Nutrimetabolomics is a field of study that focuses on the analysis of small molecules in biological

samples such as blood, urine, and tissues, to understand the relationship between diet, metabolism,
and health. It combines the disciplines of nutrition, metabolomics, and systems biology to identify
and quantify the metabolic response of an individual to dietary interventions and to gain insights into
the underlying mechanisms of how nutrients affect health and disease. It is a rapidly evolving field
with potential applications in personalized nutrition, disease prevention, and the development of
new therapeutic approaches. In this book chapter, we examine the most recent methods and
approaches for multi-omics-based nutrimetabolomics investigations. Further, we have described the
benefits of using machine learning techniques to improve the dynamics of nutrimetabolomics
analysis. We have also included various statistical tools, functional tools, modeling tools for
nutrimetabolomics, and tools for predicting chemical properties of nutrients and dietary biomarkers.
Here, we offer R scripts for chemical molecule import and visualization utilizing R packages, which
helps researchers interpret and preprocess mass spectrometry imaging (MSI) data easily. The ideas of
physiological monitoring for diet and nutrition studies with food-related disorders are also important
to comprehend. Additionally, it discusses the significance that nutrimetabolomics plays in precision
nutrition’s most recent advancements.

Metabolomics, which relates to the measurement of small molecules (<1,500 Dalton) within a
biological sample, is part of the omics sciences developed in the last 20 years. Traditionally, two
different approaches had been developed, the untargeted and targeted investigations, which differ
mainly in the identification of the molecules analyzed and the statistical data analysis. The
application of metabolomics tools in the nutrition sciences is known as nutrimetabolomics and has
shown potential in the discovery of new food intake biomarkers, validation of food frequencies
questionnaires and assessment of dietary compliance or dietary patterns. Furthermore, the
application of nutrimetabolomics within dietary interventions might help to characterize the
molecules responsible for modulating health and identifying their mechanism of action. This paper
presents an update of the applications of nutrimetabolomics toward personalized nutrition and
discusses the challenges that must be faced to become a reality the use in the clinical entourage.

Keywords:

1. Metabolomics

2. nutrimetabolomics

3. personalized nutrition

4. Metabolome

Introduction

For centuries, food has been considered an essential energy source to develop the multiple activities
of the human being, as well as a modulator of health and wellbeing. Different ancient civilizations
such as Egypt, Persia, India, and China already used food to treat and prevent the disease. In fact,
ancient Chinese doctors used ants to evaluate if the urine contained high levels of glucose, thus
making possible to diagnose diabetes (1).

Historically, nutrition science has had a reductionist approach, focusing on the analysis of single
compounds derived from food. However, technological progress has brought the ease of making
more specific and useful analyses at the molecular level, opening new horizons in nutrition research.
Therefore, a more holistic vision might be useful for understanding the interactions between the
variety of chemical compounds present in food and the biochemical networks in complex organisms.
Modern nutrition aims to characterize the relationship between diet, lifestyle, and health at the
molecular level, identifying the role played by nutrients in the organism. Hence, the application of
novel high-throughput technologies such as omics sciences will provide critical answers to these
many questions. Here we discuss the role of metabolomics in nutrition toward personalized
attention.

Metabolomics

Metabolomics is the study of global changes in the entire set of metabolites (metabolome), all
molecules of low and average molecular weight (<1,500 Dalton ), present in cells, tissues, organs and
organisms derived from the process known as metabolism (i.e., substrates, and enzymes products)
(2). Some approximations indicate that the number of metabolites present in the human being can
range between 3,000 and 20,000, whereas the number of genes is estimated at 23,000 and that of
proteins in 100,000 (3)

Metabolites are part of the structure of large macromolecules and cell membranes and are classified
as endogenous and exogenous (Figure 1). The former includes amino acids, organic acids, nucleic
acids, fatty acids, sugars, vitamins, and cofactors, among others and are products of the organism
metabolism; the latter comes from the interaction with the external environment and can include
drugs, environmental contaminants, toxins and those from the diet. Metabolites from the diet, also
known as the food metabolome, are the sum of the metabolites derived from digestion, absorption
in the intestine and biotransformation conducted in tissues or the microbiota. Moreover, we should
consider that each food has its metabolome, i.e., the metabolome of orange or fatty fish, and
therefore the human metabolome is composed of fractions of such metabolomes, partially
transformed after digestion, thus forming the human food metabolome (4). Metabolites from the
diet may act as both a source of energy or as regulators of the energy metabolism pathways; i.e., as
signaling messengers or as antioxidants, protecting the cell from oxidative stress (5).

Because the metabolites reflect the physiological state and provide a better understanding of cellular
functioning, they can be a powerful and useful tool to study the metabolism and physiology of living
organisms. The characterization of all the metabolites is commonly known as metabolomic profiling
or fingerprint. When comparing two or more metabolomic profiles is possible to determine patterns
of variation between different groups, i.e., control subjects vs. subjects in a nutritional intervention.
Furthermore, we can monitor the result of an intervention, either pharmacological or nutritional, by
observing the variation of the fingerprints through the trajectory of time. The elaboration of
classifications based on the fingerprint is known as metabotyping.

How to study the metabolome?

One of the major goals of metabolomics is the identification of a large number of metabolites
minimizing the losses or alterations in the process. Unlike the proteome, the metabolome is
composed of a wide variety of chemical compounds of a complex nature thus it is virtually
impossible to determine the entire metabolome simultaneously; consequently, the combination of
different methods and configurations for extraction makes its study more feasible. We know that the
genome remains static, however, the metabolome is in continuous change and reflects several
environmental factors, including drugs, pollutants, the activity of the intestinal microbiota and,
notably, the diet. Hence, the metabolomic profile offers a high-level description of biological systems
that transcend genetic information and reflect more accurately the human phenotype.

The complexity of the metabolome due to the intra-individual variability, the dynamic nature of the
compounds within it and the metabolic flux, make of utmost importance to select an adequate
 analytical approach. Therefore, an appropriate selection of experimental design with reliable
protocols for reproducibility will provide data of biological relevance. Traditionally, study designs on
metabolomics have been categorized into two classes: targeted and untargeted.

The untargeted analysis focuses in obtain as much data as possible in a sample; for this, platforms
with highly accurate analytical capabilities have been developed. However, the primary bottleneck is
the lack of identification of many of the metabolites that will be detected. The chemical identification
and structural elucidation of such metabolites require an intensive post-analysis work, necessary to
be able to perform a correct biological interpretation.

In a targeted analysis, the chemical identities of the metabolites sought are known before the
analysis, due to the use of pure standards and a methodology focused on providing high precision
and selectivity. The major advantage of targeted analysis is related to the knowledge of the
metabolites investigated that helps to obtain a straightforward biological interpretation. Thus, this
approach is advantageous when we want to test a hypothesis.

As of today, it is technologically impossible to determine, quantify and identify each of the

metabolites present in a biological sample. Therefore, high-throughput platforms are needed, either
separately or in combination for such analysis. Nevertheless, each platform has its advantages and
limitations on fundamental aspects such as the specificity and sensibility. The most widely used
platforms in metabolomics studies to optimize the results are nuclear magnetic resonance (NMR),
and mass spectrometry (MS) coupled to various chromatographic variants (i.e., liquid or gas). Table
1 presents the main advantages of each platform. An analysis of the technical instrumentation is
reviewed in depth by Williams et al. (6). Moreover, due to the large amount of data generated,
specific statistical approaches are needed and are reviewed elsewhere (7).

Table 1. Strengths and limitations of the most common metabolomics platforms

PLATFORM STRENGTHS LIMITATIONS

NMR: nuclear magnetic resonance; GC-MS: gas chromatography-mass spectrometry; LC-MS: liquid chromatography-
mass spectrometry; CE-MS: capillary electrophoresis-mass spectrometry

High reproducibility and reliability

NMR Minimum sample preparation Low sensitivity

Nondestructive

High reproducibility and analytical sensitivity Only volatile compounds and thermal stable
GC-MS
High identification Destructive

Chromatographic alternatives (i.e. RP-C18, HILIC, etc.) Time-consuming bioinformatics processing

LC-MS
Wide coverage Destructive

Polar charged compounds

CE-MS Minimal sample amount
Limited robustness and low reproducibility

Nutrimetabolomics
Nutrimetabolomics is the implementation of metabolomics tools in the nutritional sciences (8) and
has been used to identifying metabolic diseases influenced or modulated by the food metabolome
(9). Furthermore, the identification of metabolites that serve as food intake biomarkers might help to
assess nutritional interventions and validate dietary surveys. Technological advances and the
development of more studies in the field of nutrimetabolomics have helped to enhance the
understanding of the health-diet relationship. For instance, through the determination of
metabolomic changes after a nutritional intervention or after the exposure to a food or a dietary
pattern, or with the purpose of verifying the response to a dietary recommendation and
distinguishing between responders and non-responders.

Besides, the identification of the endogenous metabolome is paramount to determine the inter- and
intra-subject variability and allow the classification of subjects according to their nutritional status or
dietary habits. Hence, designing tailored recommendations according to nutritional characteristics or
vulnerabilities of the population in which the metabolome is determined. Hereafter,
nutrimetabolomics established as a handy tool to bring personalized nutrition into a daily basis and
here we discuss some applications.

Biomarkers of consumption to assess the exposure to the diet

Traditionally, the strategies used to assess food consumption have included conventional tools such
as food frequency diaries, 24-hour recalls or nutritional diaries, carried out in large cohorts for a few
months (10). Nutrimetabolomics might support the data validation derived from extensive studies by
providing new biomarkers, and more objective, that could offer additional information outstanding
the traditional methods aforementioned (11). Therefore, it can facilitate future research when
investigating the associations between diet and disease. Moreover, a proper metabolite
characterization provides substantial evidence when applying for nutritional claims and will aid in
offering the population better nutritional guidance.

Biomarkers of consumptions should be characterized by their specificity, which means that they are
not be influenced by the consumption of similar food or a food pattern. For example, stachydrine,
which is the methyl betaine of proline, was reported for the first time as a biomarker of orange
intake (12) and, subsequently, validated by other researchers (13). Recently, our research group
proposed a new metabolic signature including stachydrine plus betonicine, methyl glucopyranoside
(alpha+beta), dihydroferulic acid and galactonate capable of distinguishing between consumption of
orange juice with different polyphenol contents (14). Therefore, it seems that stachydrine might be
used as a suitable biomarker and its selectivity increases when combined with other metabolites.
However, other metabolites have shown their lack of specificity as shown by beta-alanyl-N-
methylhistidine and methylhistidine which were associated with oily fish consumption (15), whereas
other authors reported them as markers of chicken or meat intake (16).

Specific biomarkers for specific food might allow a better understanding of their impact on metabolic
pathways regulation. Similar foods but with different chemical composition may have different
effects on the metabolome, and this should be considered, such is the case of the isoflavones
derived from soy, which have shown a different impact according to the type of conjugate ingested
(17).

Assessing dietary patterns through nutrimetabolomics

Diet diversity, whether due to geographical or cultural reasons, has generated considerable interest
in exploring dietary patterns through metabolomics. Understanding the effect of every component of
the diet in the metabolome would help to comprehend its potential protective or harmful effects,
and furthermore their mechanisms of action. In such regard, the European Prospective Investigation
into Cancer (EPIC) cohort studied the profiles of healthy male consumers of meat, fish, vegetarians,
and vegans. The metabolic profiling allowed to differentiate between each group, particularly those
that belonged to the vegan group in front of the consumers of products of animal origin; mainly, due
to the lower concentrations of glycerophospholipids and sphingolipids that were found in the vegan
subjects (18). This method opens a door for research in which the hypothesis is whether the
differences between metabolic profiles have an impact on health, such as the reduction or the
increased risk of chronic diseases like CVD.

As of today, several dietary patterns have been profiled using metabolomics. On the one hand, the
Nordic diet has been associated with biomarkers such as trimethylamine N-oxide, hydroquinone-
glucuronide, hippuric acid, indole-3-acetic acid and 3,4,5,6-tetrahydro hippurate. On the other hand,
the Mediterranean diet has been related to 3-hydroxybutyrate, citrate and cis-aconitate; creatine,
creatinine; to different amino acids: proline, N-acetyl glutamine, glycine, amino branched-chain acids
and their derivatized metabolites; lipids, oleic and suberic acid and some microbial co-metabolites
such as phenyl acetyl glutamine and p-cresol. Therefore, the assessment of the geographical factor in
multi-centric studies should be considered as an independent variable to make an adequate
interpretation in subsequent investigations. The LIPGENE study provides a clear example (21), in
which the inclusion of subjects with metabolic syndrome from several countries in Europe, revealed
variations in urinary and plasma metabolic profiles. These differences made possible to group the
individuals according to their geographical region. For example, individuals from the northwest
region presented higher concentrations of hippurate and methyl N-nicotinate, whereas the northeast
region was characterized by elevated levels of creatinine, citrate, and EPA in plasma. Finally, the
southeastern region was defined by a higher concentration of trimethylamine and reduced levels of
eicosapentaenoic acid in plasma.

Studying the effect of food and its impact on health

Although nutrimetabolomics is becoming an exciting alternative to characterize the response to

foods or dietary, it is essential to bear in mind that there are still technical shortcomings that
complicate its clinical use. One major concern is related to the quantitation across studies. Therefore,
differences between the same metabolite in different interventions must be carefully interpreted, to
avoid wrong conclusions in regards to their mechanisms of action. Furthermore, the enhancement in
the identification and characterization of unknown compounds will provide a better understanding of
their role in the metabolism and its impact on health.

In recent years, metabolomics has been used to determine the effect of specifics nutrient or foods in
RCTs. For example, an RCT of eight weeks of duration including Finnish women compared the use of
different types of bread. A regular bread decreased the levels of leucine and isoleucine, whereas the
consumption of rye bread led to an increase of betaine and N, N-dimethylglycine concentrations.
These metabolic differences point toward a modulation in the metabolism of the branched-chain
amino acids and the one-carbon metabolism as the pathways in which rye bread exerts its protective
effects against cardiovascular diseases or certain types of cancer.

Regardless of dietary patterns, individuals have a basal metabolic trace that might be influenced by
various factors, such as metabolism, intestinal microbiota, lifestyle, physical activity, and body
composition. Therefore, all these factors should be taken into account when designing
nutrimetabolomics studies related to nutritional interventions. The determination of the basal
metabolic phenotype, as well as the establishment of the extent to which nutritional interventions
can modulate the basal phenotype, should be considered.
Microbiota, diet, and metabolomics

In recent years, interest in the gut microbiota has increased due to the presence of a large number of
microorganisms producing different metabolites that have been related to health and disease states
in humans (23,24); also, the specificity of some microbiota-related metabolites has shown to be
outstanding (4). Evidence has shown the influence of diet on diseases such as colorectal cancer, and
this might be related to the microbes’ metabolite production that might serve as modulators.
Probably, the most documented data relates to short-chain fatty acids, which are fiber fermentation
products, and their protective role impeding the growth arrest and differentiation of colorectal
cancer cells (25).

Hence, the role of the intestinal microbiota and its manipulation through food intake could be a
potential tool for the prevention and management of diseases (26,27). For example, the combination
of phytochemicals and fiber might counterattack the harmful effects of those metabolites with a pro-
carcinogen character. Because many phytochemicals, just as polyphenols, are fermented in the colon
by specific microbes producing more bioactive compounds, consequently, amplifying their protective
properties and acting together with metabolites like the short-chain fatty acids. Therefore,
comprehensive profiling of the microbiome metabolome might aid in the conception of the
interaction between microbiota, diet, disease, and treatment.

Future perspectives

The field is growing up as reflected by the increasing number of studies ongoing. Nevertheless,
several challenges must be acknowledged and solved. The standardization of the methods and
protocols is probably one of the major concerns among the community to guarantee the replication
across different labs. Fortunately, works such as the report from Wang and colleagues (28) are
helping in the identification of potential novel food biomarkers and demonstrating the validity of
early reported biomarkers in external cohorts and using different types of biofluids. Also, the use of
different types of study designs will allow identifying whether the metabolites or metabolic
fingerprints are biomarkers of intake or markers of a response to the food intake.

Finally, yet importantly, it is necessary to pursue the openness of data; there are several initiatives to
improve in this regard. Making public the raw metabolomics data will allow testing, validate,
replicate or re-analyze previous findings. Currently, there are numerous statistical approaches, and it
is time-consuming for the original researchers to apply all of them. Thus the public availability might
help to explore in depth and from different perspectives by other researchers. Altogether, filling
these gaps will approach the use of metabolomics in the clinical setting and contribute to the
development of personalized nutrition.
Diabetes is the most iconic issue in the health industry irrespective of age and vegetation. It is a
major non-communicative metabolic disorder, having a devastating impact on the healthcare and
economy (Dall et al., 2014). DM is broadly classified as type 1 (T1DM), type 2 (T2DM) and gestational
diabetes (GDM). The reason behind T1DM and T2DM are lack of insulin production due to β- cell
destruction and ability to intake insulin by cells due to insulin resistance, respectively (American
Diabetes Association, 2018, Nichols et al., 2013, Zarkogianni et al., 2015). GDM is the condition of
glucose intolerance during pregnancy (Goldenberg and Punthakee, 2013). Proper therapeutic
intervention through the external supply of insulin and systematic medication can be the remedies of
type 1 and 2 diabetes respectively (Ransom et al., 2013). Also, great efforts are taken for the
treatment of diabetic complications and the discovery of new biomarkers, which have significant
relation with diabetes. Huge rise in the number of diabetes cases reported each year to imply the
need for preventive rather than reactive approach (Zarkogianni et al., 2015) along with deployment
of point of care devices across the nations. Knowledge about the origin, diagnosis, and treatment of
DM must be spread to implement preventive measures to fight this count effectively (Ekoé et al.,
2013). In this scenario, the International Diabetes Federation has been trying to draw the attention
of countries towards early diagnosis of diabetes to ensure the diabetes-free lifecycle of future
generations.

At present, diagnosis of DM and its categorization is mainly based on the identification and
quantification of biomarkers like glycated hemoglobin and glucose in blood, urine (Gu et al., 2016;
Radhakumary and Sreenivasan, 2011), sweat (Chen et al., 2017, Liu et al., 2018), tears (Park et al.,
2018), saliva, etc. Also, parameters like fasting plasma glucose, oral glucose tolerance, postprandial
plasma glucose criteria, are executed for DM diagnosis (The Expert Committee on the Diagnosis and
Classification of Diabetes Mellitus, 2002). Moreover, food and lifestyle highly influence glucose levels,
which lead to more complications in the assessment of diabetic conditions.

In this scenario, new biomarkers (independent of the glucose levels) and techniques for accurate and
early diagnosis (prediabetic stage) of diabetes having immense importance. The identification of
prediabetes as well as prediction of associated complications has been a field, under keen
observation by many researchers and physicians across the globe. Methylglyoxal (MG) is emerging as
a biomarker of diabetes mellitus (DM) as it has a close association with protein glycation and insulin
resistance. As a biomarker, MG investigated for several other ailments. Now, the identification of the
root cause of diabetes and its complications brings MG’s potential for diabetes diagnosis into the
limelight.

Keeping this as the background, we have presented the major types of diabetes and their causes
influenced by glucose levels and insulin resistance. The biomarker MG and its origin, sources,
formation, detoxification and a major contribution to the onset of diabetic complications are
discussed briefly. Also, deliberately explained the different detection methodologies and the current
scenario of MG research and its future perspectives.