Trends in Analytical Chemistry 55 (2014) 94–116

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Analytical metabolomics-based approaches to pancreatic cancer

Iole Maria Di Gangi a,⇑, Urska Vrhovsek a, Valerio Pazienza b, Fulvio Mattivi a
a
Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM) Via E. Mach 1, 38010 San Michele all’Adige, Trento, Italy
b
Gastroenterology Unit, Fondazione I.R.C.C.S. ‘‘Casa Sollievo della Sofferenza’’ Hospital, 71013 San Giovanni Rotondo, Foggia, Italy

a r t i c l e i n f o a b s t r a c t

Keywords: We provide a comprehensive review of the analytical technologies applied in pancreatic cancer (PC)
Analytical method research, investigating tumor-associated metabolites identified as possible new biomarkers for screening
Biomarker
the population at risk and for potential early tumor diagnosis. We briefly introduce PC tumorigenesis, risk
Extraction
factors and some of the benefits of studying changes to the metabolome associated with PC. We focus on
Mass spectrometry
Metabolic profile the different matrices used in the studies and sample preparation for the analytical methods applied to PC
Metabolomics specimens. We then delineate the advantages and the disadvantages of each analytical platform, pointing
Pancreatic cancer out the importance of extraction techniques in metabolomics approaches employed in PC research. We
Pathogenesis cover a number of case studies, and then review identified metabolites associated with PC. In the final sec-
Physiopathology tion, we discuss possible approaches to understanding the link between metabolic profile and the onset of
Sample preparation PC, which may provide insights into the physiopathological mechanisms involved in PC pathogenesis.
Ó 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
2. Metabolomics and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3. Metabolomics approaches in biomarker discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4. Metabolomics-based studies in PC research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1. Biological fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.2. Analytical platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.3. NMR-based studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.4. MS-based studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5. Sample preparation in approaches using metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
6. Metabolomics data export and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
7. Commercial software and library sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
8. Analysis and summary of the results obtained in PC studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
9. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
10. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Abbreviations: CDA, Cytidine deaminase; CP, Chronic pancreatitis; CT, Computed 1. Introduction
tomography; CTPS, Cytidine 50 -triphosphate synthetase; dCK, Deoxycytidine
kinase; dFdC, 20 ,20 -difluorodeoxycytidine or gemcitabine; dFdU, 20 ,20 -difluorode-
oxyuridine; hENT, Human equilibrative nucleoside transporter; MRI, Magnetic
Pancreatic cancer (PC) is one of the leading causes of cancer-
resonance imaging; MRP, Multidrug resistance-associated protein; PC, Pancreatic related deaths worldwide, reflected by an incidence of 277,668
cancer; PCA, Principal-component analysis; PDCA, Pancreatic ductal adenocarci- new cases and almost the same mortality rate (266,029 cases)
noma; PLS-DA, Partial least squares discriminant analysis; TOF, Time-of-flight; per year [1]. The incidence of PC is constantly on the rise, especially
UHPLC, Ultra-high-performance liquid chromatography.
⇑ Corresponding author. Tel.: +39 0461 615136; Fax: +39 0461 615200. in the USA among the black male population (15/100,000) [2]
E-mail address: iolemaria.digangi@fmach.it (I.M. Di Gangi).
(Fig. 1) and increases with age, according to GLOBOCAN data [3]

http://dx.doi.org/10.1016/j.trac.2013.12.006
0165-9936/Ó 2014 Elsevier Ltd. All rights reserved.
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116 95

Fig. 1. Data for estimated 5-year prevalence for both sexes (Panel A) and the incidence of pancreatic tumor for males (Panel B) of all ages around the world. (Adapted from
GLOBOCAN 2008, IARC [3]).
96 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116

Fig. 2. Curves of estimated incidence (A) and mortality (B) for PC in males and females in southern Europe. Data are expressed as age-standardized rates per 100,000.
(Adapted from GLOBOCAN 2008, IARC [3]).

(Fig. S1). PC is also the tenth most frequent cancer in Europe,
accounting for some 2.6% of cancer in both sexes, even though
the age-specific rates of incidence and mortality in men are
one-and-a-half times higher than in women (Fig. 2).
A number of risk factors for developing PC have been estab-
lished: a family history of the disease, smoking, alcohol, age and
diabetes, overweight and Helicobacter pylori have long been known
[4–8]. Furthermore, over five decades, large independent studies
have examined ABO blood groups and the risk of PC in Western,
Asian and other populations, suggesting that blood-type A might
be associated with a higher risk of developing pancreatic tumor
[9] and observing a lower frequency of blood-type O in patients
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
with exocrine PC, compared to patients with other forms of cancer
[10]. In another study, Pelzer et al. [11] found that the majority of
patients suffering from PC had blood-group A, and they confirmed
that blood-group 0 was less frequent in patients with PC. However,
to date, the mechanisms by which ABO blood type, or closely-
linked genetic variants, may influence the risk of PC have not been
clearly defined.
Rapid tumor progression and early metastasis make it a partic-
ularly lethal solid tumor, with a 5-year survival rate of less than 5%.
Consequently, most treatment options are based on palliative care
[12].
Because early PC often does not cause symptoms and later
symptoms are usually non-specific and varied, less than 20% of
patients can have their cancer resected with a curative intent, be-
cause more than 80% of patients have a locally advanced or meta-
static tumor that is unresectable at the time of diagnosis [13–15].
The clinical symptoms of PC are usually unremarkable until the
cancer has progressed to an advanced stage. The use of imaging
studies (e.g., abdominal CT or MRI), in the context of strong clinical
suspicion of pancreatic ductal adenocarcinoma (PDAC), is inade-
quate for diagnosing PC at an early stage, as these techniques do
not reliably detect pancreatic tumors <1–2 cm in size, so, at the
moment, there is no effective method for early detection [16].
20 ,20 -Difluorodeoxycytidine (gemcitabine, dFdC) is currently
considered the preferred host chemotherapeutic drug for meta-
static and advanced pancreatic tumors, but it is far from being
the gold standard, since the overall patient-survival rate is unac-
ceptably low and new therapeutic approaches are urgently needed
[17,18]. One of the reasons for the poor response to dFdC is thought
to be acquired resistance [19], which occurs with four possible
processes, as suggested by Ohmine et al. [20], on observing the
changes in protein expression in RPK9 cells. These authors sur-
mised that the acquisition of dFdC resistance was through:
(1) attenuation of dFdC phosphorylation via suppression of dCK,
a kinase protein that has a key role in phosphorylating gem-
citabine in order to activate its function;
(2) facilitation of dFdC inactivation to dFdU by induction of
CDA;
(3) facilitation of dCTP production by induction of ribonucleo-
tide reductase and CTPS; and,
(4) attenuation of dFdC uptake or facilitation of metabolite
excretion by induction of MRP expression or by decreasing
expression of hENT1 [20].
Screening tests for the general population need to be accurate,
safe, and convenient, as well as capable of high throughput. Many
serologic markers have been found to be potential biomarkers dur-
ing screening for PC detection, belonging to different classes of
compounds, such as:
(1) tumor markers: CA 19-9, CA-242, CA-50, CEA, CEACAM1,
MIC1, MUC1, alpha-fetoprotein, DU-PAN-2 mAb, alpha4GnT;
(2) apoptosis markers: NF-kB, hTert and CK-19;
(3) cytokines, such as IL-8;
(4) adhesion molecules: ICAM-1, MMP-2, MMP-9; and,
(5) growth and angiogenesis factors, such as EGFR, IGFBP-1.
These and others serum biomarkers are summarized in
Table S1, which presents an overview of significant evaluated mol-
ecules, potentially modulated in patients diagnosed with PC.
CA19-9 is the most extensively studied of these tumor markers,
but it has poor specificity for PC, being high in the case of many
cancers of the upper gastrointestinal tract, ovarian cancer, hepato-
cellular cancer and benign conditions of the hepatobiliary system
[21]. CA19-9 is considered the standard for monitoring response
97
to chemotherapy and recurrence following surgical resection in pa-
tients with PC, but has demonstrated modest effectiveness in the
screening of symptomatic individuals on an outpatient basis, with
a median sensitivity of 79% (range 70–90%) and median specificity
of 82% (range 68–91%). However, it has been shown to be ineffec-
tive for the mass screening of asymptomatic subjects [22]
(Table S1).
Gold et al. [23] developed a monoclonal antibody (Mab PAM4),
which is highly specific for a MUC1 produced by pancreatic adeno-
carcinoma. This antigen is identified in over 90% of pancreatic car-
cinoma and its precursor lesions, but is not detected in the normal
pancreas [23]. The authors demonstrated that the sensitivity and
the specificity of the immunoassay for PC were 77% and 95%,
respectively, suggesting that PAM4 is potentially useful for the
early detection of PC [23] (Table S1).
Unfortunately, none of these markers has achieved sufficient
levels of sensitivity and specificity to be recommended for screen-
ing asymptomatic patients in the general population, so the search
for new biomarkers that would allow early detection of disease is
still an open issue.
This review has the aims of:
(1) examining the various analytical technologies that have
been applied in PC research;
(2) investigating tumor-associated metabolites identified as
possible new biomarkers for screening of the population at
risk; and,
(3) studying the potential role of metabolomics in the field of
early tumor diagnosis and personalized therapy.
2. Metabolomics and cancer
Metabolomics has developed rapidly in the past few years and it
represents a useful tool for the identification of new biomarkers,
which may help to understand tumorigenesis mechanisms in
cancer research and to develop ‘‘cancer models’’ [24]. This technol-
ogy, when used as a translational research tool, can provide a link
between the laboratory and the clinic, particularly because
metabolic and molecular imaging technologies, such as positron
emission tomography and magnetic resonance spectroscopic imag-
ing, enable non-invasive discrimination of metabolic markers
‘‘in vivo’’.
Metabolomics is one of the pillars of systems biology, providing
a quantitative snapshot of low-molecular-weight compounds (typ-
ically 1000 Da) in biofluids and other complex matrices [25,26].
Thus pancreatic tumor can represent a rich source for the discovery
of biomarkers with appreciable specificity and sensitivity. In this
context, the use of metabolomics-based approaches providing ac-
cess to biochemical-phenotype information can offer an opportu-
nity for non-invasive screening of early tumor-associated
perturbations in the cellular metabolism [27].
It also has the potential to identify new targets for diagnosis and
intervention at various stages of malignancy, which could support
early, personalized therapy [28]. A critical step in defining the
correct personalized anticancer therapy is identification of altered
genes and pathways in the patient’s tumor and elucidation of their
particular oncogenic role. The success of molecular studies in iden-
tifying potential molecular targets for therapeutic intervention is
exemplified by developments in the treatment of chronic myelog-
enous leukemia (CML) [29] and the development of clinical appli-
cations from biomarker discovery using metabolomics to
therapeutic targets in breast cancer [30], ovarian cancer [31] and
renal cell carcinoma [32].
A growing number of metabolomics-based studies have been
carried out, with more than 700 published articles on cancer re-
search (Fig. S2A). Furthermore, in the past few years, we have
 98
I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
Fig. 3. Overall flowchart of strategy for metabolomics-based investigations directed at biomarker discovery in PC (PC). The process of biomarker discovery using metabolomics begins with a well-designed experimental plan,
which most importantly must include appropriate controls for the disease concerned. A prediction model needs to be created, using independent validation datasets with a minimum of three classes: healthy control, disease group
and an additional related-disease control group (i.e. biological samples from PC versus non-PC controls versus CP patients). The analytical methods and statistical validation employed in biomarker discovery are of critical
importance. The final step is to identify and to validate new therapeutic targets, starting from the possible pathways involved in the onset and the progression of PC, which should ultimately be tested both ‘‘in vitro’’ and ‘‘in vivo’’ to
establish the utility of the biomarkers discovered in a routine clinical setting.
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116 99

observed increasing interest in the application of the metabolo- greatest potential to provide diagnostic information on the general
mics approach to PC (Fig. S2B), which represents a challenge in health of the organism. Metabolites in blood or urine reflect the
terms of survival rate and disease prognosis. This research is begin- state of the disease and the host’s response to the disease. When
ning to provide new information and search for metabolic stored at 40°C, both blood and urine samples can be kept for ex-
biomarkers using NMR, GC–MS, LC–MS, in the hope that it may tended periods without alteration for subsequent analysis [37].
allow early diagnosis and/or screening for PC in selected popula- Samples can be procured by relatively non-invasive means at mul-
tions [24]. tiple time points, permitting temporal-based analysis. It is conceiv-
able that more immediate changes in metabolites may be seen in
blood during acute physiological events. Moreover, metabolite pro-
3. Metabolomics approaches in biomarker discovery
files from serum have demonstrated less circadian variation, and
decreased inter-subject and intra-subject variability compared to
Currently, many different approaches are used for metabolo-
urine [27].
mics investigations (e.g., targeted and untargeted metabolomics
However, preparation and processing of serum or plasma is
are complementary strategies that are commonly combined in
much more complex, and even small changes in technique alter
cancer-biomarker discovery) [33]. Using the targeted method, a
the recovery of certain metabolites [38,39]. Furthermore, lipid
particular metabolite or a small group of related metabolites can
and protein present in serum specimens may induce degraded
be measured, typically focusing on one or more related pathways
spectral resolution of serum metabolites, an interference absent
of interest [34]. In other cases, an untargeted or global approach
in urine samples [40].
may be taken, in which as many metabolites as possible are mea-
The metabolomics profile of serum/plasma, urine and other
sured simultaneously and compared in biological samples without
biological fluids, such as bile, saliva, cell culture or tissue extract,
bias [35]. In contrast to targeted metabolomics results, untargeted
has been investigated for diagnostic purposes for a variety of
metabolomics datasets are exceedingly complex, but they give ac-
malignancies, including PC [41–57]. From these data, candidate
cess to semi-quantitative measurement of unknown compounds,
biomarkers, such as glutamate [50], 3-hydroxybutyrate
which is essential for generating new hypotheses [35].
[41,43,44], lysine [48,50], phenylalanine [41,44], arachidonic acid
Metabolomics also includes other types of experiments that can
[48], glucuronic acid [53], tauroursodeoxycholic acid [48] and
be applied to the field of cancer research, including metabolic pro-
deoxycholylglycine [48], have been proposed, due to the significant
filing and metabolic fingerprinting.
changes between PC patients and healthy controls.
Metabolic fingerprinting usually analyses all resectable analytes
The key pathways that have been demonstrated to behave dif-
in a given sample, with subsequent classification of samples and
ferently in pancreatic tumor and normal controls include glycolysis
identification of differentially-expressed metabolites, which define
and the pentose phosphate, nucleotide and protein biosynthesis, li-
the sample classes. Metabolic profiling instead focuses on the anal-
pid and phospholipid turnover, TCA cycle and redox stress
ysis of a specific group of metabolites or a class of compounds,
[47,54,55]. The phosphocholine metabolism has also been sug-
whose data can be integrated with pathway maps to enhance bio-
gested to be implicated in PC [54], supporting the recent finding
logical understanding.
of a new biomarker, PC-594, as an opportunity for identifying sub-
Dettmer et al. [33] provided a clear outline of these two comple-
jects with PC or at high risk of developing PC [55].
mentary methods, showing that their combination is a potential
All the observations of these metabolomics-based studies, per-
tool for metabolomics investigations.
formed in around 60% of cases on plasma or serum [41–45,48,49]
However, the number and the chemical properties of the
and in 15% of cases on urine [46,47], support the feasibility of using
metabolites to be studied are defining attributes of any metabolo-
serum/plasma or urine specimens for clinical research on the met-
mics experiment and determine the experimental design with
abolomics state of PC patients, and for biomarker discovery. The
respect to sample preparation and choice of instrumentation.
use of other biological fluids, such as saliva [56], bile [53] and pan-
Fig. 3 presents an overall flowchart of the strategy for metabolo-
creatic tissue [57], was also investigated (25% of studies) for this
mics-based investigations directed at biomarker discovery in PC.
purpose.
The process of biomarker discovery using metabolomics begins
Saliva is an important biological fluid that provides various
with a well-designed experimental plan, which includes three dif-
functions and is made up of 99% water, with minerals, mucus, elec-
ferent steps, as proposed by Wettersten and Weiss [32]:
trolytes, nucleic acids and proteins, such as enzymes, enzyme
inhibitors, growth factors, cytokines, immunoglobulins and other
 Phase 1: discovery;
glycoproteins [58]. It is a filtrate of blood, reflecting the physiolog-
 Phase 2: pre-validation and quantification; and,
ical condition of the body, so it could be used to evaluate clinical
 Phase 3: validation and application.
status and predict systemic diseases [58]. Compared to blood, sal-
iva offers distinct advantages for diagnostic or research purposes
For any biomarker discovered through metabolomics analysis
(e.g., its collection is cost effective, safe, easy and non-invasive).
to be used in routine clinical practice, sample collection and an
Indeed, many of the characteristics of biological fluids, such as
analytical methodology need to be available, allowing accurate
blood and urine, are applicable to saliva, including circadian varia-
quantification of the metabolite and offering high throughput,
tion and the presence of diverse diagnostic analytes [58].
prompt turnaround and low costs [32]. As in all biomarker studies,
A comprehensive metabolite analysis of saliva samples was
the likelihood of finding a true marker for the general population is
conducted by Sugimoto et al. [56] using capillary electrophoresis
exceedingly low. It is more likely that biofluid markers will be most
time-of-flight mass spectrometry (CE-TOF-MS). The authors identi-
useful for assessing the population at risk.
fied 48 principal metabolites, mostly amino acids, as biomarker
candidates using salivary diagnosis for the detection of PC [56].
4. Metabolomics-based studies in PC research Bile is among the other matrices that can be used to study and
to discover molecular markers. Bile composition is very complex
4.1. Biological fluids and varies according to the nutritional state of the individual.
The main components include bile acids, phospholipids (mainly
The vast majority of clinical research has been carried out on ur- phosphatidylcholine), cholesterol, bilirubin (mostly in its
ine and serum samples [36], perhaps because these fluids have the conjugated form) and inorganic salts (potassium, sodium and
100 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
bicarbonate), and very small amounts of copper and other metals.
Recent methodological developments have enabled identification
of a number of bile metabolites that have links with the biliary
tract and PCs [53,59,60]. Investigations of human bile are also con-
sidered to help the biomarker discovery process in vitro and pro-
vide avenues for translational research in detecting and following
dynamic variations of biomarkers in clinical settings using
non-invasive approaches, such as in-vivo magnetic resonance spec-
troscopy [61]. The findings of Bezabeh et al. [53], using 1H NMR
spectroscopy in human-bile samples of PC patients, clearly indi-
cated that there is an alteration in bile composition in the course
of pancreatic tumor, suggesting that elevated levels of GlcU ob-
served could be of diagnostic value in the detection of PC [53].
4.2. Analytical platforms
Metabolomics experiments in a biological sample can employ
one or more complementary analytical platforms to obtain exten-
sive coverage, because of the large number of molecules, with sub-
stantial diversity of the metabolites in terms of chemical structure
and concentration.
In PC research, metabolomics-based studies directed at bio-
marker discovery have been performed by employing nuclear mag-
netic resonance (NMR) [43,44,46,47,50,53] or MS [20,41,42,45,
48,62], the latter being coupled with a separation technique, such
as gas chromatography (GC) [41,45,48,62], liquid chromatography
(LC) [20,48,62] or CE [49,56] and with different ion sources and
mass analyzers, depending on the type of experiment.
Each of these analytical techniques has advantages and disad-
vantages, most notably differences in sensitivity, reproducibility,
accuracy and equipment costs, as well summarized elsewhere
[63,64].
NMR techniques have been applied to targeted and untargeted
studies of PC, using different kinds of experiments:
(1) One-dimensional 1H-NMR spectroscopy [43,46] with NOESY
experiments [47,50];
(2) Two-dimensional 2D-NMR spectroscopy [44,53], in particu-
lar, 1H-1H-DQF-COSY and TOCSY experiments for analysis of
D-glucuronic acid in bile samples [53];
(3) 31P-NMR spectroscopy [53];
(4) Two-dimensional J-resolved 1H and H, 13C-HSQC-NMR spec-
troscopy [47]; and,
(5) High-resolution magic angle spinning (HRMAS) 1H-NMR
spectroscopy applied to tissue samples in PC rats [52].
Of the ionization techniques, atmospheric pressure ionization
(API), especially electrospray ionization (ESI), is a popular choice
as the ion source in MS-based applications in PC research
[20,48,65]. When combined with LC, it shows good sensitivity
and a high dynamic range and versatility, but also provides soft
ionization conditions, giving access to the molecular mass of intact
molecules from complex mixtures.
Only a few targeted studies have been conducted using MS
without prior separation chromatography, by direct injection anal-
ysis [42,55], which is a high-throughput approach allowing a
sample to be processed within a few minutes, but with major lim-
itations in terms of ion suppression and loss of sensitivity. Typi-
cally, labelled standards are used in this approach to correct
matrix effects [66], but standards are not available for all com-
pounds and they are sometimes very expensive. Targeted
approaches, using a single-quadrupole or triple-quadrupole mass
analyzer, coupled with GC [41,45,62] or LC [20,62], have instead
been used to quantify particular classes of compounds, presumed
to be altered in PC in comparison to the control group, such as
amino acids, fatty acids, organic acids, sugars and enzymes and
transporter proteins.
Furthermore, the development of high-resolution and ultra-
high-resolution analyzers (TOF-MS and FT-MS, respectively) has
yielded accurate mass measurements, whereas ion-trap analyzers,
among other things, are used due to their capacity to perform two-
stage, three-stage or multi-stage MS/MS (i.e. MSn) experiments to
obtain the additional structural information needed for metabolite
identification.
Most MS-based studies have enhanced analysis for global
profiling of the PC ‘‘metabolome’’, in a non-targeted metabolomics
approach, by using the combination of UHPLC or GC with Q-TOF-
MS [57,65,67], flow injection with Fourier-transform ion-cyclotron
resonance MS (FI-FT-ICR-MS) [55], or LC with LTQ-Orbitrap [48] to
generate comprehensive metabolomics profiles containing thou-
sands of accurate mass measurements from PC patients and dis-
ease-free subjects.
Other analytical platforms showing promise for PC metabolo-
mics research include CE-MS [56] and pressurized capillary elec-
trochromatography (pCEC), which is a hybrid technique between
LC and CE, coupled with a UV detector [49]. However, CE based
techniques are still at an early stage as compared to techniques
based on GC and LC.
4.3. NMR-based studies
Recently NMR-based metabolomics was applied to toxicity,
drug efficacy and disease diagnosis both in vitro and in clinical tri-
als [25,59,68–71], providing evidence of differing spectral regions
in cancer patients and healthy controls [72]. This approach led to
discrimination of patients with PC from those with chronic pancre-
atitis (CP) and healthy controls using the metabolic profiles of rat
pancreatic tissue, demonstrating the relationship with disease pro-
gression in an animal model [70]. There is an established associa-
tion between carcinoma of the pancreas and the sporadic or
hereditary forms of CP, and most studies are designed using PC
and CP as two different classes of patients. Results from a prospec-
tive case–control study conducted by Ge et al. [73] seem to suggest
that CP may be closely related to PC, although the answer to the
question of a cause-and-effect relationship is unclear. In this
context, Zhang and colleagues [50] found different metabolomics
patterns in the plasma samples of PC and CP patients using a
NMR metabolomics-based approach. The PC group showed ele-
vated concentrations of N-acetyl glucosamine (NAG), dimethyl-
amine (DMA), very low-density lipoprotein (VLDL) and acetone
and reduced levels of 3-hydroxybutyrate, lactate, high-density
lipoprotein (HDL), low-density lipoprotein (LDL), citrate, alanine,
glutamate, glutamine, histidine, isoleucine, lysine and valine,
compared to CP patients [50]. These results suggest that PC may
develop in different ways and lead to different metabolic changes
in PC and CP patients and healthy individuals [50].
A study of targeted profiling, using the 1H-NMR spectra of urine
samples from patients with benign pancreatic conditions and
PDAC, showed a clear distinction between controls and PDAC with
early-stage or locally-advanced disease [46]. The authors observed
unique metabolite-expression patterns for PDAC, with strong pre-
dictive accuracy for cancer, which could reflect changes occurring
at both tumor-microenvironment level and the global metabolism
[46]. Recently, Napoli et al. [47] identified a discrete urinary meta-
bolomics signature for PDAC, distinguishing patients from healthy
controls and discriminating samples based on tumor localization.
The possibility of following disease evolution and deducing tu-
mor-site mapping was achieved with data extracted from NMR
spectra using multivariate statistical analysis. However, a large
proportion of the patients were at stage IV and gender effects were
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
not examined, as the profiles were developed solely from male
patients [47].
An NMR-based analytical method was applied by Bathe et al.
[44] to establish a serum-metabolomics profile capable of discrim-
inating PC patients from patients with benign pancreatic biliary
disease. The authors were able to distinguish metabolites that
are strongly related to disease alone (elevated glutamate, acetone,
and 3-hydroxybutyrate) and those related to disease and age (ele-
vated glucose, phenylalanine, formate, and mannose) [44]. These
results were congruent with serum studies limited in sample size
(17 PC) [43], which related the differences in metabolite profiles
in PC patients and healthy subjects to physiological and patholog-
ical variations.
Using 1H-NMR, Nishijima et al. [60] discovered significantly-
elevated lactate levels in the serum and the bile of patients with
malignant hepatobiliary disease. In 2009, Bezabeh et al. [53] ob-
served that PC patients had very high levels of D-glucuronic acid
(GlcU) in bile samples, whereas this was absent or negligible in
control and CP patients. The exact mechanism inducing elevated
levels of GlcU in bile remains unclear. Nevertheless, they suggest
that this metabolite could be a very promising additional marker
in these patient categories for evaluation of definitive diagnosis
[53].
Finally, another study reported successful discrimination of
malignant from benign biliary disease using bile samples, pointing
out the better performance of the metabolomics approach in the
diagnosis of biliary tract cancer, in comparison with conventional
diagnostic tools, including serum-tumor markers (CA19-9, CEA)
and bile cytology [59]. However, invasive methods are required
for the collection of bile and it is generally inconvenient to sample.
An altered blood-lipid profile was observed in plasma extracts
from a large number of PC patients (n = 82) by Beger and col-
leagues [74]. They surmised that this could be related to altered
pancreatic functionality and altered levels of pancreatic enzymes,
such as lipase, phospholipase A and cholesterol esterase, which
help the body digest fat [74].
4.4. MS-based studies
There are different applications of metabolomics-based chro-
matography-MS methods for PC. A crucial recent study, conducted
by Bi and colleagues [65], focused on the optimization of quench-
ing, harvesting and extraction of metabolites from human pancre-
atic cell line Panc-1, in order to obtain the maximum analytical
recovery of all the compounds considered in cell lysate. The most
efficient, reproducible method for extraction of Panc-1 cells was
by using 75% of a 9:1 MeOH/chloroform solution. Separation
efficiency was also evaluated by comparing HILIC and RPLC separa-
tion, using a UHPLC-QTOF-MS system [65]. HILIC provided better
separation of metabolites than RPLC, thus enhancing Panc-1 cell-
metabolome coverage and making it possible to achieve the best
reproducibility for the analytical procedure proposed [65].
A targeted approach was applied by Leichtle et al. [42] using
FIA-MS/MS analysis of 26 amino acids in the sera of PC patients,
healthy controls and pancreatitis patients [42]. This obtained data
consistent with a previous publication, in which the authors
revealed 26 significantly different metabolites in the plasma met-
abolomics profiles of 5 PC patients in comparison with 5 mixed
pancreatitis/healthy controls, by combining GC-TOF-MS, LC-ESI-
MS, and LC-LTQ-Orbitrap studies [48]. Using HILIC-LC-ESI-MS anal-
ysis, naturally-occurring isomers of polar phosphatidylcholine PCs
(34:2) were also detected in the same plasma samples [48]. Orešič
and co-workers [67] had already performed ‘‘lipidomics studies’’ in
breast-cancer research, making use of a GC-TOF-MS metabolomics
approach, demonstrating up-regulation of many membrane lipids
101
and supporting a possible link between lipid metabolism and can-
cer development.
Other possible pathways involved in PC were explored in the
past few years {e.g., MALDI-TOF-MS was used to study the fucosy-
lation pattern of glycans in MS and MS/MS modes for PC (stages IA,
IIA and B, III, and IV) versus CP and normal controls, surmising the
use of haptoglobin-fucosylation changes as a marker for disease in
pancreatic tumors [75]}. Finally, a UHPLC-TOF-MS/MS method was
applied to perform small-molecule-metabolite profiling of
matched normal and PC tissue, identifying a sub-set of known
metabolites that were found to be significantly de-regulated in
pancreas tumor tissue [57].
Nishiumi et al. [45] instead used a GC-MS-based metabolomics
approach to analyze serum samples from 21 PC patients and 9
healthy volunteers. PC patients, at early (I) and advanced (III, IVA
and IVB) stages of the disease, were shown to have significant
differences in the metabolome compared to the control group,
showing a variation in the profile of 18 out of 60 identified metab-
olites [45]. The authors later confirmed these data, creating a ser-
um metabolomics-based diagnostic model for detecting
resectable PC patients [41].
Although some published metabolome-profiling studies of pan-
creatic carcinoma are heterogeneous regarding the MS techniques
used and the metabolites studied, they all share canonical vari-
ance-based evaluation strategies with two classes (PC and healthy
subjects) or three classes (PC, CP and healthy subjects) (Table S2)
for comparison and make use of multivariate statistical analysis
of the data.
To date, only a few applications of a CE-MS based approach in
PC have been published. Using CE-TOF-MS, it was recently demon-
strated that the salivary metabolomics profile of a small number of
patients with PC was significantly different from normal controls
[56]. pCEC coupled with UV detection was instead used by Zhang
and co-workers [49] to study the plasma samples of 17 PC patients.
Metabolomics profiling, analyzed using OPLS-DA multivariate
analysis, showed different metabolite phenotypes for the PC group
and healthy controls [49]. These results suggest that pCEC can be
considered a promising technique, which, when coupled with
MS, could serve as a more powerful platform for disease-biomarker
discovery.
5. Sample preparation in approaches using metabolomics
The quality of sample preparation is an essential factor for the
success of all analytical procedures. When working with biological
matrices, such as serum, plasma, whole blood, tissue homogenates,
saliva, urine, cell pellets or cell media, analyte extraction usually
becomes very sensitive because the analytical procedure may be
affected by interference from matrix components [76]. In the met-
abolomics approach, all small molecules are the targets and only
salts and macromolecules, such as proteins or larger peptides,
can be considered as matrices. Sample preparation for the analysis
of biological samples can include liquid-liquid extraction (LLE), so-
lid-phase extraction (SPE), supercritical fluid extraction, acceler-
ated solvent extraction, protein precipitation and membrane
methods, such as dialysis or ultracentrifugation [33]. In the case
of low-abundance metabolites, the extraction procedure can
include a pre-concentration step to achieve the detection limits
for the analytical technique applied [76].
Since the polarity of biological metabolites varies widely, opti-
mization of extraction and analytical methodology is extremely
important to recover and to detect as many metabolites as possi-
ble. Moreover, sample preparation should be highly reproducible,
effective, and fast to allow high-throughput studies [77].
102 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
Nevertheless, it should be noted that any kind of sample prep-
aration would cause analyte losses. In particular, extraction proce-
dures, such as SPE or LLE, when applied to aqueous samples, result
in poor recovery of very polar compounds [33].
In the case of an NMR metabolomics-based approach for clinical
diagnostics, the advantage is that only minimal sample preparation
is necessary and it can be applied to different matrices [78]. How-
ever, some factors may affect the NMR spectra (i.e. chemical-shift
signal perturbation or inconsistency of the peak position) in a
way that subsequently decreases the efficacy of multivariate statis-
tical analysis of the data. Careful sample preparation can help to
limit peak shifts (e.g., by attempting to control the pH of biological
samples). In PC, many studies performed using NMR for serum or
plasma [43,44] and urine [46,47] treat the specimens simply by
adding NaOH or HCl [46] or phosphate-buffer solution [47] to min-
imize variations in NMR chemical shifts of metabolites arising from
differences in pH.
NMR analysis was done after derivatization with phenyl
hydrazine in order to obtain target analysis for detecting levels of
D-glucuronic acid in PC patients [53].
Analysis of volatile compounds, which can be found in urine,
plasma or exhaled air, as an important component of the metabo-
lome, is usually performed using GC-MS.
Conventional sample preparation techniques (e.g., LLE or SPE)
are often infeasible, because the analytes may be incompletely ex-
tracted and losses will occur if the extract is concentrated. More-
over, the solvent can interfere with the analytes during GC
separation, due to incomplete separation of the solvent peak from
the analyte peaks. In order to analyze organic acids, for example,
Wahl et al. [79] used headspace sampling with a gas-tight syringe,
combined with cold trapping in a temperature-controlled cold-
injection system and GC-MS for the analysis of volatile metabolites
in urine samples.
Non-polar lipid classes have also been analyzed in PC patient
biofluids using GC separation and MS, with a derivatization step
(e.g., methylation, silylation or oximation) necessary to expand fur-
ther the range of lipids accessible with this technique [41,45,67].
However, GC–MS analysis is limited to thermally-stable com-
pounds with sufficient vapor pressure for volatilization during
injection.
LC–MS experiments are often affected by deviations in reten-
tion time from sample to sample, as a consequence of column deg-
radation or sample carry-over, small fluctuations in room
temperature or mobile-phase pH, as well as other variations. Be-
fore injection, serum or plasma samples should usually be treated
with organic solvent (acetonitrile or methanol) for precipitation
protein [42,74]. Furthermore, the supernatants are sometimes
derivatized to carry out direct-injection analysis of the metabolites
concerned {e.g., amino acids [42]}, or extracted with chloro-
form:methanol mixtures for the analysis of hydrophobic com-
pounds {e.g., phospholipids [74]}.
Urine samples are usually diluted, because they contain a high
concentration of salt, which can cause ion suppression and adduct
formation in the electrospray process and may also cause rapid
deterioration of instrumental performance, due to contamination
by non-volatile residues.
For the treatment of in-vitro cell or human-tissue samples, the
procedure usually requires washing and quenching with ice-cold
methanol and the use of trypsin or cell scrapers in order to remove
adherent cells from the growth surface [20,65]. A series of freeze-
thaw cycles for metabolite extraction needs to be performed in the
solution collected and, following high-speed centrifugation,
analysis can be carried out in the supernatant [80]. A similar pro-
tocol can be applied to the preparation of tissue samples, where
freeze-thaw cycles are used in the process of tissue
homogenization.
The sample-preparation procedures used in PC metabolomics
studies are summarized in Table 1.
6. Metabolomics data export and analysis
Metabolomics is not limited to individual biomarkers, but rep-
resents a new approach to diagnostics, in which large overall data-
sets can be employed to develop new models. Both metabolic
fingerprinting and profiling can be used in the search for new
biomarkers.
The metabolic fingerprinting approach usually allows detection
of all metabolites in biological samples, with subsequent classifica-
tion into different groups and identification of differentially-ex-
pressed analytes among them. To perform this complex task,
data-analysis tools, metabolite libraries and databases are
required.
The first step in data export is to convert analytical data from
metabolomics-based experiments into a standard, uniform format
[34], which includes data pre-processing in order to reduce noise
and background, alignment of the spectra from NMR or MS and
automated picking and annotation of MS or total-ion/extracted-
ion chromatographic peaks of metabolites [81]. The data may
eventually be pre-treated (e.g., centering, and scaling) [82], and
finally processed using multivariate statistical analysis, which
facilitates data visualization through data-dimensionality reduc-
tion, and highlights relevant biological information [83].
However, for metabolomics-data analysis, it is necessary to
extract information about all compounds, including mass and
retention time in the case of chromatography, compound identifi-
ers and intensity (i.e. chromatographic height or area) as quantita-
tive representations of concentration. The process is inherently
difficult for the processing of an untargeted dataset, due to the
greater complexity of this approach, in particular as regards data
alignment, data normalization and annotation [33].
Finally, raw data from each experiment are stored in sample
files as spectra acquired at given time points or scans. There are
currently several public and proprietary software tools available
to carry out data export and subsequent statistical analysis.
Multivariate statistical techniques are the most useful tools for
investigating this array of information efficiently, by reducing the
dimensionality of complex datasets. They generally fall into two
categories of analysis – supervised and unsupervised – which
should be selected according to the aim of the study [33].
If the aim is sample classification and prior information about
the sample identity is unknown, unsupervised methods are used
[e.g., hierarchical clustering analysis (HCA), principal-component
analysis (PCA), or independent component analysis (ICA)] [35].
However, if sample identity is known, and the aim of the study
is to discover characteristic biomarkers (e.g., in order to search for
biomarkers of PC by comparing samples from healthy and diseased
subjects), supervised methods [e.g., generally discriminant analysis
(DA)], using prior information about sample class may be suitable.
In this case, particular care must be taken to ensure an appropri-
ately large number of observations, in order to reduce the possibil-
ity of generating false positives using supervised methods [63].
In any case, data mining of metabolomics datasets is generally a
complex procedure, initially requiring unsupervised methods to
investigate the underlying data structure, unbiased by knowledge
of experimental design [84]. The most popular unsupervised
data-analysis tool is PCA. Subsequent analysis of the metabolome
can assist in understanding the link between factors (e.g., genetic
mutation, natural variation, environmental conditions and the
overall characteristics of the subjects being considered).
The application of these technologies in cancer research has re-
vealed system-wide alterations of unexpected metabolic pathways
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116 103

Table 1
Metabolite-extraction techniques employed in metabolomics studies of pancreatic cancer

Sample type Authors Platforms used Sample preparation procedure Metabolites

Cell cultures
[20] LC-MS/MS (MRM) Centrifuge medium of cells and add internal standard solution (in ACN with 25 enzyme & transporter proteins
2% formic acid). Wash cells with PBS and lyse with 0.2N NaOH. Neutralize the and 6 metabolites
lysate with 1N HCl. Centrifuge, dry the supernatant and reconstitute in 0.1%
formic acid in water for analysis
[65] UHPLC-QTOF-MS Four different extraction methods investigated on the Panc-1 cells to compare Amino acids (AA), fatty acids, organic
(RPLC & HILIC) the efficiency of intracellular metabolite extractions: A) MeOH extraction acids
procedure; B) ACN extraction procedure; C) MeOH/chloroform/H2O
extraction; D) MeOH/chloroform/ACN extraction
Serum/plasma
[41] GC-MS As described by Niushiumi et al. 2010 [45] AA, fatty acids, organic acids and
sugars
[42] FIA-MS/MS Add methanol for protein precipitation and centrifuge. Dilute Supernatant 26 amino acids
with the internal standard solution. Evaporate and derivatise with 3n
butanolic-HCl. Evaporate the residual solution and reconstitute with the
mobile phase (1/1 v/v isopropanol/water)
1
[43] H-NMR Mix phosphate buffer solution (0.2 M Na2HPO4/0.2 M NaH2PO4, pH 7.4, 100% AA, trigliceride, TMAO,
spectroscopy D2O), add internal standard solution and pipe into NMR tube hydroxybutyrate, 3-
hydroxyisovalerate, etc.
1
[44] H-NMR N/A 58 metabolites (sugars, AA,
spectroscopy acylcarnitines, fatty acids)
[45] GC/MS Add solvent mixture (MeOH:H2O:CHCl3=2.5:1:1) with internal Standard, AA, fatty acids, organic acids (178
shake for 30 min at 37°C, centrifuge and transfer supernatant to a clean tube, compounds knowns and unknowns)
adding distilled water. Mix and centrifuge. Lyophilize the resultant
supernatant using a freeze dryer. Oximate using methoxyamine
hydrochloride dissolved in pyridine and shake. Add N-methyl-N-
trimethylsilyl-trifluoroacetamide (MSTFA) for derivatization incubing for
30 min at 37°C. Centrifuge and analyse the resultant supernatant
[48] GC/TOF-MS & Add methanol and homogenize the mixture, sonicate, extracte in the dark and AA, bile acids, organic acids, lipids
HPLC/ESI-MS centrifuge (13000 rpm  5 min). Transfer the supernatant to a fresh tube and
dry. Reconstitute the samples in water/acetonitrile (1:1, v/v) and transfer to a
vial with an insert and analyse
[49] pCEC-UV Mix with a solution of MeOH, ACN and acetone (1:1:1; v/v/v), to precipitate Unknowns
proteins. Kept the mixture at 4°C for 10min and then centrifuge at 13000 rpm
for 12 min. Transfer the supernatant into a clean tube and evaporate to
dryness under nitrogen. Reconstitute the dried extract in 70 lL of 50% MeOH
v/v to perform pCEC- UV analysis
[55] FI-MS/MS & Prepare serum samples for FI-FTICR-MS analysis by sequential twice with Fatty acids, phospholipids
FI-FTICR-MS EtOAc. Extract the remaining aqueous component twice with water and
remove protein by precipitation with 3:1 acetonitrile (extract B). Evaporate
under nitrogen a 1:5 ratio of EtOAc to butanol (BuOH) to the original BuOH
starting volume (extract C). Store all extracts at 80°C until analysis
Urine
1
[46] H-NMR Add a chemical shift standard (containing 5.046 mM Sodium 2,2- dimethyl-2- 59 metabolites (sugars, AA,
spectroscopy silapentane-5-sulfonate-d6 (DSS-d6) and 0.2 % NaN3 in 99.8 % w/v D20) and acylcarnitines, fatty acids)
adjuste the pH using NaOH or HCl to obtain a final pH of 6.75 ± 0.05. Transfer
to a NMR tube and analyze
1
[47] H-NMR Add potassium phosphate buffer (1.5 M K2HPO4 in 100% 2H2O, pH 7.4) Acetylated compounds, organic
spectroscopy containing 0.1% sodium 3-(trimethylsilyl)-[2,2,3,3-2H]propionate (TSP) and acids, AA, bile salts
2 mM sodium azide Centrifuge to remove any solid debris and transfer into a
NMR tube
Bile
1
[53] H-NMR Transfer to a NMR tube along with a reusable co-axial Capillary tube d-Glucuronic acid (GlcU)
spectroscopy containing TSP in D2O. Derivatization with phenylhydrazine, incubation 1 hr
at 37°C vortexing, and dilution with Millipore water
Saliva
[56] CE-TOF-MS Frozen saliva was thawed and dissolved at room temperature. Add water 48 metabolites: AA, fatty acids,
containing internal standards, mix well and analyze carnitines, polyamines, purine,
amino alchols, etc.
Tissue
[62] GC/MS & UHPLC- Remove the protein fraction using aqueous methanol extraction Process and 55 metabolites: lipids, fatty acids
MS/MS divide extract into four fractions: for +/ mode analysis by UPLC/MS/MS, for
GC/MS analysis and for backup. Place samples on a TurboVapÒ (Zymark) to
remove the organic solvent, then froze and dry under vacuum. Reconstitute
the extract for the appropriate analysis

N/A: not available.

FI-FTI CR-MS: Flow – injection Fourier transform ion cyclotron resonance mass spectrometry.

related to phenotypic perturbations [72]. Wen et al. [59] applied
NMR-based metabolomics to bile collected from individuals with
biliary tract cancer or benign biliary tract disease, including pat-
tern recognition and multivariate data analysis to note significant
differences between individuals with cancer and benign diseases,
compared to other conventional approaches.
A previous approach to classification of premalignant PC was
performed by Ge and Wong [85] using three feature-selection
schemes (Student t test, Wilcoxon rank sum test and genetic algo-
rithm) to compensate for systematic differences due to sample
loadings and instrument errors and to reduce the dimensionality
of the proteomic dataset to a manageable number [85].
104 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116

Table 2
Metabolites found differentially expressed in PC metabolomics studies included in this comprehensive review. The table shows chemical formula, molecular weight, PubChem ID,
platform(s) and matrix used for the identification of each compound

Metabolite Chemical Molecular Pubchem ID Matrix NMR GC-MS LC- CE- Fold inductiona
formula weight MS MS
Amino acids and
Peptides
Alanine (Ala) C3H7NO2 89.0932 CID 5950 Plasma [50] ;
Serum [44] [41,45] [42] 1.23 [41]
Saliva [56] 3.67
Urine [47] M
Arginine (Arg) C6H14N4O2 174.2010 CID 6322 Serum [44] [42] " [44]
Asparagine (Asn) C4H8N2O3 132.1179 CID 6267 Serum [44] [41,45] ; (x0.87) [41]; " (x1.35)
[45]; ; [44]
Aspartic acid (Asp) C4H7NO4 133.1027 CID 424 Serum [44] [41,45] [42] 0.98 [41]
C4H7NO4 133.1027 CID 424 Saliva [56] 4.1
Beta-Alanine (b-Ala) C3H7NO2 89.0932 CID 239 Saliva [56] 3.04
3-Methylhistidine (MeHis) C7H11N3O2 169.1811 CID 64969 Serum [42]
Urine [47] M
4-Hydroxyproline C5H9NO3 131.1299 CID 5810 Serum [41,45] 1.17 [41]
5-Oxoproline (Pyroglutamic acid) C5H7NO3 129.1140 CID 7405 Serum [44] [45]
Alpha-Aminobutyric acid C4H9NO2 103.1198 CID 80283 Saliva [56] 4.01
Urine [46] ""
Carnosin (Carn) C9H14N4O3 226.2325 CID 439224 Serum [42]
Citrulline (Cit) C6H13N3O3 175.1857 CID 9750 Saliva [56] 3.1
Serum [42]
Creatine C4H9N3O2 131.1332 CID 586 Plasma [50]
Serum [44] ;
Cysteine + Cystine CID 5862 + CID Serum [41] 1.02
67678
Gamma-Aminobutyric acid (GABA) C4H9NO2 103.1198 CID 119 Saliva [56] 3.31
Glutamic acid (Glu) C5H9NO4 147.1293 CID 33032 Plasma [50] ;
Serum [44] [41,45] [42] "" [44]; 1.33 [41]
Saliva [56]
Glutamine (Gln) C5H10N2O3 146.1445 CID 5961 Serum [44] [41,45] [42] ; (x0.86) [41]; ; [44]
C5H10N2O3 146.1445 CID 5961 Plasma [50] [48] ; (x1.20) [48]; ; [50]
C5H10N2O3 146.1445 CID 5961 Saliva [56] 4.96
Glutathione C10H17N3O6S 307.3235 CID 124886 Tissue [57] ; (x0.6)
Glycine (Gly) C2H5NO2 75.0666 CID 750 Serum [44] [41,45] [42] 0.98 [41]; ; (x0.76) [45]
Urine [47] ;
Saliva [56] 3.1
Glycylglycine (Gly-Gly) C4H8N2O3 132.1179 CID 11163 Serum [41] 0.76
Histidine (His) C6H9N3O2 155.1546 CID 6274 Plasma [50] ;
Serum [44] [41] [42] ; (x0.7) [41]
Saliva [56] 2.02
Hippuric acid C9H9NO3 179.1727 CID 464 Urine [47] ;
Hydroxyproline (trans) C5H9NO3 131.1299 CID 5810 Serum [42]
Isoleucine (Ile) C6H13NO2 131.1729 CID 6306 Serum [43,44] [45] ;; [34]
Plasma [50] ;
Leucine (Leu) C6H13NO2 131.1729 CID 6106 Serum [43,44] [45] " [34]
Urine [47] "
Plasma [50] ;
Leucine + Isoleucine (Leu+Ile) CID 6106 + CID Saliva [56] 7.71
6305
Serum [42]
Lysine (Lys) C6H14N2O2 146.1876 CID 5962 Plasma [50] [48] " (x1.03) [48]; ; [50]
Serum [44] [41] [42] ; (x0.78) [41]; ; [44]
Saliva [56] 3.97
Methionine (Met) C5H11NO2S 149.2113 CID 6137 Serum [44] [41,45] [42] ; (x0.86) [41]
N-Acetyltyrosine C11H13NO4 223.2252 CID 89216 Serum [45] " (x1.57)
N-Acetylglutamic acid (NAG) C7H11NO5 189.1659 CID 70914 Plasma [50] "
N-Methylalanine C4H9NO2 103.1198 CID 5288725 Plasma [48] "" (x2.81)
Ornithine (Orn) C5H12N2O2 132.1609 CID 6262 Serum [44] [41] [42] 1.11 [41]
Saliva [56] 1.97
Phenylalanine (Phe) C9H11NO2 165.1891 CID 6140 Serum [44] [41,45] [42] 0.92 [41]; " [44]
Plasma [50] [48] ; (x1.15) [48]
Saliva [56] 3.54
Pro-Gly-Pro or Pro-Pro-Gly Saliva [56] 4.27
Proline (Pro) C5H9NO2 115.1305 CID 8988 Serum [44] [41,45] [42] 0.88 [41]; ; [44]
Saliva [56] 3.99
Sarcosine (Sarc) C3H7NO2 89.0932 CID 1088 Serum [42]
Serine (Ser) C3H7NO3 105.0926 CID 5951 Serum [44] [45] [42]
CID 5951 Saliva [56] 4.34
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116 105

Table 2 (continued)

Metabolite Chemical Molecular Pubchem ID Matrix NMR GC-MS LC- CE- Fold inductiona
formula weight MS MS
Amino acids and Taurine (Tau) C2H7NO3S 125.1469 CID 1123 Serum [42]
Peptides
Urine [46] "
Saliva [56] 0.376
Tissue [57] " (x1.6)
Threonine (Thr) C4H9NO3 119.1192 CID 6288 Serum [44] [41,45] [42] ; (x0.86) [41]; ; [44];
Urine [46] "
Saliva [56] 4.75
Tryptophan (Trp) C11H12N2O2 204.2252 CID 6305 Serum [44] [45] [42] ; [44]
Urine [46] "
Saliva [56] 6.47
Tyrosine (Tyr) C9H11NO3 181.1885 CID 6057 Serum [44] [41,45] [42] ; (x0.84) [41];
Plasma [50]
Saliva [56] 2.9
Valine (Val) C5H11NO2 117.1463 CID 6287 Serum [44] [41,45] [42] ; (x0.81) [41]
Plasma [50] ;
Urine [47] M
Saliva [56] 5.92
Lipids
Cardiolipin C81H158O17P2 1466.0585 CID 449005 Plasma [56] M
Glycerophosphocholine C8H20NO6P 257.2213 CID 657272 Saliva [56] 0
Glyceryl of lipids Plasma [50] "
LDL, -CH3-(CH2)n Plasma [50] ;
Lipid, -CH2-C=O Plasma [50]
Lipid, -CH2-CH=CH Plasma [50]
LysoPC(16:0) (Palmitoyl C24H50NO7P 495.6301 CID 86554 Plasma [48] " (x1.33)
lysophosphatidyl choline)
LysoPC(18:2) (1-18:2- C26H50NO7P 519.6515 CID 11988421 Plasma [48] " (x1.59)
lysophosphatidylcholine)
Posphatidylcholine PC (34:2) C42H80NO8P 758.0603 CID 6021688 Plasma [48] " (x1.32)
Posphatidylethanolamine, PE(12:0/ C31H62NO8P 607.7996 CID 9546763 Plasma [48] " (x1.81)
14:0)
Posphatidylinositol PI (18:0,18:2) Plasma [56] ;
m/z 861
Posphatidylinositol PI (18:0,20:2) Plasma [56] ;
m/z 887
Posphatidylinositol PI (18:0,20:4) Plasma [56] ;
m/z 885
Posphatidylserine C13H24NO10P 385.3041 CID 44147587 Plasma [56] M
Triglycerides Serum [43] ""
VLDL, -CH2-CH2-C=O Plasma [50] "
VLDL, -CH3-(CH2)n Plasma [50] "
VLDL, -CH3-(CH2)n Plasma [50] "
Fatty acids
2-Aminobutyrate (Butyric acid) C4H9NO2 103.1198 CID 517460 Serum [44]
2-Phenylacetamide C8H9NO 135.1632 CID 7680 Urine [47] "
2-Hydroxybutyric acid C4H8O3 104.1045 CID 11266 Serum [44] [41,45] 0.97 [41]
2-Hydroxyglutaric acid C5H8O5 148.1140 CID 43 Serum [45]
2-Hydroxyisobutyric acid C4H8O3 104.1045 CID 11671 Serum [44]
Urine [46,47] " [46]; M [47]
2-Hydroxyisovaleric acid C5H10O3 118.1311 CID 99823 Serum [44] [45]
3-Hydroxybutyric acid C4H8O3 104.1045 CID 441 Serum [43,44] [41,45] ;; [43]; " [44]; 1.7 [41];
Plasma [50] ;
3-Hydroxyisobutyric acid C4H8O3 104.1045 CID 87 Serum [44] [45] " [44]
3-Hydroxyisovaleric acid C5H10O3 118.1311 CID 69362 Serum [43,44] ;; [43]
Urine [47] ;
3-Hydroxyphenylacetic acid C8H8O3 152.1473 CID 12122 Serum [45]
3-Hydroxypropionic acid C3H6O3 90.0779 CID 68152 Serum [45]
3-Methyl-2-oxovaleric acid C6H10O3 130.1418 CID 47 Serum [44] "
4-Hydroxyphenylacetic acid C8H8O3 152.1473 CID 127 Serum [45]
Urine [46,47] " [46]; M [47]
7-Hydroxyoctanoic acid C8H16O3 160.2108 CID 167627 Serum [45] " (x1.38)
Acetic acid C2H4O2 60.0520 CID 176 Serum [44]
Arachidonic acid C20H32O2 304.4669 CID 444899 Plasma [48] " (x1.49)
Decanoic acid C10H20O2 172.2646 CID 2969 Serum [45] ; (x0.76)
Isobutyric acid (2-Methylpropionic C4H8O2 88.1051 CID 6590 Serum [44]
acid)
Urine [47] M
Isovaleric acid (3-Methylbutyric C5H10O2 102.1317 CID 10430 Serum [44]
acid)
Lauric acid (n-Dodecanoic acid) C12H24O2 200.3178 CID 3893 Serum [41,45] 0.79 [41]; ; (x0.76) [45]
Margaric acid (n-Heptadecanoic C17H34O2 270.4507 CID 10465 Serum [45] ; (x0.80)
acid)

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106 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116

Table 2 (continued)

Metabolite Chemical Molecular Pubchem ID Matrix NMR GC-MS LC- CE- Fold inductiona
formula weight MS MS
Fatty acids Myristic acid (n-Tetradecanoic C14H28O2 228.3709 CID 11005 Serum [45] ; (x0.77)
acid)
N-Caprylic acid (n-Octanoic acid) C8H16O2 144.2114 CID 379 Serum [41,45] ; (x0.77) [41]; ; (x0.73)
[45]
Nonanoic acid (C9) C9H18O2 158.2380 CID 8158 Serum [41] ; (x0.53)
Palmitic acid (Hexadecanoic acid) C16H32O2 256.4241 CID 985 Serum [45] ; (x0.79)
Palmitoleic acid (cis-9- C16H30O2 254.4082 CID 445638 Serum [45]
Hexadecenoic acid)
Stearic acid (n-Octadecanoic acid) C18H36O2 284.4772 CID 5281 Serum [45] ; (x0.74)
Suberic acid C8H14O4 174.1944 CID 10457 Serum [44]
Sterols
Cholesterol C27H46O 386.6535 CID 5997 Plasma [48] " (x1.85)
HDL, cholesterol Plasma [50] ;
Bile acids
Bile salts Urine [47] M
Glycocholic Acid (Cholylglycine) C26H43NO6 465.6227 CID 23617285 Plasma [48] "" (x2.61)
Glycodeoxycholic Acid C26H43NO5 449.6233 CID 22833539 Plasma [48] " (x1.31)
(Deoxycholylglycine)
Taurocholic acid C26H45NO7S 515.7030 CID 6675 Plasma [48] " (x1.75)
Tauroursodeoxycholic acid C26H45NO6S 499.7036 CID 3034759 Plasma [48] "" (x2.01)
(TUDCA)
Thiodiglycolic acid (TDGA) C4H6O4S 150.1530 CID 31277 Serum [45] "" (x6.27)
Other organic acids
2-Oxoisocaproate (Alpha- C6H10O3 130.1418 CID 70 Serum [44]
ketoisocaproic acid)
Abscisic acid C15H20O4 264.3169 CID 5280896 Serum [42]
Acetoacetic acid C4H6O3 102.0886 CID 96 Urine [47] "
Aconitic acid (Cis) C6H6O6 174.1082 CID 643757 Serum [45] " (x1.54)
Urine [46] "
Aconitic acid (Trans) C6H6O6 174.1082 CID 444212 Urine [46] "
Adipic acid C6H10O 146.1412 CID 196 Serum [44]
Citraconic acid C5H6O4 130.0987 CID 643798 Serum [45]
Citric acid C6H8O7 192.1235 CID 311 Plasma [50] ;
Serum [44] [45]
Urine [47] ;
Citric acid + Isocitric acid CID 311 + CID Serum [41] 1.1
5318532
Formic acid CH2O2 46.0254 CID 284 Serum [44] "
Plasma [50]
Urine [47] M
Fumaric acid C4H4O4 116.0722 CID 444972 Serum [45]
Glucuronic acid (GlcU) C6H10O7 194.1394 CID 444791 Serum [41] 4.57
Bile [53] ""
Glucuronolactone C6H8O6 176.1241 CID 92283 Serum [45]
Glutaconic acid C5H6O4 130.0987 CID 5280498 Serum [45]
Glyceric acid C3H6O4 106.0773 CID 752 Serum [45] ; (x0.53)
Glycolic acid C2H4O3 76.0514 CID 757 Serum [41,45] 0.97 [41]
Glyoxylic acid C2H2O3 74.0355 CID 760 Serum [45]
Homogentisic acid C8H8O4 168.1467 CID 780 Serum [45] " (x1.26)
Hydrocinnamic acid (3- C9H10O2 150.1745 CID 107 Plasma [48] ; (x1.38)
phenylpropionic acid)
Isocitric acid C6H8O7 192.1235 CID 1198 Serum [45]
Lactic acid (2-hydroxypropionic C3H6O3 90.0779 CID 612 Serum [43,44] [41,45] 1.14 [41]; ; [43]; "
acid) (x1.48) [45]
Plasma [50] ;
Urine [47] M
Malic acid C4H6O5 134.0874 CID 525 Tissue [57] ; (x0.3)
Malonic acid C3H4O4 104.0615 CID 867 Serum [44] [45]
Methylsuccinic acid C5H8O4 132.1146 CID 10349 Serum [44]
Oxalic acid C2H2O4 90.0349 CID 971 Serum [45]
Pyruvic acid C3H4O3 88.0621 CID 1060 Serum [44] [45]
Pyruvic acid + Oxalacetic acid CID 1060 + CID Serum [41] 1.13
970
Succinic acid C4H6O4 118.0880 CID 1110 Serum [44]
Tissue [57] ; (x0.3)
Tartaric acid C4H6O6 150.0868 CID 875 Serum [45]
Carbohydrates
Sugars
1,5-Anhydro-D-glucitol C6H12O5 164.1565 CID 64960 Serum [41] ; (x0.55)
Arabinose C5H10O5 150.1299 CID 66308 Serum [41] 1.75
Arabitol C5H12O5 152.1458 CID 94154 Serum [41] 0.96
Erythritol (Meso-erythritol) C4H10O4 122.1198 CID 222285 Plasma [48] " (x1.53)
CID 222285 Serum [41] 2.48
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116 107

Table 2 (continued)

Metabolite Chemical Molecular Pubchem ID Matrix NMR GC-MS LC- CE- Fold inductiona
formula weight MS MS
Carbohydrates Fucose C6H12O5 164.1565 CID 19466 Urine [46] ""
Glucose C6H12O6 180.1559 CID 5793 Urine [46,47] " [46,47]
Serum [44] [41] 1.05 [41]; " [44]
Glucose-a C6H12O6 180.1559 CID 79025 Plasma [50]
Glucose-b C6H12O6 180.1559 CID 64689 Plasma [50]
Inositol C6H12O6 180.1559 CID 892 Serum [41] 1.13
Mannose C6H12O6 180.1559 CID 18950 Serum [44] [41] 0.97 [41]; " [44]
Ribulose C5H10O5 150.1299 CID 151261 Serum [41] 1.39
Xylitol C5H12O5 152.1458 CID 6912 Serum [41] 0.75
Xylose C5H10O5 150.1299 CID 644160 Urine [46] "
Nucleotides and nucleosides
50 -AMP (Adenosine C10H14N5O7P 347.2212 CID 6083 Tissue [57] ; (x0.5)
50 -monophosphate)
50 -UMP (Uridine C9H13N2O9P 324.1813 CID 6030 Tissue [57] ; (x0.4)
50 -monophosphate)
Adenine C5H5N5 135.1267 CID 190 Urine [47] M
Hypoxanthine C5H4N4O 136.1115 CID 790 Serum [44]
Urine [46] ""
Saliva [56] 3.35
Inosine (Hypoxanthosine) C10H12N4O5 268.2261 CID 6021 Plasma [48] ; (x1.40)
Nicotinamide adenine dinucleotide C21H27N7O14P2 663.4251 CID 5288979 Tissue [57] ; (x0.08)
(NAD)
UDP-N-acetyl-d-glucosamine C17H27N3O17P2 607.3537 CID 445675 Tissue [57] ; (x0.3)
(UDP-GlcNAc)
Uridine C9H12N2O6 244.2014 CID 6029 Tissue [57] ; (x0.1)
Chemicals and Drugs
Category
Alcohols
Ethanol C2H6O 46.0684 CID 702 Serum [44] ;
Glycerol C3H8O3 92.0938 CID 753 Serum [44] [41,45] 0.86 [41]; ; [44]
Methanol CH4O 32.0419 CID 887 Urine [46] ;
Serum [44] ;
Propylene Glycol C3H8O2 76.0944 CID 1030 Serum [44]
Acetylated compounds
Acetylated compounds Urine [47] "
O-Acetylcarnitine C9H17NO4 203.2356 CID 18230 Serum [44]
Urine [46] "

Amines
Betaine C5H11NO2 117.1463 CID 247 Serum [44]
Saliva [56] 0.822
Carnitine C7H15NO3 161.1989 CID 10917 Serum [44]
Saliva [56] 1.88
Choline C5H14NO+ 104.1708 CID 305 Serum [44]
Urine [46] "
Saliva [56] 2.63
Cadaverine C5H14N2 102.1781 CID 273 Saliva [56] 6.15
Dimethylamine (DMA) C2H7N 45.0837 CID 674 Serum [44] "
Urine [46,47] " [46]; M [47]
Plasma [50] "
Ethanolamine C2H7NO 61.0831 CID 700 Serum [41] 0.91
Saliva [56] 4.35
O-Phosphoethanolamine C2H8NO4P 141.0630 CID 1015 Serum [41] 0.77
Putrescine C4H12N2 88.1515 CID 1045 Saliva [56] 3.98
Trimethylamine C3H9N 59.1103 CID 1146 Saliva [56] 4.13
Trimethylamine-N-oxide (TMAO) C3H9NO 75.1097 CID 1145 Urine [46] ""
Serum [43] ;
Urine [47] M
Tryptamine C10H12N2 160.2157 CID 1150 Plasma [48] ; (x1.07)
Heterocyclic
Compounds
1-Methylnicotinamide C7H9N2O+ 137.1592 CID 457 Urine [46,47] "" [46]; M [47]
4-Pyridoxic acid C8H9NO4 183.1614 CID 6723 Urine [46] "
Serum [44] ;
Creatinine C4H7N3O 113.1179 CID 588 Serum [43,44] [41] 0.82 [41]; " [43]
Urine [47] ;
Indolelactic acid C11H11NO3 205.2099 CID 92904 Serum [45]
Methylimidazoleacetic acid C6H8N2O2 140.1399 CID 75810 Saliva [45] [56] 4.64
Pipecolic acid (PiPA) C6H11NO2 129.1570 CID 849 Serum [42]
Saliva [56] 1.11
Piperidine C5H11N 85.1475 CID 8082 Saliva [56] 3.73
Pyridine (1-Piperideine) C5H9N 83.1317 CID 68161 Saliva [56] 0.665
Pyrroline hydroxycarboxylic acid C5H7NO3 129.1140 CID 1059 Saliva [56] 2.28

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108 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116

Table 2 (continued)

Metabolite Chemical Molecular Pubchem ID Matrix NMR GC-MS LC- CE- Fold inductiona
formula weight MS MS
Heterocyclic Trigonelline C7H7NO2 137.1360 CID 5570 Urine [46,47] ; [46,47]
Compounds Urea CH4N2O 60.0553 CID 1176 Serum [44] [45] ; [44]; ; (x0.80) [45]
Uric acid C5H4N4O3 168.1103 CID 1175 Serum [45] 0.85 [41]; ; (x0.61) [45]
Inorganic chemicals
Phosphate (Triphosphoric acid) H5O10P3 257.9550 CID 983 Serum [41] 0.93
Phosphoric acid H3O4P 97.9952 CID 1004 Serum [45]
[45]
Neurotransmitter Agents
Burimamide C9H16N4S 212.3151 CID 3032915 Saliva [56] 2.56
Organic chemicals
4-Cresol (para-Cresol) C7H8O 108.1378 CID 2879 Serum [45]
Acetone C3H6O 58.0791 CID 180 Plasma [50] [45] "
Urine [46] ""
Serum [44] "
Pyrogallol (Pyrogallic acid) C6H6O3 126.1100 CID 1057 Serum [41] 0.71
Unknowns
compounds
C17H26N4O5 Saliva [56] 2.69
C18H32N6O6 Saliva [56] 6.7
C2H6N2 Saliva [56] 4.42
C30H55N27O3S Saliva [56] 5.56
C30H62N19O2S3 Saliva [56] 6.65
C32H48O13 Saliva [56] 2.03
C3H7NO2 Saliva [56] 3
C4H12N5 Saliva [56] 6.98
C4H5N2O11P Saliva [56] 0
C4H7N Saliva [56] 1.14
C4H9N Saliva [56] 2.7
C4H9NO2 Saliva [56] 5.16
C5H11NO2 (10.05 min.) Saliva [56] 2.1
C5H11NO2 (13.42 min.) Saliva [56] 2.17
C5H14N5 Saliva [56] 5.35
C6H6N2O2 Saliva [56] 2.89
C7H12N2O3 Saliva [56] 2.48
C7H8O3S Saliva [56] 0.592
C8H9N Saliva [56] 0.817
Unknown 132.0773 m/z Tissue [57] 4.4
Unknown 187.1201 m/z Plasma [48] ; (x1.11)
Unknown 188.1283 m/z Tissue [57] 2.0
Unknown 236.9944 m/z Tissue [57] 4.3
Unknown 265.0721 m/z Plasma [48] " (x1.17)
Unknown 287.2436 m/z Tissue [57] 87.9
Unknown 332.0745 m/z Plasma [48] " (x1.37)
Unknown 366.1397 m/z Tissue [57] 14.1
Unknown 414.1563 m/z Plasma [48] " (x1.69)
Unknown 459.2029 m/z Tissue [57] 3.52
Unknown 523.9791 m/z Tissue [57] 73.1
Unknown 633.1954 m/z Plasma [48] " (x1.80)
Unknown 753.1246 m/z Plasma [48] " (x1.27)

The citation number is listed in the column under the platform used to detect each metabolite.
Some metabolites were identified by more platforms in different matrices and each author reported different value of fold induction.
a
Fold induction values are reported as calculated from the PC to controls ratio. Data were not reported when not available in the study.

A recent study, conducted using an intrinsic three-class bioin-
formatics approach, allowed comparison of the metabolomics pro-
files of patients with pancreatitis and pancreatic carcinoma,
evaluating their selectivity and computing multi-marker panels
combined with the conventional tumor marker CA 19-9 to differ-
entiate PC from various benign lesions [42].
7. Commercial software and library sources
One of the major challenges of informatics is to provide tools that
link metabolomics data with other types of high-throughput molec-
ular data (e.g., transcriptomics, genomics and proteomics) and incor-
porate prior knowledge of pathways and molecular interactions.
While many tools have been developed for the analysis of gene-
expression and proteomics data, to date, there are few tools that
allow the user to analyze metabolomics data and to link them with
other ‘omics’ data [86].
General information about the physical and chemical properties
of metabolites can be obtained by searching general chemical
databases (e.g., PubChem, ChemSpider or CAS), which can also be
useful for identifying the chemical structure of unknowns by start-
ing with elemental composition, obtained from accurate mass
measurements. The development of specific metabolomics MS dat-
abases is in progress (e.g., the METLIN database catalogues metab-
olites, MS/MS spectra and the LC–MS profiles of human plasma and
urine samples) [87].
Biochemical databases can later be used to identify unknown
metabolites or to determine the biological function of identified
metabolites. Information about biochemical pathways and the
metabolites involved is available in the KEGG [88], BioCyc [89]
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
and SMPD [90] databases, which provide data visualization in the
form of individual pathway charts or an overall view of metabolic
pathways.
More specific lipidomics databases exist, such as LipidMaps
[91,92], SphinGOMAP [93] and Lipid Bank [94], containing struc-
tural and nomenclatural information as well as standard analytical
protocols.
In 2010, Gao et al. [95] developed Metscape software as a plug-
in for Cytoscape, to visualize and to interpret metabolomics data in
the context of human metabolic networks [96]. The new version,
MetScape 2 [97], provides functions for creating pathways and net-
work-level views and analyzing several types of experimental data.
It allows users to:
 upload lists of metabolites and genes with experimental
measurements;
 identify related genes, metabolites, reactions, enzymes and
pathways;
 build and analyze gene and metabolite networks;
 visualize changes in experimental data over time or experimen-
tal conditions; and, finally,
 to identify and to visualize enriched pathways from expression-
profiling data.
Metscape 2 uses an internal relational database that integrates
data from the KEGG and EHMN (Edinburgh Human Metabolic Net-
work) [98] databases. Metscape 2 has been applied to obtain a bet-
ter understanding of the underlying metabolic processes
associated with PC [97]. However, many of the molecules detected
are currently not included in databases and metabolite reposito-
ries, showing the extent to which our picture of cellular metabo-
lism in relation to PC is incomplete [99,100].
8. Analysis and summary of the results obtained in PC studies
Different classes of compounds (e.g., amino acids, organic acids,
sugars, and fatty acids) were analyzed and identified in PC as
metabolites with a relevant significance in the development and
the progression of the disease [41–57].
All the metabolites identified in PC patients using the metabolo-
mics-based approaches described in this review are listed in
Table 2.
NMR spectroscopy and GC–MS have provided identification for
most of these metabolites, which were verified using reference
standards or metabolomics databases (KEGG, PubChem or HMDB).
It is evident from Table 2 that known metabolites can be
grouped into four principal classes:
 amino acids;
 fatty acids (fatty acyls, glycerophospholipids, glycerolipids and
sterols);
 carbohydrates; and,
 bile acids.
Some metabolite classes are better detected across platforms
than others (e.g., many amino acids are usually detected by analy-
sis with NMR or MS coupled with GC or LC).
However, some metabolites (acetone, creatinine, dimethyl-
amine, glucose, glutamine, glycine, isoleucine, lactate, leucine, ly-
sine, methanol, phenylalanine, taurine, threonine, tryptophan,
tyrosine, urea and valine) identified by two or more studies
showed similar or contrasting results in terms of increased or de-
creased levels in the different matrices.
NMR techniques have contributed towards discovering about
73.7% (135) of the total number of 217 metabolites, using different
109
kinds of experiments, mainly with an untargeted approach, as
mentioned above.
GC–MS was responsible for identifying 49.8% (108) of all metab-
olites, a higher percentage than LC–MS, which identified 31.8%
(69), whereas a single CE-MS study, using CE-TOF-MS [56] identi-
fied 56 (25.8%) of the metabolites in saliva samples, mainly amino
acids, listed in Table 2.
Amino acids are also the most common metabolites identified
in plasma, serum or urine of PC subjects. Tyrosine, glutamate and
histidine were detected in serum samples using both GC–MS and
LC–MS and using NMR in serum and plasma.
The results were quite different for glutamate concentration in
serum (significantly increased) [44] and in plasma (decreased)
[50], whereas data for Tyrosine and Histidine were in agreement
across different authors. High levels of glutamate, acetone and
3-hydroxybutyrate were observed in the sera of cancer patients
[44], while increased glucose, phenylalanine, formate and mannose
were found to correlate with both disease and age. Differences
between the serum metabolic profiles of PC patients and healthy
controls have also been reported [43], although the investigation
was limited to PCA.
Ornithine, serine and proline were found to be significantly dif-
ferent in PC patients, compared to controls in sera, and valine, gly-
cine, alanine, threonine and tryptophan showed a trend towards
significance in urine, plasma or serum analyzed using NMR,
GC–MS or LC–MS.
All these amino acids were also identified by Sugimoto et al.
[56] in saliva samples using the CE-MS method of analysis
(Table 2).
Highly-significant cancer-specific increases in threonine, tryp-
tophan and 4-hydroxyphenylacetate in urine were noted by Davis
et al. [46], who surmised that this pattern reflected muscle-protein
breakdown, during which all the constituent amino acids entered
oxidative pathways. Effectively, the highest incidences of cachexia
occurred among PC patients with solid-epithelial malignancies, re-
flected in a weight loss of more than 5% at diagnosis. Cachexia re-
sults in severe metabolic disturbances in energy and protein
metabolism and takes place within a complex metabolic syndrome
characterized by anorexia and high rates of fat and skeletal muscle
degradation [101]. However, some authors instead observed
decreasing levels of threonine [41,44], tryptophan [44] and other
amino acids (valine, n-caprylic acid, nonanoic acid, methionine,
asparagine, glutamine, lysine, histidine and tyrosine) as well as
long-chain fatty acids in the serum samples of PC [55,62] and CP
patients [50], relating these significant decreases to the pathogen-
esis of pancreatic disease. These findings were supported by other
research groups, which found similar decreases in plasma samples
from both PC and CP using HPLC analysis [102].
It is well known that the uptake and the catabolism of amino
acids and fatty acids are improved to support rapid cell prolifera-
tion in cancer tissues, and these decreases may be explained as a
result of enhanced usage in tumors [103]. Patients with pancreatic
disease are also troubled by malnutrition because of pancreatic
endocrine and exocrine insufficiency [104], so there is a possibility
that decreases in their serum-metabolite levels may also reflect
malnutrition.
Schrader et al. [102] suggested mainly inflammatory effects –
apart from malnutrition – and pointed at the inverse relationship
between circulating amino-acid concentrations and the degree of
inflammation present (e.g., in hemodialysis patients) [102].
Whether increased tumor-associated proteolytic activity [105]
contributes to not only the generation of specific peptide-decay
profiles, but also the specificity of the amino-acid profiles is still
unknown.
The untargeted metabolomics study on urine, performed by Na-
poli and co-workers [47], confirmed the association of the metab-
 110
I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
Fig. 4. The interaction between metabolic pathways, drug resistance and downstream signaling pathways. K-Ras and the p53 tumor-suppressor gene are two of the main genes mutating in PC, linked to impaired metabolic
pathways. In K-Ras⁄ mutated cells, high levels of glucose are subjected to fermentation in anaerobic conditions, leading to increased production of lactic acid. Moreover, high levels of glucose concentrations and/or of arginine,
inhibit the complex formation of hCHOP-C/EBPa, down-regulating hENT1 expression and consequently inhibiting the transport of chemotherapeutic compounds. K-Ras⁄ modulates glutamine concentrations, which are involved in
cell survival, whilst, in p53⁄ cells, cell survival and cell cycle are regulated by serine concentration. For further details, please refer to the text.
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
olites produced in ketogenesis (e.g., acetoacetate) with the disease
[47], as well as a decreased level of citrate, which has often been
correlated with a down-regulated TCA cycle as a consequence of
the so-called ‘‘Warburg effect’’ [106].
A reduction in the serum level of 1,5-anhydro-D-glucitol was
observed in PC (Table 2) by Kobayashi et al. [41], using a GC–MS
metabolomics-based approach.
1,5-Anhydro-D-glucitol is usually a serum biomarker for short-
term glycemic control and a decreased serum level for this metab-
olite shows the presence of hyperglycemia and glycosuria in the
last few days [103]. These results suggest that glucose tolerance
was impaired in these patients because of pancreatic insufficiency,
reflecting systemic responses to the metabolic changes induced by
pancreatic disease [41].
High cancer-specific levels of hypoxanthine, choline, trimethyl-
amine-N-oxide (TMAO), O-acetylcarnitine and acetone were
observed in the serum or plasma and urine of PC patients using
NMR analysis, and the results of the different studies were consis-
tent, with the exception of TMAO, which showed different trends
[43,46]. These changes, perhaps indicative of metabolic distur-
bances associated with tumor metabolism, may represent an en-
hanced capacity for DNA synthesis and energy production
(hypoxanthine), cell-membrane formation (choline, trimethyla-
mine-N-oxide), as well as increased rates of lipolysis and fatty acid
metabolism (O-acetylcarnitine, acetone) seen in rapidly proliferat-
ing tumor cells [107–110].
Down-regulation of citric-acid-cycle intermediates succinate
and malate may have an overall impact on the energy metabolism
of the cell, while lower levels of uridine, 50 -uridine monophosphate
(50 -UMP) and 50 -adenosine monophosphate (50 -AMP) could reflect
rapid turnover of these nucleotides in tumor tissue [57]. The
authors also observed down-regulation of the powerful antioxi-
dant glutathione in pancreatic tumor tissue [57]. A similar de-
crease in the serum levels of glutathione has been observed in
breast carcinoma with a simultaneous increase in lipid peroxida-
tion in plasma, which becomes more pronounced with ageing of
the patients [111].
Moreover, increased levels of the amino-acid taurine in the pan-
creatic tumor tissue [57], serum and urine of PC patients [46], com-
pared to the normal controls, were found, as shown in Table 2.
Taurine is a major constituent of bile and has been reported to
be significantly higher in urinary bladder cancer [111].
Metabolomic profiling of the plasma of PC patients by Urayama
et al. [48] showed a rise in taurocholic acid and tauroursodeoxy-
cholic acid, which are also constituents of bile. The study of Beza-
beh and colleagues [53] focused on the analysis of D-glucuronic
acid, which was very high in bile samples obtained from PC
patients, whereas it was absent or negligible in control and CP pa-
tients. The presence of high levels of GlcU, observed in pancreatic
tumor, may have diagnostic implications, but, at the moment, the
reason remains unclear. D-Glucuronic acid is synthesized in the li-
ver from UDP-glucose mediated by UDP-glucose dehydrogenase,
and is involved in a number of key detoxification pathways by
removing a variety of non-polar drugs, environmental toxins and
carcinogens from the body [112]. It is also responsible for the
removal of bilirubin, a major end-product of heme catabolism. In
the liver, D-glucuronic acid is conjugated to bilirubin, forming bili-
rubin diglucuronide, and is excreted into the bile [113]. A possible
explanation can be found in a significant increase of b-glucuroni-
dase activity observed in PC tissue and CP patients, compared to
normal subjects [114]. This may be released into the bile along
with pancreatic secretions and could bring about deconjugation
of the bilirubin diglucuronide present in bile, liberating free GlcU.
The serum/plasma and tissue of PC patients has also been
shown to have an altered lipid profile based on NMR [52,74] and
FI-FT-ICR-MS analysis [55], highlighting lipid compounds
111
correlated positively with cancer. Furthermore, it has been sug-
gested that the growth-promoting effect of lipids on PC cells occurs
at multiple levels:
 transduction of signals induced by hormones;
 a structural role in cell membranes; and,
 energy supply [115].
Chronic inflammation is heavily mediated by lipid-signal-trans-
duction pathways, and correlation between chronic inflammation
and the development of cancer has been recognized in recent years
[116].
In a different study, MS metabolic profiling was applied to the
plasma samples of PC patients, proposing as candidate biomarkers
fatty-acid arachidonic acid, lipids lysoPC(18:2), PC(34:2), and
PE(26:0), and bile acids tauroursodeoxycholic acid, taurocholic
acid, deoxycholylglycine and cholylglycine. Increased levels of ara-
chidonic acid in the plasma of PC patients may be a result of sys-
temic equilibrium of the compounds involved in inflammation
[48].
In general, a high concentration of omega-6 fatty acids (e.g., ara-
chidonic acid) promotes inflammation, increasing the production
of pro-inflammatory enzymes and cytokines, including COX-2,
tumor necrosis factor (TNF-a), and interleukin 1beta (IL-1b),
whereas omega-3 fatty acids (e.g., eicosapentaenoic acid and doco-
sahexaenoic acid) have anti-inflammatory properties [117]. Fur-
thermore, TNF-a is a potent activator of nuclear transcription
factor NF-kB, which controls the expression of numerous genes in-
volved in inflammation and immune response processes, including
proliferation, invasion and adhesion, angiogenesis and apoptosis
[118]. Consistent with this, TNF-a, IL-1 and NF-kB were over-ex-
pressed and activated in PC patients [119,120].
Significant increased levels of dimethylamine (DMA), formate
and acetone were observed in urine, plasma and serum [46,47],
and significant increased levels of NAG and VLDL were reported
in plasma using NMR analysis [50]. This pattern was integrated
with significant decreased levels of HDL, LDL and citrate, confirmed
by NMR and GC-MS in the plasma, serum or urine of PC tumors
[41,50] (Table 2).
The overall significance of these results may be explained by
considering the role of possible immune-system impairment in
pancreatic disease, manifested through the increase in acetylated
glycoproteins levels (high levels of NAG in PC cases), involved in
white blood-cell recognition, especially in mammals [121,122].
The high VLDL level in the plasma of the PC group, associated with
low LDL and HDL concentrations, was related to a well-known in-
creased risk of coronary artery disease [123]. DMA, an animal
carcinogen, could instead be related to the possibility of increased
damage to the genome or disruption of cellular metabolic pro-
cesses [124].
Data on lactate levels are instead partly inconsistent for differ-
ent authors. An increase in the cancer serum levels of lactate was
reported in PDAC, using GC-MS [45], confirmed in pancreatic
tumor tissue [52] and in many human cancers [125–127], whereas
analysis of plasma serum and urine using 1H-NMR showed a con-
trasting trend [43,50]. Lactate is the product of anaerobic glycolysis
and increases in hypoxia, ischemia and poorly vascularized cancer.
These inconsistencies may be caused by metabolic patterns spe-
cific to species and tissue and partly by the small sample sizes in
the studies carried out.
Many authors have observed significantly lower levels of 3-
hydroxybutyrate {except Bathe and colleagues [44]], isoleucine
and creatine in serum and plasma using NMR or GC–MS analysis
[44,45,50], and 3-hydroxyisovalerate in serum and urine, whereas
discordant trends in the level of leucine have been observed in ur-
ine, plasma or serum from PC patients [43,44] (Table 2). Isoleucine
112 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
and leucine are essential amino acids, and changes in the serum of
PC patients could be attributed to the impaired metabolism of
these amino acids in PC. The 3-hydroxybutyric acid level is instead
higher in ketosis; indeed, in humans, beta-hydroxybutyrate is
synthesized in the liver from acetyl-CoA and can be used as an
energy source by the brain when blood glucose is low [128].
3-Hydroxyisovalerate derives from isovaleryl-CoA, a catabolic
intermediate of leucine. Interestingly, a recent study suggested
that modulation of the expression of leucine zipper tumor suppres-
sor 2 has direct effects on the proliferation of various cancer cells
[129]. Thus, a significant decrease in the 3-hydroxyisovalerate
level in the serum may suggest the development of PC.
Compared to controls, PC patients also showed significant in-
creased concentrations of serum creatinine, combined with de-
creased excretion in urine [43,47] (Table 2). An increase in
creatinine within the first 48 h is strongly associated with the
development of pancreatic necrosis [130], which, when analyzed
histologically, can be a simple, accurate and reproducible predictor
of post-operative outcome in PDAC patients [131].
9. Discussion
Cancer is commonly considered a genetic disease that is driven
by mutations of oncogenes and tumor-suppressor genes. However,
one of the major underlying purposes of these genetic and gene-
expression changes is to create a metabolic phenotype for cancer
cells that is essential for tumor-cell growth and survival
[132,133]. The metabolic phenotype of cancer includes alterations
in glycolysis, amino-acid metabolism, nucleotide metabolism and
glycerophospholipid metabolism. Indeed, over and beyond aerobic
glycolysis, also called the ‘‘Warburg effect’’ [134,135], cancer cells
exhibit increased protein and nucleotide synthesis [136,137], in-
creased fatty-acid synthesis and changes in fatty-acid metabolism
[132,138,139]. In particular, during malignant transformation in
cancer, Hilvo and co-workers observed drastic changes in the lipid
metabolism of cells, so they suggested that these alterations are a
prominent feature of solid tumors [140].
Approximately 75% of all pancreatic carcinomas occur within
the head or the neck of the pancreas, 15–20% are located in the
body of the pancreas and 5–10% in the tail [141], leading to dys-
function of this organ. As the pancreas is a dual-function gland,
being made up of two types of tissue (exocrine and endocrine tis-
sue) with two different activities (secreting the digestive enzymes
and hormones, respectively), pancreatic dysfunction due to carci-
nogenetic processes results in an imbalance of metabolic profiles
(reviewed in Table 2). Metabolomic studies have the potential both
to detect a class of altered metabolites deriving from the status of
the disease, which can predict PC at an early stage for early diagno-
sis, and to predict the follow-up of patients during chemotherapy.
It is likely that no single biomarker can selectively characterize PC,
and all metabolites with significant changes could be candidate
biomarkers for new PC diagnosis based on specific patterns of mul-
tiple metabolites.
An algorithm or Web-based risk calculator, which takes into ac-
count different and combined altered parameters/metabolites, will
probably be a better tool for metabolomics studies, enabling clini-
cians to improve understanding of the state of the disease. More-
over, identification of the small metabolites involved in PC
prediction/development may open the way for economical, com-
mercially-available kits, with the aim of carrying out extensive
screening of patients at risk. Ultimately, different metabolite con-
centrations resulting from metabolomics studies may also explain
the possible pathways involved in the onset and the progression of
PC, suggesting specific new targeted therapies {e.g., the altered
concentration of arginine found by Bathe [44] (Table 2) represents
a critical factor, by which increasing eNOS levels and NO produc-
tion in turn triggers hCHOP-C/EBPa complex formation, reducing
hENT1 expression [142]}.
hENT1 is a nucleoside transporter acting as a drug transporter,
and it mediates the cellular uptake of nucleotides (including gem-
citabine, a nucleoside analogue) in order to convert drugs into their
bioactive form. It has already been demonstrated in several studies
that hENT1 down-regulation is responsible for the poor response
to gemcitabine therapy [143–145].
Decreased hENT1 levels are also due to a higher glucose concen-
tration [142], which was found to be altered in PC patients’ urine
and serum by Napoli et al. [47], Davis et al. [46] and Bathe et al.
[44] (Table 2). Higher glucose concentrations have the same inhib-
itory effect on the complex formation of hCHOP-C/EBPa, which in
turn down-regulates hENT1 expression, consequently inhibiting
the chemotherapeutic effect of gemcitabine. These data suggest
that not only a gene therapy could be effective in improving the
therapeutic response to gemcitabine, as already reported [146],
but also an amino-acid-modified diet or a diet based on caloric/glu-
cose restriction could help to overcome chemotherapy resistance
by enhancing the effects of gemcitabine due to hENT1 modulation.
Research suggests that there are major health benefits associated
with dietary restriction.
Fig. 4 illustrates the interaction between the impaired meta-
bolic pathways mentioned in PC, chemotherapy drug resistance
and downstream signaling pathways.
Recent studies in rodent and in-vitro models uncovered a poten-
tial link between short-term starvation and the improved efficacy of
chemotherapy, which has already been demonstrated for some
types of cancer [147,148]. An interesting study performed by Safdie
et al. [149] outlined the link between fasting and increased temozol-
omide chemotherapeutic effect in a mouse model of glioma. In order
to define a potential predictor for the efficacy of chemotherapy in
patients affected by different types of cancer, a new technology,
miRNA, allowing for comprehensive and rapid mRNA-expression
profiling, has been developed and applied in recent years [150,151].
Advances in our understanding of functional genomics in the
field of cancer could be best addressed by supplementary studies
including measurement of mRNA, proteins and low-molecular-
weight metabolites over time and in varied conditions.
For this reason, connecting metabolomics studies to the differ-
ent stages of the disease would help scientists to uncover new
pathways and/or new therapeutic approaches.
10. Conclusions and perspectives
Recently, MS, NMR and multivariate statistical techniques were
incorporated into a multidisciplinary approach to profile changes
in small molecules associated with the onset and the progression
of human diseases. The aim of this research is to identify the pro-
files of metabolites that are uniquely correlated with a specific hu-
man disease, such as cancer, in order to diagnose accurately and to
treat the disease.
Efforts to discover and to distinguish specific metabolic abnor-
malities in cancer cells continue to involve scientists around the
world and changes in energy metabolism are considered an ‘‘emerg-
ing hallmark’’ of cancer [152]. Indeed, although PC is characterized
by several oncogenes and tumor-suppressor genetic aberrations
(e.g., K-Ras and p53-protein mutation), which lead to uncontrolled
proliferation and are responsible for enhanced cancer cell survival,
by modulating downstream signaling pathways (MAPK and PI3K-
mTORpathways) [153], these genetic aberrations are also closely in-
volved in modulating the main metabolic pathways (glycolysis,
amino acid metabolism and different biosynthetic pathways).
Oncogenic K-Ras mutation plays a key role in tumor initiation in
PDAC [153] and has also been shown to promote glycolysis
 I.M. Di Gangi et al. / Trends in Analytical Chemistry 55 (2014) 94–116
[154,155] to meet the increased anabolic needs of enhanced prolif-
eration. A high glycolysis rate is in accordance with the Warburg
effect, as tumor growth is mainly due to the energy generated by
non-oxidative breakdown of glucose (glycolysis) whilst ‘‘healthy’’
cells generate energy mainly from oxidative breakdown of pyru-
vate. Moreover, glucose fermentation in anaerobic conditions leads
to increased production of lactic acid, which has been confirmed to
be higher in several studies reported in Table 2 [41,45]. K-Ras and
the p53 tumor-suppressor genes are two of the main genes mutat-
ing in PC [156]. P53 mutation/inactivation, following an initial acti-
vating mutation in the K-Ras gene [157], has also been linked to an
impaired metabolic pathway, since it has been implicated in both
enhanced glycolysis and regulation of glutamine via modulation
of glutaminase expression [158]. This was found to be deregulated
in several studies, as reported in Table 2. Furthermore, Maddocks
and colleagues [159] demonstrated that human p53/ cancer
cells use exogenous serine to contribute to cell survival, to support
the Warburg effect. This may explain the high serine levels found
in the different metabolomics studies reported in Table 2. The
authors underline the therapeutic utility of serine depletion (by re-
moval from the diet, enzymatic depletion in vivo, or other means)
since serine deprivation in p53/ cancer cells accumulates cells
in G1 and reduces the S-phase and also lowers the glycolytic flux
impeding ATP production. Briefly, oncogenic signals rewire the
metabolic pathways in order to shift nutrients into anabolic path-
ways, reprogramming the metabolism in tumor cells [135].
To date, there is only a limited understanding of PC pathogene-
sis, and the challenge in the case of PC diagnostics is even bigger
due to the number of differential diagnoses, which are difficult to
discern from malignant disease, even for experienced clinicians.
In this review, we have discussed recent developments in sample
preparation, experimental techniques, and identification and quan-
tification of metabolites and chemometrics tools used in the meta-
bolomics approach in the quest for biomarkers for PC. At the
moment, this perspective is increasingly evident, in the light of
the latest recent findings by Ritchie and co-workers [55], who eval-
uated the potential clinical benefit of screening subjects based on
PC-594 to identify subjects with PC or at high-risk of developing PC.
However, the ideal biological marker for cancer-risk assessment
and early detection needs to be highly sensitive and specific, be
found in biosamples obtained using minimally-invasive proce-
dures and be possible to analyze using a high-throughput, cost-
effective and quantitative assay.
But, all metabolites with significant changes could be candi-
dates for PC biomarkers. However, in the light of current knowl-
edge, we do not expect that a single biomarker will be able to
characterize PC, and a complex profile of multiple metabolites
should instead be explored.
In the past few years, an ‘‘impetus for untargeted metabolo-
mics’’ has developed, as metabolomics can result in new putative
biomarkers that can reach the clinic as tools to diagnose health,
disease or the outcome of pharmacological treatment, following
careful validation. Moreover, an understanding of the link between
the metabolic profile and the onset of PC may provide insight into
the physiopathological mechanisms involved in the pathogenesis
of PC. Modulation of the altered metabolites involved in PC pro-
gression may be an ideal partner in therapeutic settings requiring
the inhibition of tumor-protecting mechanisms or proteins to over-
come treatment resistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.trac.2013.12.006.
113
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