms determine inter-individual fluctuation in the pharmacodynamics and
pharmacokinetics of pharmacological agents is the main concept of this approach.[17] On the
ability of an organism to respond to a specific medication pharmacogenomics identifies the
effects of individual genetic variation. Pharmacogenomics promotes the comparison of
human genomes and predicts diseases. According to individual genetic makeup it helps to
identify personalized pharmaceutical therapy.[17,18]

PHARMACOPROTEOMICS
Pharmacoproteomics basic principle is to identify and quantify the protein content in
biosamples under different pathophysiological condition and to detect the changes that occurs
in protein before and after therapy.[18]

PHARMACOMETABOLOMICS: THE METABOLOMIC APPROACH TO

PRACTICAL MEDICINE
Pharmacometabolomics studies increase the ability to predict individual variation in drug-
response phenotypes and elucidate drug effects.[1] Depending on a pre-dose mathematical
model of an individual‘s metabolic status the field of pharmacometabolomics attempts to
quantify drug interventions using complex technologies. Finding a connection between the
metabolite profile (pre-dose) and the metabolic “fate‖ of a drug (post-dose) is the basic

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principle of pharmacometabolomics. Various pharmacometabolomics studies rely on

prediction of individual drug metabolism by measuring the Cytochrome P450 activity of
drug-metabolizing enzymes.[19-22] For metabolism of many drugs Cytochrome P450 enzyme
is responsible and these enzymes have important effects on drug pharmacokinetics, efficacy,
and safety.[18]

Fig. 3: Schematic of the workflow of a metabolomics study.

SAMPLE COLLECTION, TREATMENT AND PROCESSING

Metabolomics assessment can be pursued both in vitro and in vivo using cells, fluids or
tissues.All biological samples collected for metabolic analysis require careful sample
handling, special requirements for diet, physical activities and other patient validation.Due to
high susceptibility of metabolic pathway to exogenous environment, maintaining low
temperature and consistent sample extraction is essential.Standard sample volume for
biofuids is 0.1 to 0.5 ml.

Minimal sample preparation is required for NMR (including direct analysis of intact tissue
specimen).[23] Biofluids easiest to work with are serum, plasma, urine, saliva etc.Maximum
experience with serum and urine samples. Collection of plasma and serum requires treatment
in the blood samples.For serum the whole blood is collected in test tube, allowed the blood to

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clot by leaving it undisturbed at room temperature and the clot is removed by centrifuging.
The resultant supernatant is designated serum.For plasma the whole blood is collected in the
anticoagulant treated tubes, this stimulates white blood cells to release cytokines;cells are
removed from plasma by using refrigerated centrifuge. The resulting supernatant is
designated plasma.[2]

SEPRATION TECHNIQUES
Chromatography: A process in which a chemical mixture carried by a liquid or gas is
separated into components as a result of differential distribution of the solutes as they flow
over or around a stationary liquid or solid phase is known as chromatography.[24]

Different types of chromatographic techniques can be used depending on type of sample.

Gas chromatography (GC)

Gas Chromatography involves separation of heat stable and volatile substances. A Gas
Chromatography is used to detect the components based on selective affinity of components
towards the adsorbent materials. With the help of GC syringe the sample is introduced in the
liquid/gas form into the injection port, it gets vaporized at injection port then with the help of
continuously flowing mobile phase(carrier stream) passes through column, mainly H2 and
then gets separated/detected at the detection port with suitable temperature programming. It
is visualized on computer in the form of peaks. Carrier medium can be liquid (e.g. HPLC) or
gas (e.g. GC) for the ease of separation/detection; if carrier medium is gas then it is called
Gas Chromatography.[25] At different rates different chemical constituents of the sample travel
through the column depending upon following factors.
1.Chemical properties
2. Physical properties and
3. Interaction with stationary phase (a specific column filling)[26]

The chemicals which exits the end of the column, they are electronically detected and
identified. The stationary phase in the column separate different components, causing each
one to exit the column at a different time (retention time). Fig.4

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Fig. 4: Gas chromatography.

Liquid chromatography (LC)

Liquid chromatography involves separation of involatile analytes for example amino acids,
sugars, lipids, etc.The device is usually a column packed with the porous medium made of a
granular solid material (i.e., stationary phase), such as polymers and silica, the sample is
injected and the solvent (i.e., mobile phase) passes to carry the sample. When a sample is
injected, it gets adsorbed on to the stationary phase, and when the solvent passes through the
column, it separates the compounds one by one, based on their relative affinity towards the
stationary phase and mobile phase. The component with the most affinity towards the
solvent(i.e., mobile phase) is the first to separate. The component which has less affinity
towards solvent (i.e. mobile phase) travels slower. Higher the affinity, faster is the retention
time.[27] Fig.5

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Fig.5: Liquid chromatography.

Capillary Electrophoresis (CE)

Capillary tube is placed between two buffer reservoirs, and an electric field is applied,
separation depends on electrophoretic mobility & charge. Defined volume of analyte is
introduced into the capillary by replacing one buffer reservoir with sample vial.
Electrophoretic separation is measured by detector. The tube in Capillary Electrophoresis is
typically made up of silica, which may be coated or uncoated.[28] Fig.6

Fig. 6: Capillary electrophoresis (CE).

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HYPHENATED TECHNIQUES
Various Mass spectrometry (MS) methods in combination with separation techniques such as
liquid chromatography (LC) and gas chromatography (GC) have been used in multiple
metabolomics investigations.[29-34]

Liquid Chromatography - Mass Spectrometry (LC-MS)

The LC-MS technology involves use of an HPLC and Mass Spectrometer, wherein the
individual components in a mixture are first separated in HPLC followed by ionization and
ions are further separated on the basis of their mass/charge ratio. The separated ions are then
detected by directing it towards a photo or electron multiplier tube detector, which identifies
and quantifies each ion. In any MS analysis, ion source is an important component, as this
basically aids in efficient generation of ions for analysis. For ionization of intact molecule,
the ion source could be APCI (Atmospheric Pressure Chemical Ionization), ESI (Electron
spray Ionization), APPI (Atmospheric Pressure Photo Ionization) etc. Ion sourceselection also
depends on the chemical nature of the analyte of interest i.e. polar or non-polar.[35-36] Fig.7

Fig.7: Liquid chromatography - mass spectrometry (LC-MS)

Advantages of LC-MS: Wide metabolite coverage offered, Compatibility with the analysis
of Biosamples, high sensitivity and specificity of the analytical mode, ready availability of
instrumentation.[37-41]

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Gas Chromatography-Mass Spectrometry (GC-MS)

The working principle of GC is that a mixture will separate into individual substances when
heated. The heated gases are then carried through a column with an inert gas (such as
helium). As the separated substance comes out from the column opening, they flow into the
MS. By the mass of the analytemolecule,Mass spectrometry identifies compounds. Typically
every second or so, throughout the whole run timeMass spectra are recorded.[35] Fig.8.

Fig. 8: Gas Chromatography-mass spectrometry (GC-MS).

Advantages of GC-MS: Highest available chromatographic resolution, Availability of large

mass spectral libraries (which include retention indices), Availability of robust
instrumentation and expert analysts.[18,42]

DETECTION TECHNIQUES
For biofluid metabolic profiling mass spectrometry and nuclear magnetic resonance are the
most frequently used methods.[1,43,44]

Mass Spectrometry (MS)

In Mass Spectrometry technique, molecules are bombarded with a beam of energetic
electrons. The molecules are ionized and broken up into many small fragments, some of
which are positive ions. Each type of ion has a particular ratio of mass to charge, i.e. m/e ratio

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(value). For most ions, the charge is one and thus,mass to charge ratio is simply the molecular
mass of the ion. The ion then passes through magnetic and electric fields to reach detector
where they are detected and signals are recorded to give mass spectra.[28]

Nuclear Magnetic Resonance Spectrometry (NMR)

In NMR the sample is dissolved in a solvent, usually CDCl3 (deuteron-chloroform), and
placed in a magnetic field. A radiofrequency (RF) generator then irradiates the sample with a
short pulse of radiation, causing resonance. When the nuclei fall back from higher energy
state to their lower energy state, the detector measures the energy released and a spectrum is
recorded. Protons in different environments absorb at moderately different frequencies, so
they are distinguishable by NMR. The frequency at which a specific proton absorbs is
determined by its electronic environment. The size of the magnetic field generated by the
electrons around a proton decides where it absorbs. In modern NMR spectrometers a constant
magnetic field strength B0 is used, and then to attain the resonance of all protons a narrow
range of frequencies is applied. Only nuclei that contain odd mass numbers (such as 1H, 13C,
19
F and 31P) or odd atomic numbers (such as 2H and 14N) gives NMR signals.[35]

In recent metabolomics applications various one-dimensional and two-dimensional NMR

method are used. Currently one-dimensional nuclear Overhauser enhancement spectroscopy
(NOESY) and one-dimensional selective total correlation spectroscopy (TOCSY) are most
commonly used in metabolomics studies. 1D NOESY is robust and gives a flatter baseline,
whereas 1D TOCSY is used to detect metabolites which are present even at a concentration
100 times smaller than those of the higher compound.[45,46] Most commonly used 2D NMR
methods are heteronuclear single quantum coherence (HSQC) spectroscopy, heteronuclear
multiple bond correlation (HMBC), correlation spectroscopy (COSY), 2D–J spectroscopy.
Because of its property to simplify the spectra, 2D J resolved (JRES) spectroscopy is
attractive for metabolomics studies.[47]

DATA ANALYSIS USING MULTIVARIATE ANALYSIS

The increasing complexity of the data obtained from the analytical equipment that are used in
metabolomics studies has led to the use of various multivariate data analysis methods for
biomarker identification from these data sets. There are two types of multivariate analysis
methods: ―unsupervised‖ and ―supervised‖ methods.[46]

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To get an overview of the data, certain unsupervised methods are used to highlight trends in
the data and grouping or differentiation of various sample sets, and additionally to identify
potential outlier samples and batch effects. When using these unsupervised methods, samples
are not assigned to specific groups (for example, disease and control) prior to the statistical
analysis, allowing the analyst to determine whether or not the samples are naturally
differentiated or grouped based on their analysed metabolite profiles.[45]

Principle component analysis (PCA) is the most commonly used unsupervised method. PCA
reduces the dimension of the input data matrix by calculating a sum of the compound
(metabolite) concentrations detected in each sample and in terms of principal components
(PCs) these is expressed. In this PC1 gives the most variation in the data and PC2 gives the
next highest variation, etc. On the basis of the analysed metabolomes these PCs subsequently
serve as coordinates on a scatter plot and provide an overview of the samples and how they
relate to each other.[48] Fig.9.

To identify potential biomarker candidates, supervised methods are very useful because they
helps to detect elusive differences between similar samples. To force classification,
supervised methods can be more appropriate. For recognizing supervised pattern the methods
which are used are partial least squares discriminant analysis (PLS-DA),[49] soft independent
modeling of class analogies (SIMCA), orthogonal signal correction (OSC), genetic
programming and neural networks. To extend the power of supervised methods into
metabolomics studies, OSC and PLS-DA have recently been combined.[50]

Fig. 9: Principle Component Analysis.

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APPLICATIONS
Agricultural
The development of new pesticides is critical to reach the growing demands on farming. By
allowing us to get a glimpse of genetically modified plants complex biochemistry via
informative snapshots acquired at different time points during plant development
metabolomics helps us to estimate associated risks. Plant metabolomics is
markedlyinteresting because of the range and functions of primary and secondary metabolites
in plants. About 300 definite metabolites could be routinely identified per sample a decade
ago, and the number is gradually increasing over time.[51]

Biomarker discovery
Biomarker discovery is another area where metabolomics involves decision making. Signals
of the medical state observed outside the patient which can be measured precisely and
reproducibly is biomarker. In metabolomics, biomarkers are small molecules that can be used
to differentiate two groups of samples, typically a disease and control group. For example, a
metabolite present in disease samples, but not in healthy individuals would be classed as a
biomarker. Samples collected from urine, saliva, bile, or seminal fluid contain highly
informative metabolites, therefore for the purpose of biomarker discoveryit can be promptly
analysed through metabolomics fingerprinting or profiling.[51-55]

Personalized medicine
Personalized medicine, the ultimate customization of healthcare, requires metabolomics for
rapid medical diagnosis to identify disease. In healthcare, we currently use classical
biochemical tests to estimate individual metabolite concentrations to identify disease states
(e.g. the blood-glucose level in the case of diabetes).[51] Metabolomics offers the power for
the rapid identification of hundreds of metabolites, enabling us to identify these disease states
much earlier.[48,56] By analysing the metabolite profile of a patient prior to dosing one can
predict some aspects of drug metabolism.[57]

CASE STUDY
Hypertension[58,59]
One of the most common chronic diseases, which affect about one billion people worldwide,
is hypertension.[60] Although there are various types of antihypertensive drugs available in
market, but only about 40% of hypertensive patients have optimally controlled blood
pressure.[61] Between individuals and even in races the efficacy of antihypertensive drugs

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varies greatly. However, little information about the mechanisms underlying the different
responses of individuals to antihypertensive drugs is known. Currently, the emerging
metabolomicstudy is helping to reveal the reasons for the variations in drug efficacy. This
metabolomic study is carried out by using GC-MS method. A metabolomic assessment of the
individual variations to antihypertensive drugs, i.e. for atenolol and hydrochlorothiazide, was
performed with non-targeted metabolomics approach in white and black subjects.[62]

Atenolol belongs to the beta-receptor blocker family and is a classical antihypertensive drug
and Hydrochlorothiazide is a thiazide diuretic.[63] Plasma samples at baseline and post-
treatment of atenolol, and hydrochlorothiazide therapy were collected. As expected, the
majority of the hypertensive subjects experienced an obvious reduction in blood pressure;
there were significant differences in blood pressure reduction between white and black
subjects with both treatments. The metabolomic study revealed that the baseline levels of 5-
methoxytryptamine were negatively correlated with the change in blood pressure of white
participants with atenolol treatment, while seven metabolites at baseline were associated with
that in black participants. The baseline levels of arachidonic acid and other unknown
metabolites were positively correlated with the change in blood pressure of white participants
with hydrochlorothiazide treatment. Multivariable models were constructed to predict the
antihypertensive response for all participants treated with either atenolol or
hydrochlorothiazide on the basis of baseline metabolomic profiles.

From both the discovery and validation datasets, statistically significant results were obtained
in the multivariable models.With increase in number of subjects it was observed that there is
increase in predictive power in models.The subsequent analysis revealed that there are
numbers of metabolic pathways that were altered jointly or individually in both white and
black participants after antihypertensive drug therapy implying mechanisms causing the
different outcomes of drug therapy.

CONCLUSION
The knowledge obtained from metabolomics studies are used for development of
personalized medicines. Metabolomics provides the information of pathogenesis of diseases,
effects of diet or applied drugs. One of the most popular aim of metabolomic study is
biomarker discovery. By combination of biochemistry, analytical chemistry, bioinformatics,
medicine etc, metabolomics represents a rapidly expanding and interdisciplinary field of

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science. This metabolomics study provides fresh insight into pathogenesis of diseases, effects
of diet or applied drugs.

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