Polarity-Tuning Derivatization-LC-MS Approach for Probing Global Carboxyl-Containing Metabolites in Colorectal Cancer

Article abstract of DOI:10.1021/acs.analchem.8b01873

Carboxyl-containing metabolites (CCMs) widely exist in living systems and are the essential components for life. Global characteristics of CCMs in biological samples are critical for the understanding of physiological processes and the discovery for the onset of relevant diseases. However, their determination represents a challenge due to enormous polarity differences, structural diversity, high structural similarity, and poor ionization efficiency in mass spectrometry. Herein, 5-(diisopropylamino)amylamine (DIAAA) derivatization coupled with liquid chromatography-mass spectrometry (LC-MS) was developed for mapping the CCMs. With this methodology, the sensitivity was significantly enhanced. More importantly, the hydrophobicity of polar CCMs, amino acids, TCA cycle intermediates, and short-chain fatty acids and the hydrophilicity of low-polar CCMs, long-chain fatty acids, and bile acids were significantly increased, resulting in a remarkable separation efficiency for which 68 CCMs can be simultaneously determined. Furthermore, the polarity-tuning effect was confirmed to be induced by the different impacts of aliphatic chains and nitrogen atom in DIAAA, the latter existing as a cation in the acidic mobile phase, using different derivatization reagents. Finally, this derivatization method was utilized to hunt for the potential biomarkers in colorectal cancer (CRC) patients and 52 CCMs, related with several key metabolic pathways, including amino acids metabolism, TCA cycle, fatty acid metabolism, pyruvate metabolism, and gut flora metabolism were identified. This innovative polarity-tuning derivatization-LC-MS approach was proved to be a valuable tool for probing global metabolome with high separation efficiency and sensitivity in various biological samples.

Full text of DOI:10.1021/acs.analchem.8b01873

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Technical Note
Polarity-tuning Derivatization-LC-MS Approach for Probing
Global Carboxyl-containing Metabolites in Colorectal Cancer
Xiqing Bian, Na Li, Binbin Tan, Baoqing Sun, Ming-Quan Guo,
Guoxin Huang, Li Fu, W. L. Wendy Hsiao, Liang Liu, and Jian-Lin Wu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01873 • Publication Date (Web): 07 Sep 2018
Downloaded from http://pubs.acs.org on September 8, 2018
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Polarityꢀtuning DerivatizationꢀLCꢀMS Approach for Probing Global
Carboxylꢀcontaining Metabolites in Colorectal Cancer
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Xiqing Bian,† Na Li,*† Binbin Tan,‡ Baoqing Sun,§ MingꢀQuan Guo,∥ Guoxin Huang,† Li Fu,‡ W. L.
Wendy Hsiao,† Liang Liu,*† JianꢀLin Wu,*†
† State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and
Health, Macau University of Science and Technology, Macao, China
‡ Guangdong Key Laboratory for Genome Stability & Disease Prevention, Department of Pharmacology and Carson Internaꢀ
tional Cancer Research Centre, Shenzhen University School of Medicine, Shenzhen 518060, China.
§ State Key Laboratory of Respiratory Disease, National Clinical Center for Respiratory Diseases, Guangzhou Institute of
Respiratory Diseases, First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong Province, China.
∥Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, SinoꢀAfrica Joint
Research Center, Chinese Academy of Sciences, Wuhan 430074, China.
ABSTRACT: Carboxylꢀcontaining metabolites (CCMs) widely exist in living system and are the essential components for the life.
Global characteristics of CCMs in biological samples are critical for
the understanding of physiological processes and the discovery for
the onset of relevant diseases. However, their determination repreꢀ
sents a challenge due to enormous polarity difference, structural
diversity, high structural similarity, and poor ionization efficiency
in mass spectrometry. Herein, 5ꢀ(diisopropylamino)amylamine
(DIAAA) derivatization coupled with liquid chromatographyꢀmass
spectrometry (LCꢀMS) was developed for mapping the CCMs. With
this methodology, the sensitivity was significantly enhanced. More
importantly, the hydrophobicity of polar CCMs, amino acids, TCA
cycle intermediates and shortꢀchain fatty acids, and the hydrophilicꢀ
ity of lowꢀpolar CCMs, longꢀchain fatty acids and bile acids, were
significantly increased, resulting in a remarkable separation efficiency for which 68 CCMs can be simultaneously determined. Furꢀ
thermore, the polarityꢀtuning effect was confirmed to be induced by the different impacts of aliphatic chains and nitrogen atom in
DIAAA, the latter existing as a cation in the acidic mobile phase, using different derivatization reagents. Finally, this derivatization
method was utilized to hunt for the potential biomarkers in colorectal cancer (CRC) patients and 52 CCMs, related with several key
metabolic pathways including amino acids metabolism, TCA cycle, fatty acid metabolism, pyruvate metabolism and gut flora meꢀ
tabolism, were identified. This innovative polarityꢀtuning derivatizationꢀLCꢀMS approach was proved to be a valuable tool for
probing global metabolome with high separation efficiency and sensitivity in various biological samples.
Carboxylꢀcontaining metabolites (CCMs) widely exist in
living system and are the essential components for the life.
CCMs mainly contain amino acids (AAs), TCA cycle interꢀ
mediates (TCAs), shortꢀchain fatty acids (SCFAs), longꢀchain
fatty acids (LCFAs), bile acids and so on. AAs have been cenꢀ
tral to the study of cancer metabolism and are required for the
synthesis of protein, nucleotide and cell growth.1,2 TCA cycle
is an important route for oxidative metabolism and also assoꢀ
ciated with highly proliferating cells, like tumour cells.3
SCFAs, such as butyric acid (BA) and propionic acid (PA),
have beneficial effects on antiꢀinflammation and are the key
determinants of diseases in intestinal microbiota.4 Longꢀchain
fatty acids (LCFAs), for example, hydroxyeicosatetraenoic
acids (HETEs, ωꢀ6), prostaglandins (PGs, ωꢀ6), thromboxanes
(TXs, ωꢀ6), and hydroperoxyeicosapentaenoic acids (HEPEs,
ωꢀ3), play vital roles in cancer,5,6 inflammation,7 and other
biological activities.8 Bile acids influence the composition of
the gastrointestinal microbiota and host metabolic pathways
through the reciprocal microbeꢀhost crosstalk.9 Moreover,
some SCFAs and TCAs are the precursors of biosynthesis of
LCFAs.10 Therefore, the global determination for CCMs proꢀ
file is critical for the understanding of physiological processes
and the discovery for the onset of relevant diseases.
Colorectal cancer (CRC) is the second most common cancer
in the world.11 The diet, inflammation, and loss of tumor supꢀ
pressor gene are the key risk factors contributing to the high
incidence of CRC. Recent studies further indicated that
dysbiosis of commensal gut microbiota and its metabolites
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also play an important role in the development of CRC.12
of other endogenous components in serum on the determinaꢀ
tion of CCMs, the recovery and matrix effects were assessed
according to the previously described procedure (Supporting
Information).20 As well, the stability was also evaluated.
However, it is unclear which metabolites and how they influꢀ
ence the process of CRC development. Thus, there is an urgent
need to clarify the metabolic variations in CRC to improve the
diagnosis and treatment of the disease.
Derivatization. The reaction solutions were firstly prepared
as follows, i.e. HOBt and HATU were separately dissolved in
DMSO at the concentration of 20 mM, while the diamineꢀTEA
solution, i.e. DMEEAꢀ, DEEEAꢀ, DMAPAꢀ DMABAꢀ,
DEABAꢀ and DIAAAꢀTEA solution, was prepared by dissolvꢀ
ing 100 ꢁmol of corresponding diamine and 200 ꢁmol of TEA
in 1 mL of DMSO. Thereafter, the dried residue of real samꢀ
ples was sequentially mixed with 5 µL HOBt, 5 µL DIAAAꢀ
TEA solution, and 5 µL HATU followed by 1 min incubation
at room temperature. As well, the dried residue of standards
were sequentially mixed with 5 µL HOBt, 5 µL DMEEAꢀ,
DEEEAꢀ, DMAPAꢀ DMABAꢀ, DEABAꢀ or DIAAAꢀTEA
solution, and 5 µL HATU followed by 1 min incubation at
room temperature. Finally, 35 µL acetonitrile was added to
make up to the final volume of 50 µL, and 1 µL was directly
injected into UHPLCꢀQꢀTOF/MS.
Though gas chromatography (GC) and liquid chromatogꢀ
raphy (LC) coupled with mass spectrometry (MS) have been
widely used for CCMs analysis,13,14 the thermal instability of
some CCMs hinders the use of GCꢀMS,15 and LCꢀMS has to
face the problems of retention and separation on the column
owing to the marked polarity differences between hydrophilic
CCMs (AAs, TCAs and SCFAs) and hydrophobic CCMs
(LCFAs and bile acids), high polarity similarities among difꢀ
ferent hydrophilic or hydrophobic CCMs, as well as poor ioniꢀ
zation efficiency.16 Various derivatization methods, e.g. diisoꢀ
propylphosphoryl,17 pꢀdimethylaminophenacyl bromide18 for
hydrophilic CCMs; N,Nꢀdimethylaminobutylamine,19 cholaꢀ
mine20 and 2ꢀdimethylaminoethylamine21 for hydrophobic
CCMs have been developed to solve these difficulties. Howꢀ
ever, until now, the comprehensive analysis of global CCMs
could not be reached yet.
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UHPLCꢀQꢀTOF/MS analysis. An Agilent 1290 Infinity
LC system (UHPLC, Santa Clara) and binary pump with Waꢀ
ters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 ꢁm)
was employed for the separation of CCMs. The mass specꢀ
trometry was conducted on an Agilent 6550 UHD accurateꢀ
mass QꢀTOF/MS system with a dual Jet stream electrospray
ion source (dual AJS ESI). The instrument was operated in
positive (POS) and negative (NEG) ion modes. For the deꢀ
tailed parameters of UHPLCꢀQꢀTOF/MS, please see the Supꢀ
porting Information.
In this research, 5ꢀ(diisopropylamino)amylamine (DIAAA)
was first applied to establish a highly sensitive derivatization
ultraꢀhigh performance liquid chromatographyꢀquadrupleꢀtime
of flight mass spectrometry (UHPLCꢀQꢀTOF/MS) approach,
that achieved the simultaneous determination of 68 CCMs
belonging to 6 different types, which have a large polarity
differences (Scheme 1 and Figure 1). Furthermore, utilizing
the newly developed approach, the potential biomarkers in the
sera of CRC patients were determined and the influence of
microbial metabolites on the process of CRC was disclosed.
EXPERIMENTAL SECTION
Chemicals and reagents. Amino acids mixtures, TCA cyꢀ
cle intermediates, shortꢀchain fatty acids, bile acids, and 4ꢀ
chloroꢀDLꢀphenylalanine were purchased from SigmaꢀAldrich
Laboratories, Inc. (St. Louis, MO). All longꢀchain fatty acid
standards mixtures and the isotope labeled internal standards
were provided by Cayman Chemical (Ann Arbor, MI). The
detailed information of CCMs was shown in supporting inꢀ
formation.
Scheme 1. Derivatization of carboxylꢀcontaining metaboꢀ
lites with different reagents.
5ꢀ(Diisopropylamino)amylamine
dimethylethylenediamine
(DIAAA),
(DMEEA),
N,Nꢀ
N,Nꢀ
diethylethylenediamine (DEEEA), 3ꢀ(dimethylamino)ꢀ1ꢀ
propylamine (DMAPA), 4ꢀdimethylaminobutylamine
(DMABA), 4ꢀ(diethylamino)butylamine (DEABA), Oꢀ(7ꢀ
azabenzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀtetramethyluronium hexꢀ
afluorophosphate (HATU), 1ꢀhydroxybenzotriazole hydrate
(HOBt), triethylamine (TEA) and DMSO were bought from
SigmaꢀAldrich Laboratories, Inc. LCꢀMS grade acetonitrile
and HPLC grade methanol were obtained from Anaqua Chemꢀ
icals Supply Inc., Ltd. (Houston, TA). Other reagents were
purchased from SigmaꢀAldrich Laboratories, Inc. Deionized
water was prepared using a Millipore water purification sysꢀ
tem. DIAAA was purified by prepared HPLC before use.
Sample preparation. Fifty ꢁL of serum was first mixed
with 4 times volume of cold methanol to remove the proteins
by centrifugation at 13,000 rpm for 5 min at 4 °C. The extracꢀ
tion was repeated 3 times and the combined supernatants were
dried under a nitrogen stream. The residue was stored at ꢀ20
°C prior to derivatization. In addition, to assess the influence
Figure 1. Representative structures of carboxylꢀcontaining
metabolites.
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Linearity. The calibration curves were established by plotꢀ
ting the peak area ratios of the standards to the corresponding
internal standard against the concentrations.
these compounds. Over all, the DIAAAꢀderivatization not
only achieved the simultaneous determination of hydrophilic
and hydrophobic metabolites, but also improved the separation
resolution (Figure 2).
CRC and healthy human serum. All the experiments were
performed in accordance with the first affiliated hospital, Sun
Yatꢀsen University Ethics Committee's guidelines and regulaꢀ
tions. The serum samples of 20 healthy human and 20 CRC
patients were recruited and CRC was diagnosed by the histoꢀ
pathological methods according to the Union Internationale
Contre Le Cancer (UICC), while the healthy volunteers were
selected by a routine physical examination. The samples were
stored at −80 °C before use.
Animals and experimental design. ApcMin/+ mice were
purchased from Jackson Laboratory (Bar Harbor, ME) for
breeding. Mice were housed in a 12ꢀh lightꢀdark cycle facility
with controlled room temperature and humidity and were fed
with PicoLab®Rodent Diet 20ꢀ5053 (LabDiet, St. Louis, MO).
Blood was collected from the main celiac vein of 14ꢀweeks
old ApcMin/+ and wide type C57BL/6J mice as described.22
Fresh blood sample was centrifuged at 4,000 rpm for 10 min at
4 °C. The supernatant was taken out and stored at −80 °C for
later usage. The mice were sacrificed for ploys count in the
intestines. The ApcMin/+ mice yielded 30 polyps in average,
while the wildꢀtype mice produced none detectable polyp.
Good linearities of calibration curves were obtained for
most of CCMs with the coefficients of determination (R2)
greater than 0.99 (Table S2). The derivatized metabolites were
stable in 4 °C for at least 3 days. Other endogenous compoꢀ
nents in serum were found to have no significant influence on
the determination of CCMs from the matrix effect and recovꢀ
ery (Table S3).
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Mechanism of polarity tuning induced by DIAAAꢀ
derivatization. However, why did DIAAAꢀderivatization
reverse the retentions of hydrophobic and hydrophilic CCMs
on C18 column?
As shown in Scheme 1, DIAAAꢀderivatization introduced a
new amino group into the CCMs. The introduced amino group
could be protonated in the acidic mobile phase, which made
the LCFAs and bile acids derivatives acted as the cationic
surfactants23,24 and reduced their interaction with C18 solid
phase. Compared to the large lipophilic groups in LCFAs and
bile acids, the introduction of small hydrophobic groups in
DIAAA should have relatively low contribution. Finally, the
retention times shortened (Figure S2).
RESULTS AND DISCUSSION
Differently, an aliphatic chain with five methylene and two
side chains of Nꢀisopropyls in DIAAA have big influence on
the hydrophobicity of polar CCMs (AAs, TCAs and SCFAs),
which might be the major reason for the increase of retention
time. To confirm it, similar diamine derivatization reagents
with various main chain lengths and side chains were investiꢀ
gated, and they were DMEEA (a, C4H12N2), DEEEA (b,
C6H16N2), DMAPA (c, C5H14N2), DMABA (d, C6H16N2), and
DEABA (e, C8H20N2) (Scheme 1). The results showed that the
derivatization with DMEEA, DMAPA, or DMABA, which
had two NꢀCH3 and a chain with 2ꢀ4 carbons, decreased the
retention times for all hydrophilic CCMs as expected. When
derivatizing with DEEEA and DEABA, which containing two
NꢀCH2CH3 groups and a 2ꢀ or 4ꢀcarbon chain respectively, the
retention times were longer than that of DMEEA, DMAPA or
DMABA derivatives, and the retention for most of DEABAꢀ
derivatized AAs and TCAs were even longer than that of nonꢀ
derivatized CCMs, while they were still shorter than that of
corresponding DIAAA derivatives (Table 1 and Figure S3).
Consequently, the lipophilic groups in diamine reagents inꢀ
deed influenced the retentions of hydrophilic CCMs on C18
column. Moreover, the side chains had more effect than the
main chain, and more carbons the chain had, longer the retenꢀ
tions for most of hydrophilic CCMs were. Finally, DIAAA
with two NꢀCH(CH3)2 and five methylene chain were found to
be the best choice (Table 1). On the other hand, the change of
lipophilic groups in the derivatization reagents had the similar
influence on the retentions of LCFAs and bile acids derivaꢀ
tives, while all derivatizations induced the shorter retentions
compared to nonꢀderivatization metabolites. The above experꢀ
iments confirmed that the contributions of the lipophilic and
hydrophilic groups in derivatization reagents resulted in the
polarityꢀtuning of DIAAAꢀderivatized CCMs (Figure S2).
Improvement of sensitivity and separation efficiency of
CCMs by DIAAAꢀderivatization. As mentioned in Introducꢀ
tion, CCMs play pivotal roles in various physiological funcꢀ
tions and may serve as potential biomarkers in many diseases,
thus a simple, rapid, sensitive and global method is required to
quickly detect the metabolites in biological samples. In this
research, a derivatization method using DIAAA, was develꢀ
oped. First, the reaction conditions were optimized using the
representative CCM standards (Figure 1), phenylalanine (Phe),
threonine (Thr), histidine (His), fumaric acid (Fum), succinic
acid (Suc), malic acid (Mal), PA, BA, valeric acid (VA), 11ꢀ
dehydro TXB2, 15(S)ꢀHETE, PGE2, 13(S)ꢀHOTrE, 15(S)ꢀ
HEPE, 5(S)ꢀHEPE, cholic acid (CA), chenodeoxycholic acid
(CDCA), and glycocholic acid (GCA). It was found that the
reaction could complete within 1 min at room temperature
(Figure S1). And, no evident byꢀproduct was observed.
Then, using the above optimized method, 68 CCMs were
derivatized with DIAAA followed by UHPLCꢀQꢀTOF/MS
analysis. It was found that the ionization efficiency of
DIAAAꢀderivatized CCMs remarkably enhanced, which inꢀ
duced the improvement of detection sensitivity up to 2000
times (Table S1). More importantly, the retention times for
hydrophilic CCMs, AAs, TCAs and SCFAs, were greatly proꢀ
longed from 0.5ꢀ3 min to 0.5ꢀ16.5 min (Figure 2, from region
A1 to B1) after derivatization. Conversely, the retention times
for hydrophobic CCMs, LCFAs and bile acids, shortened from
27ꢀ41 min to 21.5ꢀ39 min (Figure 2, from region A2 to B2).
As a result, 68 CCMs can be well separated within 40 min,
otherwise, only 45 CCMs can be detected with very weak MS
signals and heavy peak overlap using nonꢀderivatization
method. For example, the isomers of HETEs and epoxyeicosaꢀ
trienoic acids (EETs) could reach to baseline separation,
which is essential for the identification and quantification of
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Figure 2. LCꢀMS chromatograms of carboxylꢀcontaining metabolite standards (A) and DIAAAꢀderivatives (B). After derivatizaꢀ
tion, the carboxylꢀcontaining metabolites in regions A1 and A2 appeared in regions B1 and B2, respectively.
Table 1. Retentions of carboxylꢀcontaining metabolite standards and corresponding derivatives on HSS T3 (C18) column.
derivatization
reagent
Classification
Name
No.
derivatization reagent
Retention time (min)
Classification
Name
No.
Retention time (min)
1
2
3
Nonꢀderivatization
DMEEA
1
2
3
Nonꢀderivatization
DMEEA
DEEEA
DEEEA
11ꢀ
Phe
dehydro
ꢀTXB2
4
DMAPA
4
DMAPA
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
PUFAs
ωꢀ6
15(S)ꢀ
HETE
AAs
Thr
His
Suc
PGE2
13(S)ꢀ
HOTrE
15(S)ꢀ
HEPE
PUFAs
ωꢀ3
Fum
TCAs
DMABA
DEABA
DMABA
DEABA
7
DIAAA
7
DIAAA
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
DIAAA
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
5(S)ꢀ
HEPE
Mal
BA
CA
PA
CDCA
SCFAs
Bile acids
DMABA
DEABA
DMABA
DEABA
7
DIAAA
7
DIAAA
1
2
3
4
5
6
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
1
2
3
4
5
6
Nonꢀderivatization
DMEEA
DEEEA
DMAPA
DMABA
DEABA
VA
GCA
7
DIAAA
7
DIAAA
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Application in sera of CRC patients. Finally, the DIAAA
Figure 3. Comparisons and ROC curves of SCFAs in CRC
human serum. All SCFAs decreased in the sera of CRC paꢀ
tients, compared to the normal controls.
derivatization was applied to probe the changes of CCMs beꢀ
tween CRC patients and healthy volunteers. By comparison
with the corresponding standards, 52 CCMs were determined,
as well, 30 CCMs in CRC patients including 11 AAs, 3 TCAs,
5 SCFAs, 9 PUFAs, and 2 bile acids were significantly deꢀ
creased. Furthermore, the detected CCMs were found to be
involved in several key metabolic pathways including amino
acids metabolism, TCA cycle, fatty acid metabolism, pyruvate
metabolism and gut flora metabolism (Table S4).
Additionally, DIAAAꢀderivatization method was also sucꢀ
cessfully applied to the determination for the metabolites in
cell line, serum, feces, cerebrospinal fluid and tea. Compared
with nonꢀderivatization method, more metabolites could be
sensitively detected after DIAAAꢀderivatization (Figure S5).
9
CONCLUSION
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In summary, DIAAAꢀderivatization coupled with an
UHPLCꢀQꢀTOF/MS analysis was developed for mapping
global CCMs. The aliphatic chains and nitrogen atom in
DIAAA induced the polarity tuning of polar and lowꢀpolar
CCMs, which facilitated the high separation efficiency. At the
same time, the sensitivities were also enhanced up to 2000
times. This method was successfully applied for the investigaꢀ
tion on the potential biomarkers in CRC patients and animal
model. This innovative CCMs probe was proved to be a valuꢀ
able tool for inꢀdepth study on global metabolome, especially
for CCMs with high polarity differences.
Among the CCMs, the SCFAs group has profound effects
on the health of gut microenvironment. It regulates gut epitheꢀ
lial proliferation and immune responses. The decrease of
SCFAs in feces and the SCFAꢀproducing bacteria were found
to be associated with the etiology of CRC.25,26 Our results
showed that five SCFAs, PA, BA, isobutyric acid (IBA), isoꢀ
valeric acid (IVA) and VA, were all decreased in the sera of
CRC patients compared to the normal controls (Figure 3),
which well echo the previous findings.25,26 The area under the
receiver operating characteristic (ROC) curves of PA (0.81),
BA (0.80), IBA (0.79), IVA (0.80) and VA (0.80) indicated
they should be potential markers. To further confirm the relaꢀ
tionship between SCFAs and CRC, SCFAs in ApcMin/+ mice, a
wellꢀestablished animal model of intestinal cancer, were exꢀ
amined and compared with that in wild type mice. In this
model, Clostridiaceae, Lachnospiraceae and Ruminococca-
ceae bacteria, which generate SCFAs, were found to be deꢀ
creased.27 Similarly to CRC patients, IBA, IVA and VA sigꢀ
nificantly decreased in ApcMin/+ mice compared with wild type
C57BL/6J mice (Figure S4). The contents of PA and BA were
lower than that in control group, while the difference was not
statistically significant. Collectively, these data presented here
clearly indicated that variations of these SCFAs had a close
relationship with CRC. This significant differentiation beꢀ
tween the normal and CRC in the levels of SCFAs may proꢀ
vide another nonꢀinvasive diagnosis for CRC. In addition, this
is the first report on the SCFAs in the sera of CRC patients.
ASSOCIATED CONTENT
Supporting Information
Additional information as noted in text. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Email: nli@must.edu.mo. Tel: +853 8897 2405. Fax: +853 2882
5886 (N Li); Email: lliu@must.edu.mo. Tel: +853 8897 2799.
Fax: +853 2882 7222 (L Liu); Email: jlwu@must.edu.mo. Tel:
+853 8897 2406. Fax: +853ꢀ2882ꢀ2799 (J.ꢀL.W.)
Author Contributions
All authors have given approval to the final version of the manuꢀ
script.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work is supported by Macau Science and Technology Develꢀ
opment Fund [009/2017/A1] and the National Natural Science
Foundation of China (81772957).
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