Metabolomics, Lipidomics and Pharmacometabolomics of Human Hypertension

AnthonyAu1, Kian-Kai Cheng1,2 & Loo Keat Wei3,4

Institute of Bioproduct Development and Department of Bioprocess Engineering,

Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81300 Johor,

Malaysia.
Innovation Centre in Agritechnology, Universiti Teknologi Malaysia, 81300 Johor,

Malaysia.
Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Bandar Barat,

31900 Kampar, Perak, Malaysia.

Department of Biological Science, Faculty of Science, Universiti Tunku Abdul

Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia.

Corresponding author: Anthony Au (auzlanthony@gmail.com)

Abstract

Hypertension is a common but complex human disease, which can lead to a heart
attack, stroke, kidney disease or other complications. Since the pathogenesis of
hypertension is heterogeneous and multifactorial, it is crucial to establish a
comprehensive metabolomic approach to elucidate the molecular mechanism of
hypertension. Although there have been limited metabolomic, lipidomic and
pharmacometabolomic studies investigating this disease to date, metabolomic studies
on hypertension have provided greater insights into the identification of
disease-specific biomarkers, predicting treatment outcome and monitor drug safety
and efficacy. Therefore, we discuss recent updates on the applications of
metabolomics technology in human hypertension with a focus on metabolic
biomarker discovery.

Keywords: Metabolomics, Lipidomics, Pharmacometabolomics, Hypertension

Introduction

Hypertension is a symptomless disease which can lead to serious complications,

including metabolic syndrome, blurred vision, memory loss, kidney failure, stroke and
damage to the heart and coronary arteries (Messerli et al. 2007). Hypertension can be
classified into primary (essential or idiopathic), secondary (non essential), and to a
lesser extent, isolated systolic hypertension, malignant hypertension, white coat
hypertension, and resistant hypertension. Around 90-95% of hypertensive patients are
primary hypertension, as a result of interaction between genetic and lifestyle factors;
whereas the remaining 5-10% are categorized as secondary hypertension, due to renal
diseases, endocrine disorders, or side effect of medications (Poulter et al. 2015).

Diagnosis of hypertension is based on sphygmomanometer to measure the

pressures exerted during the heart beats (systolic) and resting between beats
(diastolic). Normal blood pressure reading is under 120/80 mm Hg (systolic over
diastolic), while 140/90 mm Hg and above are considered high blood pressure.
Hypertension can be managed with several anti-hypertensive drugs, such as
beta-blockers (e.g. atenolol), thiazide diuretics (e.g. hydrochlorothiazide), angiotensin
converting enzyme inhibitors (e.g. captopril), calcium channel blockers (e.g.
amlodipine) and alpha-1 antagonist (e.g. terazosin) (Chobanian et al. 2003). Other
treatments may include lipid-lowering drugs (e.g. statins and fibrates) that can prevent
dyslipidemia, diabetes mellitus, cardiovascular and cerebrovascular diseases in

hypertensive patients (Jacobson and Zimmerman 2006).

Since pathogenesis and pathophysiology of hypertension may changes during the

course of hypertension, it is important to establish a new omics approach for early
prediction and elucidate the molecular mechanism of hypertension (Thongboonkerd
2005; Nikolic et al. 2014). Consequently, this would be beneficial for the prevention
and management of hypertension as well as promote the development of new
therapeutic strategies and improved diagnostic methods to meet future demands.

Metabolomics, Lipidomics and Pharmacometabolomics

Omics approaches, including genomics (DNA), transcriptomics (RNA),

proteomics (proteins), and metabolomics (metabolites), play a pivotal role in our
understanding of human biology and diseases such as hypertension (Figure 1).
Metabolomics is one of the omics approaches that study the global metabolic profile
in biological samples (e.g. cells, tissues and body fluids), by using analytical
techniques with the combination of cheminformatics, bioinformatics and statistics
analyses (Madsen et al. 2010). Lipidomics is a subfield of metabolomics, which
focuses on the identification and quantitation of the complete set of lipids on a
metabolic

basis

and

further

associated

with

diseases

or

treatments.

Pharmacometabolomics is an emerging field that positively contributes to

personalized medicine, involves the identification of metabolic patterns that can

predict the response of drug treatment in human diseases (German et al. 2007).
Pharmacometabolomics also helps to investigate the effects of drugs on metabolism
and identify the metabolic pathways that correspond to drug-response phenotype and
metabolic adverse effects (Kaddurah-Daouk et al. 2014).

Human metabolome consisted of approximately 4229 endogenous metabolites in

serum sample (Psychogios et al. 2011). Metabolites represent the intermediate and
end products of metabolic reactions and cellular processes, and may have diverse
functions in different organisms. Metabolites usually have low molecular weight of
less than 1500 Dalton, and biological molecules including carbohydrates, fatty acids,
and amino acids can be considered as metabolites. Metabolites also vary depending
on their polarity, solubility and volatility. For instance, amino acids are hydrophilic
polar metabolites, while lipids are hydrophobic non-polar metabolites.

Targeted and Untargeted Metabolomics

An appropriate experimental design is required to ensure that the metabolomic

data is able to answer the questions of interest. Two distinct metabolomic strategies,
namely targeted and untargeted approaches have been applied to the analysis of
metabolites. Targeted metabolomic approach is on the basis of hypothesis-driven

manner and also used for biomarkers validation (Griffiths et al. 2010; Wei et al. 2010).
Targeted metabolomics is the measurement of a select group of metabolites, typically
focusing on one or more related pathways of interest (Dudley et al. 2010). It is
normally performed using mass spectrometry (MS) based approach. By taking this
approach, a novel association between metabolites and diseases may be revealed and
enables a more comprehensive understanding on metabolic functions and pathways
(Roberts et al. 2012).

In contrast to targeted metabolomics, untargeted approaches allow the unbiased

detection of full set of metabolites in the biological samples. This approach offers the
opportunity for the discovery of novel metabolites and metabolic pathways without
prior knowledge of the identified metabolites (Patti et al. 2012). Therefore, untargeted
metabolomics is classified as discovery-phase or hypothesis-generating research.
Untargeted metabolomics can be performed by nuclear magnetic resonance (NMR)
and MS approaches, in order to measure and quantify as many metabolites as possible
(Alonso et al. 2015). However, the challenging part of this approach is build upon the
complexity and pleiotropic nature of metabolomic data as well as the protocol and
time required to analyze the massive data sets (Patti et al. 2012; Roberts et al. 2012).

Sample Preparation

The initial step in a metabolic profiling is the extraction of metabolites from

biological samples. Metabolite extraction varies profoundly depending on study
objectives, sample availability (cell, tissue or body fluid) and analytical platform to be
used. During sample acquisition, several factors have been identified to interfere with
metabolomic outcomes. For instance, gender, age, diurnal variations, diet, exercise
and drug consumption may influence the metabolic profile of an individual (Yin et al.
2015). In addition, type of blood-collection tubes (such as lithium heparin, potassium
EDTA and citrate), duration between sample collection and processing, transportation,
storage and repeating freeze-thaw cycles of samples may also affect the analytical
results (Yin and Xu 2014; Yin et al. 2015).

In the consideration of complex, heterogeneous and dynamic nature of

metabolome, metabolite extraction can be performed in order to separate and
maximize the total number of metabolites detected. The procedure of metabolite
extraction is specific to samples types and metabolites of interest (Patti, 2011).
Liquid-liquid extraction and solid phase extraction are the most commonly used
methods for metabolites extraction (Yin and Xu 2014). Metabolites should be
quenched immediately to stop metabolism for example by perchloric acid treatment or
freezing at -80C or liquid nitrogen prior to extraction. During extraction process,
different solvents such as methanol, isopropanol, chloroform, acetonitrile or their
combinations are used, thus allow the separation of polar and non-polar metabolites
and also protein depletion (Bruce et al. 2008; Sellick et al. 2011; Sapcariu et al. 2014).

Lipids are usually extracted with chloroform-methanol-aqueous mixtures (Jung et al.

2011). As a result, the upper aqueous soluble phase is consisted of polar metabolites,
while non-polar metabolites are remained in the lower organic phase. However, it has
been suggested that minimal sample preparation may lead to the production of
accurate, reliable, and unbiased data (Dunn et al. 2011).

Analytical Instrumentation

High-throughput analytical platforms allow the simultaneous separation and

comprehensive detection of metabolites present in biological samples, mainly based
on either NMR spectroscopy or MS technologies. These including, but are not limited
to 1H-NMR spectroscopy, 13C-NMR spectroscopy, 15N-NMR spectroscopy, 31P-NMR
spectroscopy,

gas

chromatography-MS

(GC-MS),

gas

chromatography-flame

ionization detection (GC-FID), capillary electrophoresis-MS (CE-MS), direct

infusion-MS (DI-MS) and liquid chromatography-MS (LC-MS). High-resolution
magic-angle spinning NMR spectroscopy can be used for metabolic profiling of intact
tissues without any pretreatment of samples (Beckonert et al. 2010).

Nuclear Magnetic Resonance Based Method

NMR spectroscopy is a rapid and effective technique for untargeted

metabolomics, as it can identify and quantify a wide range of metabolites in biological
samples. This technique is a non-destructive method and requires only minimal
sample preparation. 1H-NMR spectroscopy is the one of most common used methods
for metabolomic analysis, and to a lesser extent

13

C-,

N- and

15

31

P-NMR (Keun and

Athersuch, 2011). The principle of NMR involved the absorption of radiant energy by
atomic nuclei (1H) in the presence of a magnetic field (Lane, 2012). At the same field,
different atomic nuclei within a molecule will generate different resonance
frequencies. These signals will provide the chemical and structural information on the
detected molecule.

However, NMR has relatively low analytical sensitivity that detects only
high-abundance metabolites, thus leads to a requirement for greater initial sample
volume (Bothwell and Griffin, 2011). Moreover, complex datasets may result in peaks
overlapping or similar coupling constants in one-dimensional NMR. This can be
overcome by applying two- and three-dimensional NMR spectroscopy methods such
as J-resolved spectroscopy, spin-echo correlated spectroscopy, diffusion ordered
spectroscopy and ultrafast J-Resolved correlation spectroscopy (Ludwig and Viant
2009; Dunn et al. 2011; Larive et al. 2015). Furthermore, high-resolution magic angle
spinning (HRMAS) NMR spectroscopy can be performed on intact tissues without
sample preparation (Bothwell and Griffin, 2011; Larive et al. 2015). During the
process, sample is spun at magic angle (54o44) to the magnetic field with high speed

(2,000-6,000 Hz), in order to reduce or remove the chemical shift anisotropy and
dipole-dipole couplings (Salek et al. 2011).

Mass Spectrometry Based Method

MS coupled with liquid chromatography (LC) or gas chromatography (GC),

enables the ionization and subsequent separation of the ions and fragment ions
according to their mass-to-charge (m/z) ratio (Pitt, 2009). In LC-MS, several types of
atmospheric pressure ionization methods are used for the ionization of different
categories of metabolites. Of which, the most commonly used electrospray ionization
is used for the initial screening of unknown metabolites (Xiao et al. 2012).
Atmospheric-pressure chemical ionization and atmospheric-pressure photoionization
are suitable for the detection of non-polar metabolites and have been widely applied
in lipidomic studies (Xiao et al. 2012). There are different types of MS analyzers,
including ion trap, time-of-flight, orbitrap, and quadrupole MS (Fuhrer and Zamboni,
2015).

The high resolution of GC combined with the MS detection, and more recently
GCGC-MS, provides an excellent system for performing global metabolic profiling,
with the help of metabolome databases in the identification of unknown compounds
(Patti et al. 2012). However for GC-MS based method, sample derivatization is

required prior to data acquisition. For aqueous metabolites, the common practice is to
use a two-step derivatization processes (methoximation followed by silylation) prior
to GC-MS analysis to reduce polarity and increase thermal stability and volatility
(Cheng et al. 2014). The benefits of GC-MS based strategies may include very high
resolution, good sensitivity and robustness; these are further enhanced by a two
dimensional GCGC-MS. The metabolites elute from the first column are separated
based on volatility and followed by polarity in the second column (Lenz and Wilson
2007).

Compared to NMR and GC-MS, LC-MS based method is able to detect a larger
numbers (200-500) of metabolites, and therefore has been recommended for global
metabolite profiling (Patti et al. 2012). In many ways, LC-MS analysis can be
performed using reversed-phase gradient chromatography, electrospray ionization
probe and Z-spray ion source in order to obtain the most comprehensive metabolic
profile (Cheng et al. 2014). LC-MS may reduce the interference between analytes and
background by improving gaps (ion suppression) or peaks (ion enhancement) signals
(Lenz and Wilson 2007). However, it is suggested that identified metabolites have to
be confirmed and validated with authentic metabolic standards.

The sensitivity of MS is determined by metabolites pK, hydrophobicity and

ionization potential (Want et al. 2007). Therefore, quenching and metabolite
extraction method as well as sample handling and storage may affect the amount of

variability in the sample results. Another limitation of MS is the lower reproducibility

as compared with NMR method. Despite these limitations, MS platform still
outperforms NMR-based method for the detection of low-abundance and large
quantity of metabolites (Theodoridis et al. 2011). MS possesses a greater sensitivity
than NMR spectroscopy that capable to detect very low-abundance metabolites at
picogram level (Dunn et al. 2011). By showing their respective pros and cons, the
combination strategy of MS with NMR spectroscopy has proven to be effective and
becomes increasingly popular in metabolomic studies.

In addition, advancement of new technology has been made in utilizing

triple quadrupole (QqQ) MS for the quantitatively measurement of metabolites with
higher sensitivity and specificity (Patti et al. 2012). QqQ MS consists of precursor ion
scan, neutral loss scan, product ion scan, and selected reaction monitoring to filter and
detect the metabolites with more sensitive and specific to their molecular weight and
structure (Pitt, 2009). QqQ MS is coupled with other separation techniques (LC, GC,
and CE) and multiple reaction monitoring, in order to enhances its selectivity and
sensitivity. Hence, QqQ MS technology provides excellent depth and breadth of
metabolome coverage, and is well-suited for targeted metabolome analysis and
quantification of metabolites.

Data Acquisition and Analysis

Data acquisition and analysis are conducted to identify the particular metabolites
that could potentially serve as biomarkers for diseases. Putative identification of
metabolites is initially carried out by matching the actual m/z values and theoretical
m/z values in various databases such as Human Metabolome Database
(http://www.hmdb.ca/),
(http://www.lipidmaps.org/),

HumanCyc
MassBank

(http://humancyc.org/),

LipidMaps

(http://www.massbank.jp/?lang=en)

and

Metlin (https://metlin.scripps.edu/index.php) (Johnson et al. 2015). Metabolomic data

acquisition and processing can be performed by metabolomic softwares such as
AMDIS, CODA, HighChem MassFrontier, LECO ChromaTOF, MathDAMP,
MetAlign, MZMine, SpectralWorks AnalyzerPro, TargetSearch, WMSM and XCMS
(Shulaev 2006; Kind and Fiehn 2010; Patti et al. 2012).

Metabolomics data has been analyzed with a wide range of statistical analyses, in
order to reduce the number of variables and examine the difference between groups
(Bartel et al. 2013). These analyses can be classified into either univariate analysis of
t-test and ANOVA (analysis of variance) or multivariate analysis such as the widely
used PCA (principal components analysis), PLS (Partial least squares) regression, and
PLS-DA (PLS-discriminant analysis). In addition, metabolomics data have also been
analysed

using

MANOVA

(multivariate

analysis

of

variance),

ASCA

(ANOVA-simultaneous component analysis), OPLS (orthogonal-PLS), OPLS-DA

(OPLS-discriminant analysis), SIMCA (Soft independent modelling of class

analogies), HCA (hierarchical cluster analysis),

SOMs (self-organizing maps), SVM

(Support vector machines) and Random Forest (Sugimoto et al. 2012; Bartel et al.
2013). Differential metabolic profiles between two groups can be compared using
parametric Student t-tests and Wilcoxon rank sum nonparametric tests, while ANOVA
test is applied for multiple groups (Bartel et al. 2013). In contrast, multivariate
methods analyzed and compared between different metabolic features and interactions
between them (Bartel et al. 2013; Alonso et al. 2015). These multivariate analyses can
be classified into unsupervised (PCA, ASCA, HCA and SOMs) and supervised (PLS
regression, OPLS, SIMCA and SVM) methods (Sugimoto et al. 2012; Bartel et al.
2013). Unsupervised methods analyze data irrespective to the type of study samples;
whereas supervised methods categorize the study samples according to the
phenotypes prior to analysis and more appropriate for constructing risk prediction
models (Alonso et al. 2015).

Bioinformatic tools have contributed to metabolomics field by transforming the

metabolomic raw data obtained into biologically meaningful information (Johnson et
al. 2015). A number of bioinformatic databases are currently publicly available for
metabolomic

data

analysis

and

(http://www.genome.ad.jp/kegg/),
(http://medicago.vbi.vt.edu),
MetaCyc (http://metacyc.org/),

visualization

BioCyc

MapMan

tools,

including

(http://biocyc.org/),

KEGG
DOME

(http://gabi.rzpd.de/projects/MapMan/),

MetNet (http://metnet.vrac.iastate.edu/)

KaPPA-View (http://kpv.kazusa.or.jp/kappa-view/)

and

(Shulaev 2006; Johnson et al.

2015; Sas et al. 2015). Other bioinformatics tools for pathway mapping and network
visualization include Paintomics, VANTED (Visualization and Analysis of Networks
containing Experimental Data), and MetaboAnalyst. In order to build a network of
genes-metabolites pathways interaction, several network tool such as MetScape,
MBRole (Metabolites Biological Role), MSEA (Metabolite Set Enrichment Analysis),
MetaMapp, 3Omics, ProMeTra and mummichog attempted to extend this approach to
metabolite biomarkers discovery (Shulaev 2006; Johnson et al. 2015; Sas et al. 2015).

Metabolomic Biomarkers

As indicated in Table 1, a number of metabolites have been discovered in

metabolomics studies associated with incident hypertension (Holmes et al. 2008; Liu
et al. 2011; Zheng et al. 2013; Zhong et al. 2014; Nikolic et al. 2015; van Deventer et
al. 2015; Wang et al. 2015). These metabolites may serve as biomarkers for early
diagnosis, prevention and treatment of this disease.

Carbohydrate metabolism alterations were found to be associated with

hypertension, and patients with essential hypertension demonstrated impaired glucose
tolerance, insulin resistance and abnormal glucose metabolism (Garca-Puig et al.
2006). In conjunction with the elevated level of glucose, altered levels of other
monosaccharides, disaccharides and polysaccharides were observed in hypertensive

patients. Of which, the levels of lactate, galactose, glucosamine, glycerol,

4-hydroxyphenyllactate, 2-hydroxyvaleric acid, 2-ketoglutaric acid, oxalic acid,
sorbose, sucrose, sorbitol, inosose and myo-inositol were increased, whereas fructose,
cellobiose, methyluric acid, lactobionic acid, indole carboxylic acid, tricarballylic acid
formate and glucuronide were decreased (Liu et al. 2011; Zhong et al. 2014; van
Deventer et al. 2015; Wang et al. 2015). These results suggest that carbohydrate
metabolism dysregulation may play an important role in hypertension, through
sodium retention, renal tubular sodium reabsorption, sympathetic nervous system and
adverse effects of anti-hypertensive drugs (Gambardella et al. 1993; Sechi et al. 1997;
Savica et al. 2000).

Several lines of evidence also indicated that free fatty acids were significantly
associated with the development of hypertension (Fagot-Campagna et al. 1998; Wang
et al. 2008), possibly by regulating vascular tone and microvascular function,
inhibiting endothelium-dependent vasodilatation and increasing blood pressure (de
Jongh et al. 2004; Spijkers et al. 2011). Furthermore, increased levels of free fatty
acids may contributed to hypertension through the alteration of membrane
microviscosity and lipid metabolism, by modulating ion transport, pH regulation,
intracellular Ca2+ handling and signaling pathway (Zicha et al. 1999). Interestingly,
findings from metabolomic studies indicated that the levels of very low-density
lipoprotein, low-density lipoprotein, acetone, 1-stearoylglycerol, 1-palmitoylglycerol,
and free fatty acids such as oleic acid, nonanoic acid, ecosanoic acid, hexanoic acid,

and heptanoic acid were significantly higher among hypertensive patients compared
to normotensive controls (Liu et al. 2011; Zhong et al. 2014).

Another metabolic pathway that is repeatedly found to be perturbed in

hypertensive patients is amino acid metabolism. Amino acids, including alanine,
histidine,

isoleucine,

lysine,

homocysteine,

ornithine,

tyrosine,

p-hydroxyphenylalanine, methylhistidine, aspartic acid, glutamine, glutamic acid and

pyroglutamic acid were observed at higher concentrations in blood samples of
patients with hypertension (Liu et al., 2011; Zhong et al. 2014). However, isoleucine,
glycine, threonine, phynylalanine, methionine, ornithine, asparagine, glutamine,
citrulline,

lysine,

tyrosine,

tryptophan,

cystin,

hexenoylcarnitine

and

trimethyl-L-lysine were found to have lower concentrations in other metabolomic

studies (van Deventer et al. 2015; Wang et al. 2015). Alanine is found in dietary
sources of proteins, but is particularly concentrated in meats. Alanine was positively
associated with high blood pressure, which in agreement with several metabolomic
studies (Holmes et al. 2008; Liu et al. 2011; Zhong et al. 2014). In addition, other
amino acids, branched chain amino acids and sex steroid metabolites such as
5-androstan-3,17-diol sulfate, androsterone sulfate and epiandrosterone sulfate,
were independently associated with increased risk of hypertension (Zheng et al.
2013).

Urea, the main metabolite of protein metabolism, has become an independent

predictor of hypertension (De Meyer et al. 2008; Liu et al. 2011). Likewise, uric acid
and allantoin were observed at higher levels in hypertensive patients, in contrast to
dimethyluracil and methyluric acid (Liu et al. 2011; van Deventer et al. 2015; Wang et
al. 2015). The underlying mechanism could be explained by the regulation of urinary
sodium (Na) homeostasis via an effect of diuresis and natriuresis (Cirillo et al. 2002).
In hypertensive patients, urinary urea was inversely associated with blood pressure, as
the higher urea level may reduce glomerular filtration rate and impaired renal function
(Cirillo et al. 2002; Martin et al. 2005).

Researchers also highlighted changes in metabolites originated from gut

microorganism, indicating a possible link between gut microflora activities with
hypertension.

Notably,

microbial

metabolites

(such

as

formate,

hippurate,

4-hydroxyhippurate and lyxose) detectable blood and urine in humans were found
significantly changed and can be associated with to the development of hypertension
(Hao et al. 2016, Holmes et al. 2008; Zheng et al. 2013). Together, these finding
shows a promising future research direction for hypertension.

Lipidomic Biomarkers

The number of lipidomic studies related to hypertension is limited (Graessler et al.

2009; Hu et al. 2011; Kulkarni et al. 2013). As shown in Table 2, the identified lipid

metabolites were

ether

phosphatidylcholine,

ether

phosphatidylethanolamine,

lysophosphatidylcholine, phosphatidylcholine, sphingomyelin, cholesteryl ester,

triacylglycerol, monohexosylceramide, phosphatidylinositol and diacylglycerol,
which mainly involved in lipid signaling pathway. Dysregulation of this signaling
pathway may contribute to the pathogenesis of human diseases such as inflammation,
cancer and metabolic disease (Wymann and Schneiter 2008). For instance,
sphingomyelins consisted of phosphocholine or phosphoethanolamine as their
phospholipid polar head group and served as substrates for the hydrolysis of
ceramides through action of sphingomyelinases (Hannun and Obeid 2008).
Sphingomyelinases enzymes further break down sphingomyelin to generate ceramide
and phosphocholine, which may, in turn, accelerate the formation of diacylglycerol
from phosphocholine (Hannun and Obeid 2008). Therefore, diacylglycerol (final
product of lipid signaling pathway), together with the second messenger
inositol-1,4,5-triphosphate, play important roles in regulating protein kinase C activity
and calcium release (Hannun and Obeid 2008). Moreover, it has been reported that the
accumulation of triacylglycerol lipid species may induce lipotoxicity in hypertension
(Hu et al. 2010). Therefore, there is a strong biological plausibility for the role of lipid
signaling molecules in pathophysiology of hypertension (Kulkarni et al. 2013).

Pharmacometabolomic Biomarkers

Recently, the promise of pharmacometabolomics for hypertensive patients has

been delivered in several studies (Wikoff et al. 2013; Altmaier et al. 2014; Rotroff et
al. 2015). These studies investigated the effects of the five major classes of
anti-hypertensive drugs on human metabolism and identified the metabolic pathways
response to drug treatment. The potential pharmacometabolomic markers associated
with these outcomes were further highlighted in Table 3.

Beta blockers, or known as beta-adrenergic blocking agents, may lower the blood
pressure by blocking the effects of epinephrine (adrenaline) and norepinephrine
(noradrenaline) hormones on b-adrenergic receptors (Frishman 2003). Free fatty acids
such as oleic acid, linoleic acid, palmitoleic acid, palmitic acid, 3-hydroxybutanoic
acid, arachidonic acid, 3-hydroxybutyrate methyl-hexadecanoic acid, dihomolinoleate,
10-nonadecenoate, stearic acid, myristic acid and heptadecanoic acid were
significantly decreased in patients treated with beta-blockers. Meanwhile, elevated
levels of sugar alcohol (including threitol, arabitol, allo-inositol) and amino acids
(including pyroglutamine, homocitrulline, salicylate, hydroxyisovaleroyl-carnitine,
2-methylbutyroyl-carnitine) were observed. Beta-blockers (both propanolol and
atenolol) may attenuate lipolysis by lowering the plasma free fatty acid levels
(Deacon, 1978). Lipolysis is stimulated by catecholamine hormones, including
adrenaline and noradrenaline, and is regulated by the beta-adrenergic receptors (Millet
et al. 1998). Therefore, it seems that beta-blockers may cause metabolic changes in
fatty acid biosynthesis and glycerolipid metabolism pathways. Moreover, these

metabolic perturbations are greatly influenced by ethnicity (eg. Causcasians and

African Americans) (Wikoff et al. 2013; Rotroff et al. 2015).

Thiazide diuretic (hydrochlorothiazide) is used to treat high blood pressure, by

inhibiting sodium reabsorption within the kidneys distal tubule, which leads to
increased excretion of sodium and water in urine (Duarte and Cooper-DeHoff 2010).
Decreased levels of dipeptide and amino acids (such as phenylalanyl-phenylalanine,
threonine, glutamine, and serine) have been reported in diuretics-treated patients
(Altmaier et al. 2014; Rotroff et al. 2015). Interestingly, this drug may impaired the
glucose and lipid metabolism, resulted from the increased metabolite concentrations
of ribonic acid, 1-hexadecanol, erythritol, glycero-gulo-heptose, dihydroabietic acid,
2-ketoisocaproic acid, aconitic acid, behenic acid, glucose 1-phosphate and glycine
(Rotroff et al. 2015). Thiazide diuretic-induced hyperglycemia has been reported as
one of the side-effects of this drug and associated with the increased risk of type 2
diabetes mellitus (Ellison and Loffing 2009). Thiazide diuretic-based treatments also
induced

the

formation

of

tryptophan

related

metabolites,

including

C-glycosyltryptophan, pseudouridine and kynurenine (Altmaier et al. 2014; Rotroff et

al. 2015). The role of tryptophan in hypertension is further supported by several in
vivo and human studies, in which oral L-tryptophan administration successfully
improve blood pressure, glucose metabolism and kidney function (Feltkamp et al.
1984; Wolf and Kuhn 1984; Fregly et al. 1989; Yonemura et al. 2004; Ardiansyah et al.
2011; Niewczas et al. 2014). Furthermore, an increased of urate and uric acid

concentration are direct side-effect of thiazide diuretics, which may affect the risk of
gout among patients with hypertension (McAdams DeMarco et al. 2012).

Angiotensin converting enzyme (ACE) inhibitors reduce blood pressure by

blocking the conversion of angiotensin I to angiotensin II, a nature substance that
induces vasoconstriction. Thus, ACE inhibitors help to dilate the blood vessels and
consequently improve the blood circulation in heart (Sweitzer 2003). In hypertensive
patients treated with ACE inhibitors, lower levels of aspartyl-phenylalanine and
phenylalanylphenylalanine formed during ACE inhibition (Altmaier et al. 2014).
Aspartyl-phenylalanine is a metabolite of aspartame that inhibits ACE activity in
humans who consumed large amount of aspartame (Grobelny and Galardy 1985). On
the other hand, the level of des-Arg9-bradykinin was increased after taking ACE
inhibitors. Bradykinin and its active metabolite des-Arg9-bradykinin are both the
selective substrates for ACE (Skidgel and Erdos 1987; Cyr et al. 2001). Bradykinin
potentiation is a metabolic process mediated by ACE inhibitors, via the inhibition of
Bradykinin receptor B2 binding and prevents the degradation of des-Arg9-bradykinin
(Tom et al. 2002). Therefore, the anti-hypertensive mechanisms of ACE inhibitors
may act through the activation of bradykinin-releasing pathway, where the increase of
bradykinin activity can dilates blood vessels and further lowering blood pressure
(Sharma 2009).

Statins and fribates are both lipid-lowering drugs that can prevent the

complications of hypertension. Several metabolic perturbations have been observed in

hypertensive patients under these medications (Altmaier et al. 2014). Lipid levels
such as lathosterol, glycochenodeoxycholate, 7-alpha-hydroxy-3-oxo-4-cholestenoate
and 1-palmitoyl-glycero-phosphoinositol were significantly reduced in patients on
statins, which further confirmed the direct action of this drug (Altmaier et al. 2014).
For

fibrates

users,

several

intermediate

metabolites

of

fenofibrate

(2-hydroxyisobutyrate and 3-Dehydrocarnitine), amino acid derivative (indolelactate,

carnitine and pipecolate) as well as vitamins and cofactors (riboflavin, pantothenate
and uridine) were detected (Altmaier et al. 2014). Pyroglutamine level was decreased
after fibrates treatment, but the opposite effect was observed among patients treated
with beta-blockers (Altmaier et al. 2014). Pyroglutamine, a cyclic derivative of
glutamine, has been reported in association with heart failure incident (Zheng et al.
2013). Therefore, it is questionable whether the combination of beta-blockers and
fibrates intake could balance the metabolic effects of these anti-hypertensive drugs.

Conclusions

Metabolomics has been applied in hypertension studies to gain new insights into
pathophysiological processes underlying hypertension, discover metabolite markers
for early risk prediction and monitor the efficacy and side effects of drugs treatment.
A number of studies had highlighted perturbation in amino acids metabolism in

hypertensive patients, notably increased concentration of alanine in blood and urine,

as well as reduced concentrations of threonine and phenylalanine as risks for
hypertension. In addition, the potential role of gut microflora warrants further
investigation as microbial metabolites including lyxose, formate, hippurate and
4-hydrocyhippurate were found significantly changed in hypertensive patients. Taken
altogether, future metabolomic, lipidomic and pharmacometabolomic studies in
combination with other omics technologies may act as a powerful platform to
intensively study the molecular mechanisms of hypertension.

Acknowledgment

The current work is funded by Ministry of Education, Malaysia under Fundamental

Research Grant Scheme (Vot. No. R.J130000.7844.4F483).

Figure 1 Omics approaches

Figure 2 Metabolomics workflow

Table 1 Association of metabolic changes with incident hypertension

Metabolomic markers
Increased concentrations

Decreased concentrations

Technique Statistical
s
analysis
employed

Reference
s

Alanine

Formate, hippurate and

N-methylnicotinate

1H-NMR

Choline

Urea, -1 Acid glycoprotein

D-glucose, D-galactose, glucosamine, L-sorbose, sucrose,

D-sorbitol, inosose, myo-inositol, heptanoic acid,
1-stearoylglycerol, oleic acid, 1-palmitoylglycerol, nonanoic
acid, eicosanoic acid, hexanoic acid, pipecolic acid,
L-ornithine, L-lysine, pyroglutamic acid, L-histidine,
L-alanine, glutamine, L-isoleucine, -aminoadipic acid,
N-acetylglycine, L-tyrosine, homocysteine, L-aspartic acid,
glutamic acid, L-tryptophan, allantoin, 3-amino-2-piperidone,
urea and 2-ketoglutaric acid

D-Fructose, D-cellobiose, lactobionic

acid, glycerol 3-phosphate

GC-TOFMS

PCA, PLS Liu et al.

and OPLS 2011

4-Hydroxyhippurate, 5-androstan-3,17-diol disulfate,

androsterone sulfate and epiandrosterone sulfate

GC-MS
and

PCA

H-NMR

OPLS-DA Holmes et
al. 2008
OPLS and
PLS-DA

De Meyer
et al. 2008

Zheng et
al. 2013

LC-MS
Very low density lipoprotein, low density lipoprotein, lactic
acid, acetone and acetylformic acid

Valine, alanine, glucose, inose,

p-hydroxyphenylalanine and
methylhistidine

1H-NMR

PLS-DA, Zhong et
OPLS-DA al. 2014
and t-test

Oxalic acid, fumaric acid, glycerol, adenine, pyrophosphate

and uric acid

L-Valine, L-isoleucine, glycine,

L-threonine, L-methionine, ornithine,
L-asparagine, L-glutamine, citrulline,
L-lysine, L-tyrosine, L-tryptophan,
L-cystine and capric acid

GC-MS

PCA,
Mann-Wh
itney-Wil
coxon

Wang et
al. 2015

3-OH-Sebacic acid,2-OH-isovalerate, 4-OH-phenyl-lactate,

tricarballylic acid and lactic acid

Hesperetin, hexenoylcarnitine, fumaric

acid, methylguanosine,
N-acetylarylamine, kynurenic acid,
phenylglyoxylate, indole carboxylate
glucuronide, methyluric acid,
dimethyluracil and trimethyl-L-lysine

GC-MS
and
LC-MS

PCA

van
Deventer
et al. 2015

Talose, lyxose, glucose-1-phosphate, methylmalonic acid,

malonic acid, shikimic acid

Threonine, nicotinoyl glycine,

phenylalanine, aspartic acid, Gly-Pro,
galactose, thymol, noradrenaline,
methyl-beta-D-galactopyranoside,
2-methoxyestrone, alpha-tocopherol

GC-MS

t-test/
Wilcoxon
rank sum
test

Hao et al.
2016

1H-NMR

= Proton nuclear magnetic resonance

GC-TOF-MS = Gas chromatography time-of-flight mass spectrometry

GC-MS = Gas chromatography mass spectrometry
LC-MS = Liquid chromatography mass spectrometry
OPLS = Orthogonal partial least square
PLS-DA = Partial least square discriminant analysis
OPLS-DA = Orthogonal partial least square discriminant analysis
PCA = Principal component analysis
PLS = Partial least square

Table 2 Association of lipid metabolites with incident hypertension

Lipidomic markers

Statistical
analysis

Reference
s

Ether phosphatidylcholine(36:4, 36:5, 38:4, 38:5, 34:2, 36:3, 34:1, 40:5) and ether LC-ESIphosphatidylethanolamine(38:5, 38:6, 40:5)
MS/MS

SOLAR
software

Graessler
et al. 2009

Lysophosphatidylcholine(22:6), phosphatidylcholine(40:6), sphingomyelin(16:1, 24.2), LC-IT-TO

cholesteryl ester(20:4, 22:6) and triacylglycerol(48:0, 48:1, 48:2, 48:3, 50:0, 50:1, 50:2, 50:3, F-MS
50:4, 50:5, 52:1, 52:2, 52:3, 52:4, 52:5, 52:6, 54:2, 54:3, 54:4, 54:5, 54:6, 56:5, 56:6, 56:7,
56:8, 56:9)

PCA,
ANOVA

Hu et al.
20011

Monohexosylceramide(22:0, 24:0, 24:1), phosphatidylcholine(34:4, 34:1, 36:2, 40:6), LTQ

phosphatidylinositol(34:1, 36:2, 40:6) and diacylglycerol(16:0/18:0, 16:0/20:3, 16:0/22:5, Orbitrap
16:0/22:6, 16:1/18:1, 18:0/18:1, 18:0/24:0)
hybrid
MS

PCA,
MANCO
VA,
ANOVA

Kulkarni
et al. 2013

LC-ESI-MS/MS = Liquid chromatography-electrospray ionization-tandem mass spectrometry

LC-IT-TOF-MS = Liquid chromatography ion trap time-of-flight mass spectrometry
LTQ Orbitrap hybrid MS = LTQ Orbitrap hybrid mass spectrometry

Technique
s
employed

PCA = Principal component analysis

ANOVA = Analysis of variance
MANCOVA = Multivariate analysis of variance

Table 3 Association of metabolic changes with anti-hypertensive drugs

Anti-hyp
ertensive
drugs
Beta-bloc
kers

Pharmacometabolomic markers
Increased concentrations

Statistical
analysis

Reference
s

Alpha ketoglutaric acid, threitol, arabitol,

dihydroabietic acid,
conduritol-beta-epoxide and allo-inositol

Oleic acid, linoleic acid, palmitoleic acid,

palmitic acid, 3-hydroxy-butanoic acid,
arachidonic acid, methyl-hexadecanoic acid,
myristic acid, threonine, stearic acid,
glycerol-alpha-phosphate

GC-TOFMS

Wilcoxon
signed
rank

Wikoff et
al. 2013

Pyroglutamine, homocitrulline,

Serotonin, dihomolinoleate,
3-hydroxybutyrate, 10-nonadecenoate,
margarate, and eicosenoate

UHPLCMS-MS
and
GC-MS

Linear
regression
test

Altmaier
et al. 2014

6-deoxyglucitol, threitol, uracil, talose,

1-monoolein, hydroxycarbamate,
allo-inositol and 2,3,5-trihydroxypyrazine

Linoleic acid, oleic acid, palmitic acid,

palmitoleic acid, 3-hydroxy-butanoic acid,
arachidonic acid, methyl-hexadecanoic acid,
heptadecanoic acid and threonine

GC-TOFMS

Wilcoxon

Rotroff et
al. 2015

Pseudouridine, c-glycosyl-tryptophan,
glutarylcarnitine, homocitrulline and urate

Phenylalanyl-phenylalanine

UHPLCMS-MS
and

Linear
regression

salicylate, hydroxyisovaleroyl-carnitine and

2-methylbutyroyl-carnitine

Thiazide
diuretics

Decreased concentrations

Technique
s
employed

signed-ran
k test

Altmaier
et al. 2014

GC-MS

test

Uric acid, ribonic acid, 1-hexadecanol, Threonine, aminomalonic acid, glutamine, GC-TOFerythritol,
kynurenine, serine, phytol, phosphoethanolamine and MS
glycero-gulo-heptose,
dihydroabietic methoxytryosine
acid, 2-ketoisocaproic acid, aconitic acid,
behenic acid, glucose 1-phosphate and
glycine

Wilcoxon

ACE
inhibitors

Des-Arg9-bradykinin

Statins

1-Arachidonoyl-glycerophosphocholine,
7-alpha-hydroxy-3-oxo-4-cholestenoate,
1-arachidonoylglycerol-phosphoethanolami 1-palmitoyl-glycero-phosphoinositol,
ne, isobutyrylcarnitine,
lathosterol and glycochenodeoxycholate
1-docosahexaenoyl-glycerophosphocholine,
alpha-tocopherol and uridine

Linear
regression
test

Fibrates

2-Hydroxyisobutyrate, 3-dehydrocarnitine,
riboflavin, pantothenate, indolelactate,
carnitine, pipecolate and uridine

Phenylalanyl-phenylalanine and
aspartyl-phenylalanine

Pyroglutamine

UHPLCMS-MS
and
GC-MS

signed-ran
k test

Rotroff et
al. 2015

Altmaier
et al. 2014

GC-TOF-MS = Gas chromatography-time-of-flight mass spectrometry

UHPLC-MS-MS = Ultra-high performance liquid chromatography tandem mass spectrometry
GC-MS = Gas chromatography mass spectrometry

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