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ScienceDirect
Metabolome responses to physiological and nutritional
challenges
Francesc Puiggròs1,2, Núria Canela3,4 and Lluı́s Arola1,2,3
Nutrition-related pathologies need a broader nutritional view to
address the multitude of involved homeostatic perturbations.
Nutrition challenge tests combined with non-targeted
metabolomics, as a scientific methodology to obtain new
information on the dietary components, will accelerate the
discovery of new biomarkers, thus preventing diet-related
pathways in early disease stages or in healthy individuals.
Addresses
1
Centre Tecnològic de Nutrició i Salut (CTNS), TECNIO, Reus, Spain
2
Departament de Bioquı́mica i Biotecnologia, Nutrigenòmics Research
Group, Universitat Rovira i Virgili, Tarragona, Spain
3
Centre for Omic Sciences (COS), Universitat Rovira i Virgili (URV), Reus,
Spain
4
Group of Research on Omic Methodologies (GROM), URV-CTNS,
Reus, Spain
Corresponding author: Puiggròs, Francesc
(francesc.puiggros@ctns.cat)
Current Opinion in Food Science 2015, 4:111–115
This review comes from a themed issue on Foodomics technologies
Edited by Alejandro Cifuentes
For a complete overview see the Issue and the Editorial
Available online 14th June 2015
http://dx.doi.org/10.1016/j.cofs.2015.06.001
2214-7993/# 2015 Elsevier Ltd. All rights reserved.
Introduction
Nutrition research has evolved during recent years by
transitioning from the targeted nutritional biochemical
approaches to non-targeted metabolomic approaches
aimed at obtaining personalized nutritional responses
and needs [1]. The former approach is aimed at searching
the response of a subset of metabolites of a given popu-
lation, whereas the latter approach provides nutritional
data using biological models as multiple interacting func-
tional networks rather than unrealistic ‘reductionist’
cause–effect models [2].
Theoretically, metabolome profiling may allow a com-
plete description of the physiological and biochemical
response to a dietary intervention, thus making the suc-
cessful development of functional foods and nutraceuti-
cals easier. Metabolomics and complementary omics
integration should overcome the medical challenges of
the 21st century, such as controlling the worldwide spread
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of non-communicable disorders and ensuring healthy
ageing [3].
However, at present, the successful rate of launching new
functional foods to the market is still poor. The causes are
a reductionist approach that is still driving the knowledge
applied to the food industry added to due to the restric-
tive legal framework of each country. The required
rationale to substantiate the binomial food component
health benefit should evolve towards a holistic approach
and should overcome the limitations of considering the
traditional approach based on assessing the effect of one
component on a single physiological target [4].
Nutritional rationale for facing chronic
diseases
Traditionally, new functional food development has tar-
geted a single physiological process to modulate a marker
and/or even has combined several ingredients to obtain
synergistic, complementary or even additive effects for
more than one physiological target. Indeed, food compa-
nies often adopt an industrial strategy that uses specific
biological effects together to beneficially modulate or
reverse isolated homeostatic alterations belonging to a
single multifactorial situation [5].
This approach for the management of multifactorial pa-
thologies is incomplete, and there is an emerging need for a
broader nutritional view to address the multitude of ho-
meostatic perturbations involved in a chronicle disease or a
given health condition, such as menopause or ageing [6,7].
In addition to an unbalanced nutrition, the molecular
pathways causing most of the chronicle disease progres-
sion and final phenotypes, such as obesity, diabetes,
inflammatory bowel disease and some types of cancer,
are initiated by imbalances of major ‘overarching process-
es’ where nutrition has a function in maintaining homeo-
stasis [8]. These imbalanced processes are focused on (i)
metabolic stress, (ii) oxidative stress, (iii) inflammation
and (iv) psychological stress, which are closely related on
the cellular, organ and organism levels [8]. In addition, the
host–microbiome interactions would be considered as the
new process to be included in the overarching concept of
the organism as a whole [9].
Homeostasis challenge to amplify biological
responses
The ability to react appropriately to the abovementioned
stresses is critical in maintaining optimal health. In fact,
Current Opinion in Food Science 2015, 4:111–115
112 Foodomics technologies
human metabolome responses to nutritional and func-
tional stressors and/or challenges are an innovative way to
assess the biological concept of resilience, which links
health to the capacity of the body to address daily stress-
ors that challenge homeostasis [10]. Nutrition research is
evolving by developing biological models and methodol-
ogies used to assess resilience, which provides a meta-
bolomic profile that depicts the disturbance and
restoration of homeostasis, thereby providing a method
to measure health and wellbeing [11].
Nutrition induces a subtle effect that is barely detectable
using static homeostatic biomarkers, which may be of less
value than the same biomarkers measured in a condition
of homeostatic perturbation. Further, metabolomic anal-
ysis measured under stress may identify new key bio-
markers that are more adequate in describing healthy and
compromised conditions [8,12].
There are two types of challenges applied in nutrition
research as follows: (i) nutritional challenge (i.e., oral
glucose tolerance test or short-term intakes of high fat,
carbohydrates or antioxidants); and (ii) functional chal-
lenge testing the robustness and flexibility of functions
involved in maintaining homeostasis (e.g., hypoxia, en-
durance and learning tests) [11,13–15].
Nutrition studies performing the use of foodomics —
defined as a discipline that studies the food and nutrition
domains through the application of omics technologies
[16–18] — and challenge tests have only recently been
published and have been the BIOCLAIMS FP7 Europe-
an project pipeline.5 BIOCLAIMS approach has signifi-
cantly contributed to gain insight into the molecular
machinery behind the physiological observations related
to the control of homeostasis in a systems perspective
with the following components: (i) perturb homeostasis to
quantify the system robustness; (ii) measure all relevant
components that describe the system and discriminate
between individuals; and (iii) assess the nutritional inter-
vention able to reduce the magnitude of perturbation.
Innovation of ingredients
Because the postprandial response reveals multiple
aspects of metabolic health that would not be apparent
from studying the fasting (homeostatic) parameter, the
metabolome responses to nutrition challenges with/with-
out a bioactive compound allow the detection and quan-
tification of molecular changes related to metabolic
flexibility, robustness of homeostatic mechanisms and
adaptive responses, thus increasing the knowledge about
the molecular aspects of the postprandial response of a
given ingredient. As shown in Figure 1, this knowledge
provides an explanation for the biological efficacy of a
bioactive ingredient avoiding the onset and progression
5
http://www.bioclaims.eu.
Current Opinion in Food Science 2015, 4:111–115
due to its ability to maintain resilience [19]. Thus, the
diet-derived metabolome is highly complex and variable,
and it constitutes a unique and extremely rich source of
information on dietary experiments that show promise in
accelerating the discovery of new biomarkers and diet-
related pathways.
An example challenge protocol is the oral glucose toler-
ance test, which can uncover early alterations in metabo-
lism preceding chronic diseases, and another challenge
protocol is the fat overload protocol, which induces a pro-
inflammatory (IL6, IL18, factor VII and TNF-a) and
oxidative profile and reduces homeostatic vascular func-
tion [19,20]. Thus, ingredients that can control low-grade
inflammation and improve metabolic flexibility in elderly
people [7] or that can strengthen the metabolic flexibility
of insulin response are promising ingredients to prevent
both type 2 diabetes (T2D) and metabolic syndrome
[17,18].
For example, resveratrol and muscadine polyphenols
suppress the postprandial increase in oxidative and in-
flammatory stress markers (such as expression of NQO-1
and GST-P1 antioxidant genes) induced by fat and car-
bohydrate load, thus demonstrating the acute antioxidant
and anti-inflammatory effects of these ingredients in
humans in the postprandial state [21].
Innovation that profits foodomics
To analyze this more dynamical view of the human
metabolome in response to diet stimuli, an untargeted
methodology is the most suitable strategy. This method
compares global metabolomic profiles between groups
without prior knowledge of the biomarkers [22]. The
hypothesis-driven traditional approach allows the mea-
surement of known biomarkers, but it is restricted to
specific families of metabolites or a particular pathway
[21,23].
The main technologies used in metabolomics are nuclear
magnetic resonance (NMR) spectroscopy-based and mass
spectrometry (MS)-based techniques [24]. NMR is a
fast, reproducible, non-sample destructive technique that
requires minimal sample preparation and does not require
separation, but it has lower sensitivity than MS instru-
ments [25]. MS-based metabolomics has grown through
the rapid development of high resolution MS technolo-
gies coupled to chromatographic techniques, mainly gas
(GC) and liquid chromatography (LC) and Capillary
Electrophoresis (CE). MS is highly sensitive and allows
high-throughput and rapid determination of masses and
structure information [24,25,26]. However, MS is not as
reproducible as NMR as it requires pre-treatment and
chromatographic separation steps [27].
In biofluids or tissues, metabolomics identifies and quan-
tifies the final entities of the reaction chain along the
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Figure 1
Health biomarkers
(non-predictable to risk factor)
Healthy
homeostasis
Oc
ch cas Challenged
all ion
en a homeostasis
ge l
s
Diagram showing different metabolome theoretical profiles that mimic (a) integration of omic technologies (b) the sense to test nutrition responses
by challenging homeostasis to measure metabolic flexibility (as a spring).
paradigm of biology as follows: from genes to transcripts
to proteins to metabolic intermediates. By contrast to the
other profiling technologies, such as transcriptomics or
proteomics, metabolomics data can be better interpreted
on the basis of biochemical knowledge gained throughout
almost a century [28].
Today, foodomics is used not only to ensure the presence
of nutrient markers in human samples with cutting edge
analytical methods but also to improve the assessment of
the biological effects of bioactive compounds in humans
using properly developed nutrition trials. This issue has
been and remains of key importance for research institu-
tions, food industries, agencies and regulatory laboratories
[29].
In fact, foodomics is intended to be a global discipline
that includes all emerging working areas in which food
plays a role, including food safety, quality, traceability,
bioactivity and nutrition.
Using foodomics, nutrition researchers are able to report
any changes in human biofluid metabolomic profiles
after the consumption of dietary phytochemicals from a
holistic point of view [30]. Thus, it is more likely to prove
a real effect when considering the entire metabolome.
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Foodomic challenges Puiggròs, Canela and Arola 113
Chronic Disease
Disease
biomarkers
en us
s
all uo
ge
ch ntin
Co
Health biomarkers
(predictable to risk factor)
Current Opinion in Food Science
Moreover, the adoption of these tools is unavoidable if we
hope to detect the differences between a healthy physio-
logical status and a multifactorial risk factor human status.
Comprehensive omics integration
Emerging high-throughput omics technologies allow the
analysis of biologic phenotypes at multiple molecular
levels. Data from the genome, epigenome, transcriptome,
proteome, metabolome, lipidome or the microbiome can
be combined using systems biology tools [28,31], which
provide a full integrative interpretation [29,30,32,33,
34,35].
In fact, no single analytical technique is able to simulta-
neously determine all the metabolites in a sample due to
the enormous complexity of these molecules. Thus, the
best approach to obtain a complete metabolome view is
the combination and integration of data from different
analytical platforms [36].
Untargeted workflow can be divided in sample prepara-
tion, separation and detection as well as data analysis,
biomarker identification and biological interpretation.
Due to the large variation in physicochemical properties
of metabolites, different extraction and separation proto-
cols are applied assuring high coverage and yield of
Current Opinion in Food Science 2015, 4:111–115
114 Foodomics technologies
metabolites. After extraction, compounds can be deter-
mined by coupling GC, LC and CE to MS or directly by
NMR. Metabolites can be identified with the measured
accurate mass using high resolution MS or specific chem-
ical shifts in NMR. Fragmentation patterns in MS/MS
spectra are specific for each compound allowing them to
be matched with curated databases.
The complex information retrieved in a single experi-
ment requires bioinformatic tools to evaluate the raw data
and generate meaningful results [37,38]. Before statistical
analysis, data are subjected to preprocessing, including
data conversion, scaling, normalization and peak align-
ment. For metabolic profiling, multivariate statistical
methods are most commonly used to analyze data [39],
but other analysis methods are also used including prin-
cipal components analysis (PCA), partial least squares
discriminant analysis (PLS-DA) [39], orthogonal PLS-
DA (O-PLS-DA) or probabilistic principal component
and covariate analysis (PPCCA) [40]. Metabolites that
significantly vary between the sampled groups create
specific phenotypes or traits that can potentially be con-
sidered as biomarkers.
The last steps in metabolite profiling are crucial because
they connect the evaluation of changed metabolites with-
in the biological context and help to visualize the global
results. Pathway analyses tools [41,42] aid in the visuali-
zation of all measurements on pathway maps to provide
better interpretability.
Conclusion
We propose a new nutritional approach based on intro-
ducing healthy biological models to an acute but subclin-
ical homeostatic alteration, called a challenge test,
coupled with analysis via foodomics (metabolomics) to
measure all (and new) biomarkers that would indicate the
evolved disease along time. The metabolomic profile of
challenge responses can deliver new early biomarkers
while also broadening the inclusion criteria to study
nutrient effects on a given risk factor for a given disease.
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