Download - gutjnl-2021-325189-R1 Supplemental materials
1
Supplementary materials Table of contents
Supplementary Methods ......................................................................................................... 2
S Figure 1 Quality control checks. ........................................................................................... 9
S Figure 2 The PCA score plots for all samples containing QC samples. .............................. 10
S Figure 3 Metabolome alterations in HCC were found in various samples. ......................... 11
S Figure 4 Pro-HCC effect of L-tryptophan was validated in vitro. ....................................... 12
S Figure 5 Checking the assumption of proportional hazards by using log minus log function
plot ........................................................................................................................................... 13
S Figure 6 The relationship between age and potential harmful metabolites in portal vein serum
of HCC patients and healthy controls. ..................................................................................... 14
S Table 1 Study participant's characteristics for HCC patients and healthy controls in the
discovery cohort. ..................................................................................................................... 15
S Table 2 Study participant's characteristics for HCC patients and healthy controls in the
validation cohort. ..................................................................................................................... 17
S Table 3 The raw data of metabolomics assay for portal and central vein serum, liver tissues
and stool samples in HCC patients and healthy controls in the discovery and validation cohorts.
................................................................................................................................................. 19
S Table 4A In ESI- model, the mean relative change values for all identified metabolites in
portal vein and central vein serum of HCC patients and normal controls. .............................. 20
S Table 4B In ESI+ model, the mean relative change values for all identified metabolites in
portal vein and central vein serum of HCC patients and normal controls. .............................. 25
S Table 5A Mean relative abundance of 28 elevated metabolites in portal vein in HCC patients
compared to healthy controls in ESI- model ............................................................................ 33
S Table 5B Mean relative abundance of 28 elevated metabolites in portal vein in HCC patients
compared to healthy controls in ESI+ model ........................................................................... 34
S Table 6 The mean value of elevated metabolites in the discovery cohort was also increased
in portal vein of HCC patients in the independent validation cohort compared with healthy
controls. ................................................................................................................................... 35
S Table 7 The follow-up information for 52 HCC patients. ................................................... 36
S Table 8A In the discovery cohort, the relative concentration of linoleic acid and phenol in
portal vein serum of HCC patients and healthy controls. ........................................................ 38
S Table 8B In the discovery cohort, the relative concentration of linoleic acid and phenol in
stool samples of HCC patients and healthy controls. .............................................................. 41
S Table 8C In the validation cohort, the relative concentration of linoleic acid and phenol in
portal vein serum of HCC patients and healthy controls. ........................................................ 44
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
2
Supplementary Methods
Study population
For healthy subjects, all clinical examinations were within normal ranges, including physical
examination, blood tests (e.g., liver function, renal function, coagulation function, tumour
markers test), stool and related imaging examinations (e.g., liver ultrasound, abdominal CT
scan). Donors with systematic diseases (including hypertension, diabetes and metabolic
disease), irritable bowel syndrome (IBD), coeliac disease, and who received antibiotics or
probiotics or antifungal treatment within one month before sample collection were excluded.
For HCC cases, the diagnosis was diagnosed by comprehensive integration of imaging,
clinical symptoms, laboratory tests, medical history and further confirmed with
histopathological examination of specimens from surgical resection in the discovery and
validation cohort. The distributions of age and gender were matched as much as possible.
Collection of clinical data
Clinical data were collected by reviewing hospital electronic medical records. The following
data were recorded: age, gender, height, body weight, and blood pressure were measured.
Alpha-fetoprotein (AFP), aspartate aminotransferase (AST), alanine aminotransferase (ALT),
alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), total serum bilirubin and
serum albumin, platelets, prothrombin time (PT), tumour size and tumour differentiation. HCC
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
3
stage was determined according to the Barcelona Clinic Liver cancer (BCLC) staging system1
and liver function was assessed by Child-Pugh-Turcotte.2
Metabolome profiling of human serum, tissue and stool samples
Sample preparations. For portal vein and central vein serum, 100 µL of serum sample was
transferred to an EP tube and 300 µL extract solution (methanol) containing internal standard
(L-2-Chlorophenylalanine, 2 µg/mL). After 30s vortex, samples were sonicated for 10min in
ice-water bath. Then, samples were incubated at -40˚C for 1h and centrifuged at 12000 r.m.p.
for 15min at 4˚C. 100 µL of the supernatant was transferred to a fresh glass vial for LC-MS
analysis. For tissue and faecal samples, 25mg of tissue sample and 50mg of faecal sample were
weighted to an EP tube. 500 µL tissue extract solution (Acetonitrile: methanol: water = 2: 2: 1)
containing internal standard (L-2-Chlorophenylalanine, 4 µg/mL) and 1000𝜇L faecal extract
solution (Acetonitrile: methanol: water = 2: 2: 1) containing internal standard (L-2-
Chlorophenylalanine, 2 µg/mL) were added. After 30s vortex, the samples were homogenized
at 40Hz for 4min and sonicated for 5min in ice-water bath. The homogenization and sonication
cycle were repeated for 3 times. Then, the samples were incubated at -40˚C for 1 hour and
centrifuged at 10000 r.p.m. for 15min at 4˚C. 400 µL of supernatant was transferred to a fresh
tube and dried in a vacuum concentrator at 37˚C. Then, the dried samples were reconstituted
in 200 µL of 50% acetonitrile by sonication on ice for 10min. The constitution was then
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
4
centrifuged at 13000 r.p.m. for 15min at 4˚C. 75 µL of the tissue and faecal supernatant was
transferred to a fresh glass vial for LC-MS analysis. The QC sample was prepared by mixing
an equal aliquot of the supernatant from all of samples.
Metabolomics analysis
LC-MS/MS analysis were carried out using UHPLC System (1290, Agilent Technologies,
Santa Clara, CA), equipped with a UPLC BEH Amide column (2.1*100 mm, 1.7 µm, Waters).
The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonium
hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The analysis was carried with elution
gradient as follows: 0~0.5 min, 95% B; 0.5~7.0 min, 95%~65% B; 7.0~8.0 min, 65%~40% B;
8.0~9.0 min, 40% B; 9.0~9.1 min, 40%~95% B; 9.1~12.0 min, 95% B. The column
temperature was 25˚C. The auto-sampler temperature was 4˚C, and the injection volume was
2 µL (ESI+) or 2 µL (ESI-), respectively. A 6550 QTOF mass spectrometer (Agilent
Technologies) was utilized to acquire the full scan MSI data. The scan range was 60-1200 Da.
The ESI source conditions were set as following: Gas Temperature as 250˚C, Gas Flow as 16
L/min, Sheath Gas Temperature as 350˚C, Sheath Gas Flow as 12 L/min, Nebulizer as 20 psi,
Fragmentor as 175V, Capillary Voltage as 3000V. A triple TOF 6600 mass spectrometers (AB
Sciex) were used for its ability to acquire MS/MS spectra on an information-dependent
acquisition (IDA) during an LC/MS experiment. In this mode, the acquisition software
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
5
(Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects
and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle,
the most intensive 12 precursor ions with intensity greater than 100 were chosen for MS/MS
at collision energy (CE) of 30 eV. The cycle time was 0.56s. ESI source conditions were set as
following: Gas 1 as 60 psi, Gas 2 as 30 psi, Curtain Gas as 35 psi, Source Temperature as
600˚C, Declustering potential as 60V, Ion Spray Voltage Floating (ISVF) as 5000V or -4000V
in ESI+ or ESI- model, respectively.
Data analysis
MS raw data files were converted to the mzXML format by ProteoWizard and processed by R
package XCMS (version 3.2). The process includes peak deconvolution, alignment and
integration. Minfrac and cut off are set as 0.5 and 0.3 respectively. In-house MS2 database was
applied for metabolites identification. The metabolomics data analysis was conducted with
Python and R. The Pearson correlation coefficient between QC samples based on the relative
quantification of metabolites was set as a standard in the assessment of stability in
metabolomics data sets.3 An unsupervised model of PCA and supervised model of PLS-DA4
were applied to establish a model of the relationship between the level of metabolites and
sample category to assess the metabolome alterations among groups. A permutation test was
performed 200 times to assess the risk of overfitting for the PLS-DA model.5 The model
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
6
evaluation parameters (R2, Q2) obtained after seven-cross-validation. The is more stable and
reliable if R2 and Q2 are closer to 1. When the R2 in the model is greater than Q2 and the
intercept between Q2 regression line and Y- axis is less than 0, indicating the model is not
overfitting. Differential metabolites were identified on the basis of variable important in the
projection (VIP)> 1, P< 0.05 and fold change > 1.5 or < 0.667.3, 6-8 VIP scores estimate the
importance of each variable in the projection used in PLS-DA model and is often used for
variable selection. VIP scores more than one are considered relevant for group discrimination.
HCA was used for differential metabolites clustering analysis.9 A Wilcoxon Mann-Whitney
test with Benjamini-Hochberg-based false discovery rate was used for the statistical analysis,
setting P< 0.05 as the significance levels.10 A heatmap was used to express the results of
correlation analysis. The KEGG (https://www.genome.jp/kegg/pathway.html),11 HMDB
(https://hmdb.ca/metabolites) and LIPID MAPS databases were used for metabolites
annotation and pathway analysis. Correlation analysis of the metabolites with clinical
parameters was performed using SPSS.
Colony formation assay
For the cell colony formation assay, Hep3B, Huh7 and MIHA cells (1000 cells per well) were
plated and cultured with or without indicated metabolites in six-well plates for 5 to 7 days.
Cells were fixed with 70% ethanol and stained with 0.5% crystal violet solution. Colonies were
counted and photographed. All experiments were conducted three times.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
7
Apoptosis assay
Apoptosis was assessed using PE Annexin V Apoptosis Detection kit I (BD Pharmingen, San
Jose, CA, USA). Cells were harvested at 12h after indicated metabolites treatment. After
washing with PBS and resuspended in 100 µL annexin-binding buffer (10 mmol/L HEPES,
140 mmol/L NaCl, and 2.5 mmol/L CaCl2, pH 7.4) containing 2 µL Annexin V and 2 µL PI
(50 µg/mL) staining. Each sample was added with another 400 µL of 1Í annexin-binding
buffer and analysed by using FACSAria cell sorter (BD Biosciences, Franklin Lakes, NJ) after
incubating for 15 minutes at room temperature. The apoptotic cells were compared, and
apoptosis profiles were determined by Flowjo software. Experiments were performed three
times.
REFERENCES
1. Forner A, Reig M, Bruix J. Hepatocellular carcinoma. The Lancet 2018;391:1301-
1314.
2. Rimassa L, Personeni N, Czauderna C, et al. Systemic Treatment of Hcc in Special
Populations. J Hepatol 2020.
3. Rao G, Sui J, Zhang J. Metabolomics reveals significant variations in metabolites and
correlations regarding the maturation of walnuts (Juglans regia L.). Biol Open
2016;5:829-36.
4. Boulesteix AL, Strimmer K. Partial least squares: a versatile tool for the analysis of
high-dimensional genomic data. Brief Bioinform 2007;8:32-44.
5. Wang JB, Pu SB, Sun Y, et al. Metabolomic Profiling of Autoimmune Hepatitis: The
Diagnostic Utility of Nuclear Magnetic Resonance Spectroscopy. J Proteome Res
2014;13:3792-3801.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
8
6. Heischmann S, Quinn K, Cruickshank-Quinn C, et al. Exploratory Metabolomics
Profiling in the Kainic Acid Rat Model Reveals Depletion of 25-Hydroxyvitamin D3
during Epileptogenesis. Sci Rep 2016;6:31424.
7. Haspel JA, Chettimada S, Shaik RS, et al. Circadian rhythm reprogramming during
lung inflammation. Nat Commun 2014;5:4753.
8. Sreekumar A, Poisson LM, Rajendiran TM, et al. Metabolomic profiles delineate
potential role for sarcosine in prostate cancer progression. Nature 2009;457:910-4.
9. Chen X, Xie C, Sun L, et al. Longitudinal Metabolomics Profiling of Parkinson's
Disease-Related alpha-Synuclein A53T Transgenic Mice. PLoS One
2015;10:e0136612.
10. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate - a Practical and
Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series
B-Statistical Methodology 1995;57:289-300.
11. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic
Acids Res 2000;28:27-30.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
9
S Figure 1. Quality control checks. A-C, The Pearson correlation of serum ESI-
and ESI+
QC samples
(A), tissue ESI-
and
ESI+
QC samples (B) and faecal ESI-
and
ESI+
QC samples (C). ESI-
, negative
electrospray ionization; ESI+
, positive electrospray ionization. QC, quality control.
1 0.993 0.991 0.989 0.99 0.992 0.992 0.998 0.998 0.993 0.972 0.996 0.993
0.993 1 0.992 0.981 0.98 0.994 0.991 0.997 0.994 0.992 0.96 0.991 0.993
0.991 0.992 1 0.989 0.989 0.995 0.991 0.993 0.99 0.99 0.974 0.995 0.997
0.989 0.981 0.989 1 0.998 0.992 0.991 0.984 0.989 0.991 0.991 0.995 0.991
0.99 0.98 0.989 0.998 1 0.991 0.994 0.985 0.991 0.993 0.993 0.996 0.992
0.992 0.994 0.995 0.992 0.991 1 0.997 0.994 0.994 0.996 0.979 0.996 0.996
0.992 0.991 0.991 0.991 0.994 0.997 1 0.992 0.997 0.999 0.985 0.997 0.995
0.998 0.997 0.993 0.984 0.985 0.994 0.992 1 0.996 0.993 0.967 0.995 0.994
0.998 0.994 0.99 0.989 0.991 0.994 0.997 0.996 1 0.998 0.977 0.997 0.994
0.993 0.992 0.99 0.991 0.993 0.996 0.999 0.993 0.998 1 0.982 0.997 0.995
0.972 0.96 0.974 0.991 0.993 0.979 0.985 0.967 0.977 0.982 1 0.986 0.979
0.996 0.991 0.995 0.995 0.996 0.996 0.997 0.995 0.997 0.997 0.986 1 0.998
0.993 0.993 0.997 0.991 0.992 0.996 0.995 0.994 0.994 0.995 0.979 0.998 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
0.96
0.97
0.98
0.99
1.00
R2
Pearson correlation between neg QC samples
1 0.996 0.993 0.993 0.991 0.994 0.993 0.994 0.993 0.995 0.994 0.993 0.994 0.994 0.988 0.976 0.993 0.992 0.993 0.994 0.993 0.989
0.996 1 0.997 0.994 0.994 0.995 0.995 0.996 0.996 0.996 0.996 0.996 0.996 0.996 0.989 0.979 0.996 0.995 0.996 0.995 0.996 0.99
0.993 0.997 1 0.993 0.994 0.997 0.996 0.997 0.996 0.996 0.998 0.997 0.997 0.997 0.987 0.981 0.997 0.996 0.997 0.997 0.997 0.992
0.993 0.994 0.993 1 0.992 0.997 0.998 0.998 0.997 0.996 0.997 0.997 0.997 0.997 0.988 0.982 0.997 0.995 0.997 0.997 0.997 0.991
0.991 0.994 0.994 0.992 1 0.994 0.996 0.994 0.994 0.993 0.994 0.994 0.995 0.995 0.989 0.976 0.993 0.993 0.994 0.994 0.994 0.989
0.994 0.995 0.997 0.997 0.994 1 0.997 0.998 0.997 0.997 0.998 0.997 0.998 0.998 0.988 0.98 0.998 0.997 0.997 0.998 0.998 0.992
0.993 0.995 0.996 0.998 0.996 0.997 1 0.999 0.997 0.997 0.998 0.997 0.998 0.998 0.989 0.981 0.998 0.997 0.998 0.999 0.998 0.993
0.994 0.996 0.997 0.998 0.994 0.998 0.999 1 0.998 0.998 0.999 0.998 0.998 0.998 0.989 0.982 0.999 0.997 0.998 0.999 0.999 0.993
0.993 0.996 0.996 0.997 0.994 0.997 0.997 0.998 1 0.995 0.997 0.997 0.998 0.997 0.988 0.979 0.997 0.996 0.997 0.997 0.997 0.991
0.995 0.996 0.996 0.996 0.993 0.997 0.997 0.998 0.995 1 0.998 0.997 0.997 0.997 0.988 0.981 0.997 0.995 0.997 0.998 0.998 0.993
0.994 0.996 0.998 0.997 0.994 0.998 0.998 0.999 0.997 0.998 1 0.997 0.998 0.998 0.988 0.982 0.999 0.997 0.998 0.999 0.998 0.994
0.993 0.996 0.997 0.997 0.994 0.997 0.997 0.998 0.997 0.997 0.997 1 0.997 0.998 0.988 0.982 0.997 0.997 0.997 0.997 0.998 0.993
0.994 0.996 0.997 0.997 0.995 0.998 0.998 0.998 0.998 0.997 0.998 0.997 1 0.997 0.989 0.982 0.998 0.997 0.997 0.998 0.998 0.992
0.994 0.996 0.997 0.997 0.995 0.998 0.998 0.998 0.997 0.997 0.998 0.998 0.997 1 0.991 0.982 0.998 0.997 0.998 0.998 0.998 0.992
0.988 0.989 0.987 0.988 0.989 0.988 0.989 0.989 0.988 0.988 0.988 0.988 0.989 0.991 1 0.969 0.988 0.986 0.988 0.989 0.988 0.983
0.976 0.979 0.981 0.982 0.976 0.98 0.981 0.982 0.979 0.981 0.982 0.982 0.982 0.982 0.969 1 0.985 0.98 0.977 0.983 0.98 0.989
0.993 0.996 0.997 0.997 0.993 0.998 0.998 0.999 0.997 0.997 0.999 0.997 0.998 0.998 0.988 0.985 1 0.996 0.997 0.999 0.998 0.995
0.992 0.995 0.996 0.995 0.993 0.997 0.997 0.997 0.996 0.995 0.997 0.997 0.997 0.997 0.986 0.98 0.996 1 0.997 0.996 0.997 0.991
0.993 0.996 0.997 0.997 0.994 0.997 0.998 0.998 0.997 0.997 0.998 0.997 0.997 0.998 0.988 0.977 0.997 0.997 1 0.998 0.998 0.991
0.994 0.995 0.997 0.997 0.994 0.998 0.999 0.999 0.997 0.998 0.999 0.997 0.998 0.998 0.989 0.983 0.999 0.996 0.998 1 0.999 0.995
0.993 0.996 0.997 0.997 0.994 0.998 0.998 0.999 0.997 0.998 0.998 0.998 0.998 0.998 0.988 0.98 0.998 0.997 0.998 0.999 1 0.993
0.989 0.99 0.992 0.991 0.989 0.992 0.993 0.993 0.991 0.993 0.994 0.993 0.992 0.992 0.983 0.989 0.995 0.991 0.991 0.995 0.993 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC21
QC22
QC1QC2QC3QC4QC5QC6QC7QC8QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC21
QC22
0.97
0.98
0.99
1.00
R2
Pearson correlation between neg QC samples
1 0.995 0.995 0.985 0.994 0.992 0.995 0.99 0.995 0.992 0.992 0.994 0.994 0.992 0.994 0.996 0.994 0.993 0.986 0.959 0.989 0.993
0.995 1 0.999 0.991 0.998 0.997 0.999 0.99 0.994 0.998 0.996 0.997 0.998 0.995 0.999 0.999 0.998 0.996 0.98 0.964 0.994 0.998
0.995 0.999 1 0.99 0.999 0.996 0.999 0.992 0.996 0.998 0.997 0.998 0.997 0.998 0.998 0.999 0.999 0.997 0.983 0.967 0.995 0.997
0.985 0.991 0.99 1 0.987 0.993 0.992 0.992 0.983 0.987 0.983 0.987 0.987 0.991 0.993 0.99 0.987 0.992 0.969 0.955 0.979 0.986
0.994 0.998 0.999 0.987 1 0.993 0.997 0.991 0.996 0.999 0.999 0.999 0.998 0.997 0.996 0.999 0.999 0.998 0.982 0.967 0.998 0.999
0.992 0.997 0.996 0.993 0.993 1 0.998 0.987 0.991 0.994 0.989 0.993 0.994 0.992 0.998 0.996 0.994 0.993 0.977 0.962 0.986 0.992
0.995 0.999 0.999 0.992 0.997 0.998 1 0.99 0.994 0.997 0.995 0.997 0.997 0.995 0.999 0.999 0.998 0.996 0.98 0.964 0.992 0.996
0.99 0.99 0.992 0.992 0.991 0.987 0.99 1 0.992 0.991 0.99 0.991 0.99 0.994 0.99 0.991 0.991 0.995 0.987 0.956 0.989 0.991
0.995 0.994 0.996 0.983 0.996 0.991 0.994 0.992 1 0.995 0.994 0.995 0.994 0.992 0.994 0.995 0.996 0.994 0.992 0.958 0.993 0.995
0.992 0.998 0.998 0.987 0.999 0.994 0.997 0.991 0.995 1 0.998 0.998 0.997 0.996 0.996 0.998 0.999 0.997 0.982 0.965 0.996 0.997
0.992 0.996 0.997 0.983 0.999 0.989 0.995 0.99 0.994 0.998 1 0.999 0.997 0.997 0.993 0.997 0.999 0.997 0.981 0.967 0.999 0.998
0.994 0.997 0.998 0.987 0.999 0.993 0.997 0.991 0.995 0.998 0.999 1 0.998 0.998 0.995 0.998 0.999 0.998 0.981 0.972 0.998 0.998
0.994 0.998 0.997 0.987 0.998 0.994 0.997 0.99 0.994 0.997 0.997 0.998 1 0.995 0.997 0.998 0.998 0.997 0.98 0.963 0.996 0.999
0.992 0.995 0.998 0.991 0.997 0.992 0.995 0.994 0.992 0.996 0.997 0.998 0.995 1 0.993 0.996 0.997 0.998 0.978 0.969 0.995 0.994
0.994 0.999 0.998 0.993 0.996 0.998 0.999 0.99 0.994 0.996 0.993 0.995 0.997 0.993 1 0.998 0.997 0.995 0.98 0.962 0.99 0.996
0.996 0.999 0.999 0.99 0.999 0.996 0.999 0.991 0.995 0.998 0.997 0.998 0.998 0.996 0.998 1 0.999 0.997 0.981 0.965 0.995 0.998
0.994 0.998 0.999 0.987 0.999 0.994 0.998 0.991 0.996 0.999 0.999 0.999 0.998 0.997 0.997 0.999 1 0.998 0.982 0.967 0.997 0.998
0.993 0.996 0.997 0.992 0.998 0.993 0.996 0.995 0.994 0.997 0.997 0.998 0.997 0.998 0.995 0.997 0.998 1 0.981 0.965 0.996 0.997
0.986 0.98 0.983 0.969 0.982 0.977 0.98 0.987 0.992 0.982 0.981 0.981 0.98 0.978 0.98 0.981 0.982 0.981 1 0.94 0.979 0.982
0.959 0.964 0.967 0.955 0.967 0.962 0.964 0.956 0.958 0.965 0.967 0.972 0.963 0.969 0.962 0.965 0.967 0.965 0.94 1 0.965 0.958
0.989 0.994 0.995 0.979 0.998 0.986 0.992 0.989 0.993 0.996 0.999 0.998 0.996 0.995 0.99 0.995 0.997 0.996 0.979 0.965 1 0.997
0.993 0.998 0.997 0.986 0.999 0.992 0.996 0.991 0.995 0.997 0.998 0.998 0.999 0.994 0.996 0.998 0.998 0.997 0.982 0.958 0.997 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC21
QC22
QC1QC2QC3QC4QC5QC6QC7QC8QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC21
QC22
0.94
0.95
0.96
0.97
0.98
0.99
1.00
R2
Pearson correlation between pos QC samplesA
1 1 0.999 0.999 0.999 0.997 0.997 0.999 0.999 0.997 0.999 0.999 0.996 0.999 0.999 0.999 0.999 0.999 0.996 0.995
1 1 0.999 0.999 0.999 0.997 0.997 1 0.999 0.997 0.999 0.999 0.996 0.999 0.999 0.999 0.999 1 0.996 0.995
0.999 0.999 1 0.999 0.998 0.997 0.996 0.999 0.999 0.997 0.999 0.999 0.996 0.999 0.999 0.999 0.999 0.999 0.996 0.994
0.999 0.999 0.999 1 0.998 0.996 0.996 0.999 0.998 0.996 0.999 0.999 0.996 0.999 0.999 0.999 0.999 0.999 0.996 0.994
0.999 0.999 0.998 0.998 1 0.998 0.998 0.999 0.998 0.998 0.999 0.999 0.998 0.999 0.999 0.999 0.999 0.999 0.997 0.997
0.997 0.997 0.997 0.996 0.998 1 0.994 0.997 0.996 0.999 0.997 0.997 0.999 0.996 0.996 0.997 0.996 0.996 0.996 0.999
0.997 0.997 0.996 0.996 0.998 0.994 1 0.997 0.997 0.995 0.997 0.997 0.994 0.997 0.997 0.997 0.997 0.997 0.994 0.992
0.999 1 0.999 0.999 0.999 0.997 0.997 1 0.999 0.996 1 0.999 0.996 0.999 0.999 0.999 1 1 0.997 0.995
0.999 0.999 0.999 0.998 0.998 0.996 0.997 0.999 1 0.996 0.999 0.999 0.996 0.998 0.999 0.999 0.999 0.999 0.996 0.994
0.997 0.997 0.997 0.996 0.998 0.999 0.995 0.996 0.996 1 0.997 0.997 0.999 0.997 0.996 0.997 0.996 0.996 0.995 0.998
0.999 0.999 0.999 0.999 0.999 0.997 0.997 1 0.999 0.997 1 0.999 0.997 0.999 0.999 1 0.999 1 0.996 0.995
0.999 0.999 0.999 0.999 0.999 0.997 0.997 0.999 0.999 0.997 0.999 1 0.997 0.998 1 0.999 0.999 0.999 0.995 0.995
0.996 0.996 0.996 0.996 0.998 0.999 0.994 0.996 0.996 0.999 0.997 0.997 1 0.995 0.996 0.996 0.995 0.996 0.996 0.999
0.999 0.999 0.999 0.999 0.999 0.996 0.997 0.999 0.998 0.997 0.999 0.998 0.995 1 0.998 0.999 0.999 0.999 0.995 0.993
0.999 0.999 0.999 0.999 0.999 0.996 0.997 0.999 0.999 0.996 0.999 1 0.996 0.998 1 0.999 0.999 0.999 0.996 0.994
0.999 0.999 0.999 0.999 0.999 0.997 0.997 0.999 0.999 0.997 1 0.999 0.996 0.999 0.999 1 0.999 1 0.997 0.995
0.999 0.999 0.999 0.999 0.999 0.996 0.997 1 0.999 0.996 0.999 0.999 0.995 0.999 0.999 0.999 1 1 0.996 0.994
0.999 1 0.999 0.999 0.999 0.996 0.997 1 0.999 0.996 1 0.999 0.996 0.999 0.999 1 1 1 0.996 0.995
0.996 0.996 0.996 0.996 0.997 0.996 0.994 0.997 0.996 0.995 0.996 0.995 0.996 0.995 0.996 0.997 0.996 0.996 1 0.994
0.995 0.995 0.994 0.994 0.997 0.999 0.992 0.995 0.994 0.998 0.995 0.995 0.999 0.993 0.994 0.995 0.994 0.995 0.994 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
0.992
0.994
0.996
0.998
1.000
R2
Pearson correlation between neg QC samples
1 0.989 0.98 0.979 0.981 0.994 0.986 0.99 0.989 0.988 0.993 0.99 0.996 0.996 0.995 0.995 0.995 0.993 0.994 0.993
0.989 1 0.959 0.961 0.972 0.991 0.986 0.991 0.987 0.986 0.992 0.99 0.994 0.993 0.994 0.991 0.994 0.988 0.992 0.991
0.98 0.959 1 0.995 0.991 0.984 0.965 0.982 0.976 0.975 0.981 0.975 0.981 0.983 0.983 0.983 0.977 0.989 0.981 0.983
0.979 0.961 0.995 1 0.997 0.984 0.963 0.982 0.974 0.971 0.98 0.978 0.981 0.983 0.983 0.979 0.98 0.989 0.983 0.981
0.981 0.972 0.991 0.997 1 0.989 0.97 0.988 0.981 0.976 0.984 0.986 0.986 0.988 0.988 0.983 0.986 0.993 0.989 0.985
0.994 0.991 0.984 0.984 0.989 1 0.98 0.997 0.985 0.984 0.998 0.995 0.997 0.998 0.998 0.995 0.996 0.997 0.996 0.996
0.986 0.986 0.965 0.963 0.97 0.98 1 0.976 0.997 0.997 0.979 0.981 0.988 0.985 0.989 0.985 0.989 0.986 0.989 0.987
0.99 0.991 0.982 0.982 0.988 0.997 0.976 1 0.983 0.981 0.998 0.992 0.996 0.996 0.996 0.994 0.993 0.996 0.994 0.995
0.989 0.987 0.976 0.974 0.981 0.985 0.997 0.983 1 0.997 0.984 0.986 0.992 0.99 0.993 0.99 0.991 0.992 0.993 0.991
0.988 0.986 0.975 0.971 0.976 0.984 0.997 0.981 0.997 1 0.984 0.983 0.991 0.988 0.992 0.989 0.99 0.99 0.991 0.99
0.993 0.992 0.981 0.98 0.984 0.998 0.979 0.998 0.984 0.984 1 0.991 0.997 0.997 0.996 0.997 0.993 0.996 0.993 0.997
0.99 0.99 0.975 0.978 0.986 0.995 0.981 0.992 0.986 0.983 0.991 1 0.993 0.995 0.995 0.99 0.995 0.992 0.995 0.99
0.996 0.994 0.981 0.981 0.986 0.997 0.988 0.996 0.992 0.991 0.997 0.993 1 0.998 0.999 0.996 0.998 0.997 0.997 0.998
0.996 0.993 0.983 0.983 0.988 0.998 0.985 0.996 0.99 0.988 0.997 0.995 0.998 1 0.999 0.997 0.997 0.997 0.997 0.996
0.995 0.994 0.983 0.983 0.988 0.998 0.989 0.996 0.993 0.992 0.996 0.995 0.999 0.999 1 0.997 0.998 0.998 0.998 0.997
0.995 0.991 0.983 0.979 0.983 0.995 0.985 0.994 0.99 0.989 0.997 0.99 0.996 0.997 0.997 1 0.993 0.995 0.994 0.996
0.995 0.994 0.977 0.98 0.986 0.996 0.989 0.993 0.991 0.99 0.993 0.995 0.998 0.997 0.998 0.993 1 0.995 0.999 0.994
0.993 0.988 0.989 0.989 0.993 0.997 0.986 0.996 0.992 0.99 0.996 0.992 0.997 0.997 0.998 0.995 0.995 1 0.996 0.997
0.994 0.992 0.981 0.983 0.989 0.996 0.989 0.994 0.993 0.991 0.993 0.995 0.997 0.997 0.998 0.994 0.999 0.996 1 0.994
0.993 0.991 0.983 0.981 0.985 0.996 0.987 0.995 0.991 0.99 0.997 0.99 0.998 0.996 0.997 0.996 0.994 0.997 0.994 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC14
QC15
QC16
QC17
QC18
QC19
QC20
0.96
0.97
0.98
0.99
1.00
R2
Pearson correlation between pos QC samplesB
C
1 0.989 0.99 0.99 0.9910.9910.9920.9910.9890.9880.9910.9880.984
0.989 1 0.9920.9920.9910.9920.9940.992 0.99 0.99 0.9910.9890.986
0.99 0.992 1 0.991 0.99 0.99 0.992 0.99 0.9890.9870.9890.9860.983
0.99 0.9920.991 1 0.9920.9950.9950.9930.9920.9910.9930.9910.988
0.9910.991 0.99 0.992 1 0.9930.9940.9920.9920.9910.9930.9910.988
0.9910.992 0.99 0.9950.993 1 0.9940.9920.9920.9920.9930.9910.987
0.9920.9940.9920.9950.9940.994 1 0.9950.9940.9930.9950.9930.991
0.9910.992 0.99 0.9930.9920.9920.995 1 0.9950.9920.9930.9920.989
0.989 0.99 0.9890.9920.9920.9920.9940.995 1 0.9920.9930.9920.989
0.988 0.99 0.9870.9910.9910.9920.9930.9920.992 1 0.9940.9930.992
0.9910.9910.9890.9930.9930.9930.9950.9930.9930.994 1 0.9940.993
0.9880.9890.9860.9910.9910.9910.9930.9920.9920.9930.994 1 0.993
0.9840.9860.9830.9880.9880.9870.9910.9890.9890.9920.9930.993 1
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
QC1
QC2
QC3
QC4
QC5
QC6
QC7
QC8
QC9
QC10
QC11
QC12
QC13
0.984
0.988
0.992
0.996
1.000
R2
Pearson correlation between pos QC samples
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
10
S Figure 2. The PCA score plots for all samples containing QC samples. A-C, The PCA score plots for
ESI-
and ESI+
serum (A), tissue (B) and stool (C) samples containing QC samples and the QC samples
clustered tightly together. C_PV, portal vein of patients with HCC; N_PV, portal vein of normal liver donors;
C_CV, central vein of patients with HCC; N_CV, central vein of normal liver donors; C_TT, HCC tissue of
patients with HCC; C_ANT, adjacent non-cancerous tissue of patients with HCC; N_NT, normal tissue of
normal liver donors; C_S, stool samples of patients with HCC; N_S, stool samples of normal liver donors.
PCA, principal component analysis.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2021
22
23
24
25 26
27
28
29
30
31
32
33
34
35
36
37
38
3940
41
42
43
4445
46
47
48
4950
51
52
5354
55
56
5758
59
60
61
62
63
64
65
66
67
68
69
7071
72
73
74
75
76
7778
79
80
81
82
8384
8586
87
888990
91
92
9394
95
96
97
9899
100
101
102
103104105106107108
109110
111112113114115
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●●
●●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●●
●
●●
●●●●●
−20
−10
0
10
−20 −10 0 10 20
PC1 (12.02%)
PC
2 (
10.0
9%
) class
a●
a●
a●
C_S
N_S
QC
A
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
2122
23
24
252627
28
2930
31
3233
34
35
36
37
38
39
40
4142
43
44
4546
4748
49
50
51
52
53
54
55
5657
58
59
60
61
62
63
64
65 66
6768
69
70
71
72
73
74
75
76
77
78
79
8081
82
83
84
85
86
87
8889
90
91
92
93
9495
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112113
114115116
117
118
119120
121
122
123
124
125
126
127
128
129
130
131
132
133134
135
136
137
138139
140
141142
143144
145
146147
148
149
150
151152
153
154
155
156157
158
159
160
161
162
163
164
165166
167
168
169
170
171
172173
174
175
176
177
178179
180181
182
183184 185
186
187
188
189190 191
192
193
194
195
196
197198
199
200
201
202203
204
205206207208
209210211212
213
214215216217218219220221222223224225226
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●
●
● ●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●●
●●●●
●
●
●●●●
●
●●
●
●●●
●
−5
0
5
−10 0 10
PC1 (13.24%)
PC
2 (
6.2
3%
)
class
a●
a●
a●
a●
a●
C_CV
C_PV
N_CV
N_PV
QC
1
2
3
4
5
6
7
8
9
10
1112
13
1415161718
19
2021
22
23
2425
2627
28
29
30
31
32
33
34
35
36
3738
39
40
414243
44
45
46
47
484950
51
52
53
54
5556
57
58
59
60
61
62
63
64
65
666768
69
7071
72
73
74
75
76
7778
79
80
81
82
8384
85
86
87
8889
90
91
92
93
94
95
96
97
98
99100
101102
103
104
105
106
107108
109
110
111
112
113
114115116
117
118119120
121
122
123
124125
126
127
128129
130
131
132
133134
135
136
137
138
139140
141
142143144
145
146147
148
149
150
151
152
153154
155156157158159160
161
162
163
164165166
167168
169
170
171
172173
174
175
176
177178
179
180181
182
183
184
185
186187
188
189190
191192
193
194
195196
197
198199
200
201202
203204
205206207208209210211212213214215216217218219220221222223224225226
●
●
●
●
●
●
●
●
●
●
●●
●
●●●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●●
●
●●
●
●
●
●
●
●
●
●●
●●●●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
● ●
●
● ●
●
●
●
●
●
●●●●●●●●
●
●●●●●●●●●●●●●
−10
−5
0
5
10
−20 −10 0 10 20
PC1 (14.70%)
PC
2 (
7.9
6%
)
class
a●
a●
a●
a●
a●
C_CV
C_PV
N_CV
N_PV
QC
1234
567
8
9
10
111213
14
15
16
1718
19
20
21
22
23
24
25
26
2728
29
30
3132
33
34
35
363738
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
585960
61
62
63
64
6566
67
68
69
7071
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
878889
90
91
92
93
94
95
9697
98
99
100
101
102
103104
105
106107108
109110
111
112113
114115116
117
118
119
120
121
122
123124
125126
127
128129
130
131132
133
134135
136
137138139
140 141142143
144145146
147
148
149150
151152153
154155156157158159160161162163164165166167168169170171172173174
●●●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●●
●
●
●
●●
●●
●
●
●
●
●
●
●
●●
●
●
●
●●
●
●●
●
● ●
●
●
●●
●●
●
●
●
●●
●
●
●●
●●●
●
●●●●●●●●●●●●●●●●●●●●
−20
−10
0
10
0 20 40 60
PC1 (24.81%)
PC
2 (
11.8
6%
) class
a●
a●
a●
a●
C_ANT
C_TT
N_NT
QC
1 234
56
7
8
9
1011
1213
14
1516
17
18
19
20
21
22
23
24
25 262728
29
30
3132
3334 35
3637
38
39
40
41
42
43
4445
46
4748
49
50
51
52
53
54
55
56
57
58
5960
61
62
63
64
65
66
67
68
69
70
71
72
73 7475
76
7778
79
80
8182
83
8485
86
87
88
89
9091
9293
9495
96
97
98
99
100
101
102
103
104
105106107
108
109110
111
112113114
115
116
117
118
119
120
121
122
123124
125126
127
128129130131
132
133
134135
136
137
138139
140141
142
143144
145146147148
149150
151152153154
155156157158159160161162163164165166167168169170171172173174
●●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●
●
●●
●●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●●●●●●●●●●●●●●●●●●●●
−20
−10
0
10
20
−40 −20 0 20
PC1 (21.17%)
PC
2 (
12.7
1%
) class
a●
a●
a●
a●
C_ANT
C_TT
N_NT
QC
1
234
5
67
8
9
10
11
12
1314
15
16
17
18
19
20
21
2223
24
25
26
27
28
29
30
31
3233
34
35
36
37
3839
40
41
42
43
44
4546
47
4849
5051
52
53
54
55
5657
58
59
60
61
6263
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
8081
82
83
84
85
86
87
888990
91
92
939495
96
97
9899
100
101
102
103104105106107108109110111112113114115
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●●●
●
●
●
●
●
●
●
●
●●●●●●●●●●●●
−20
−10
0
10
−20 0 20 40 60
PC1 (26.89%)
PC
2 (
6.8
7%
) class
a●
a●
a●
C_S
N_S
QC
ESI- ESI
+
B
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
12
S Figure 4. Pro-HCC effect of L-tryptophan was validated in vitro. A-C, L-tryptophan significantly
inhibited the cell viability of HCC cells Hep3B (A) and Huh7 (B), but not normal hepatocyte cell MIHA (C),
as determined by MTT assay. Data are expressed as mean± SD. Statistical significance was determined by
two-way analysis of variance. * P< 0.05, **** P< 0.0001, ns, no significant.
1 2 3 4 5 day0.0
0.2
0.4
0.6
0.8
1.0Hep3B-L-tryptophan
Cell v
iab
ilit
y (
OD
=570)
Control
60uM*
****
1 2 3 4 5 day0.06
0.08
0.10
0.12
0.14
0.16
Huh7-L-tryptophan
Cell v
iab
ilit
y (
OD
=570)
Control
60uM
*
1 2 3 4 5 day0.0
0.5
1.0
1.5
2.0MIHA-L-tryptophan
Cell v
iab
ilit
y (
OD
=570)
Control
60uM
A
B
C
ns
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
15
S Table 1. Study participant's characteristics for HCC patients and healthy controls in
the discovery cohort
Donor (n=50)
HCC (n=52)
P-Value
Age (year) 33.18±6.67 57.9±10.14 <0.0001
Gender
Female 21 (42.0%) 10 (19.2%) 0.012
Male 29 (58.0%) 42 (80.8%)
BMI 23.51±2.47 23.25±3.27 0.656
AFP
≤7 50 (100%) 17 (32.7%) ——
>7 0 (0%) 35 (67.3%) ——
Tumor size (cm)
2≤&≤5 —— 22 ——
5<&≤10 —— 16 ——
>10 —— 14 ——
Tumor differentiation
I-II —— 30 ——
III-IV —— 22 ——
Child-Pugh-Turcotte
A —— 50 (96.2%) ——
B —— 2 (3.8%) ——
ALT (U/L) 19.00 [12.00-28.50] 32.50 [24.75-56.00] <0.0001
AST (U/L) 19.00 [15.00-26.00] 43.50 [31.00-65.75] <0.0001
GGT (U/L) 19.50 [13.37-32.50] 83.00 [39.50-159.15] <0.0001
ALP (U/L) 84.00 [70.75-93.25] 96.00 [73.00-133.75] 0.044
Total protein (g/L) 74.42±4.67 69.48±6.84 <0.0001
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
16
Albumin (g/L) 47.01±3.19 41.39±4.53 <0.0001
Globulin (g/L) 27.41±3.31 28.09±6.21 0.0846
Total bilirubin
(µmol/L)
11.35 [8.60-14.22] 13.45 [9.22-18.01] 0.115
Direct bilirubin
(µmol/L)
3.80 [2.80-5.05] 4.80 [3.46-6.77] 0.013
Prothrombin time (s) 10.52±0.57 12.09±1.44 <0.0001
Platelets (10^9/L) 259.26±61.94 156.73±78.04 <0.0001
Aetiological factors
HBV+ —— 34 (65.4%) ——
BCLC stage
A —— 38 (73.1%) ——
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
17
S Table 2. Study participant's characteristics for HCC patients and healthy controls in
the validation cohort
Donor (n=50)
HCC (n=50)
P-Value
Age (year) 33.56±7.60 57.64±13.56 <0.0001
Gender
Female 21 (42.0%) 13 (26.0%) 0.091
Male 29 (58.0%) 37 (74.0%)
BMI 23.50±2.75 23.13±2.88 0.522
AFP
≤7 50 (100%) 6 (12.0%) ——
>7 0 (0%) 44 (88.0%) ——
Tumor size (cm)
2≤&≤5 —— 18 ——
5<&≤10 —— 20 ——
>10 —— 12 ——
Tumor differentiation
I-II —— 35 ——
III-IV —— 15 ——
Child-Pugh-Turcotte
A —— 49 (98.0%) ——
B —— 1 (2.0%) ——
ALT (U/L) 18.5 [11.00-28.25] 30.5 [23.00-43.50] <0.0001
AST (U/L) 19.00 [17.00-22.25] 32.00 [24.00-53.50] <0.0001
GGT (U/L) 19.50 [15.00-38.00] 101.50 [66.00-174.50] <0.0001
ALP (U/L) 83.50 [70.50-97.50] 95.00 [77.75-121.25] 0.032
Total protein (g/L) 73.97±5.33 68.08±6.79 <0.0001
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
18
Albumin (g/L) 46.96±4.66 38.44±4.73 <0.0001
Globulin (g/L) 27.00±3.95 29.64±5.41 0.007
Total bilirubin
(µmol/L)
11.45 [8.68-15.18] 15.50 [11.10-18.28] 0.008
Direct bilirubin
(µmol/L)
4.05 [2.80-5.45] 5.80 [3.68-6.95] 0.001
Prothrombin time (s) 11.04±0.80 12.33±2.06 <0.0001
Platelets (10^9/L) 243.84±55.47 176.12±66.22 <0.0001
Aetiological factors
HBV+ —— 35 (70.0%) ——
BCLC stage
A —— 37 (74.0%) ——
Notes: Continuous variables were expressed in mean ± standard deviation or median (range)
as appropriate. Qualitative and quantitative differences between subgroups were analyzed
using Chi-squared test or Fisher’s exact test for categorical parameters, and Student’s t test or
Mann-Whitney test for continuous parameters as appropriate. P values of <0.05 were
considered as being statistically significant. AFP, alpha-fetoprotein; BMI, body mass index;
ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, glutamyl
transpeptidase; ALP, alkaline phosphatase; BCLC, Barcelona Clinic Liver Cancer; HCC,
hepatocellular carcinoma; HBV, hepatitis B virus.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
19
S Table 3. The raw data of metabolomics assay for portal and central vein serum, liver
tissues and stool samples in HCC patients and healthy controls in the discovery and
validation cohorts.
3A, In ESI- model, 156 metabolites were annotated in portal and central vein serum of HCC
patients and healthy controls in the discovery cohort. C_PV, portal vein of patients with HCC; N_PV,
portal vein of normal liver donors; C_CV, central vein of patients with HCC; N_CV, central vein of normal liver donors.
3B, In ESI+ model, 285 metabolites were annotated in portal and central vein serum of HCC
patients and healthy controls in the discovery cohort.
3C, In ESI- model, 305 metabolites were annotated in HCC, non-cancerous and normal liver
tissues in the discovery cohort. C_TT, HCC tissue of patients with HCC; N_NT, normal tissue of normal liver
donors; C_S, C_ANT, adjacent non-cancerous tissue of patients with HCC.
3D, In ESI+ model, 535 metabolites were annotated in HCC, non-cancerous and normal liver
tissues in the discovery cohort.
3E, In ESI- model, 440 metabolites were annotated in stool samples of HCC patients and
healthy controls in the discovery cohort. C_S, stool samples of patients with HCC; N_S, stool samples of
normal liver donors.
3F, In ESI+ model, 703 metabolites were annotated in stool samples of HCC patients and
healthy controls in the discovery cohort.
3G, In ESI- model, 146 metabolites were annotated in portal and central vein serum of HCC
patients and healthy controls in the validation cohort.
3H, In ESI+ model, 229 metabolites were annotated in portal and central vein serum of HCC
patients and healthy controls in the validation cohort.
*For the data of S Table 3, we would like to submit through offline method because of its
large file size.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
20
S Table 4A. In ESI- model, the mean relative change values for all identified metabolites
in portal vein and central vein serum of HCC patients and normal controls
Name Mean of abundance
((portal vein-central vein)/central vein)
Normal HCC
Sebacic acid 0.2592 5.5975
Glycocholic acid 29.4513 5.0856
Taurocholate 42.3702 4.7756
sn-Glycerol 3-phosphoethanolamine 0.7585 3.2620
Glycerol 3-phosphate -0.0013 2.4819
Glycochenodeoxycholate 10.0648 2.2258
Traumatic Acid 0.0215 1.6378
Phenylacetylglycine 0.2496 1.2721
Xanthine 0.3498 0.9551
Glycolithocholic acid 8.2446 0.8770
L-Citrulline 0.7646 0.8634
Xanthosine -0.0945 0.8398
N-Acetylneuraminic acid -0.0734 0.7860
L-Galactono-1,4-lactone 0.1478 0.7831
3-Methoxy-4-Hydroxyphenylglycol Sulfate 0.1711 0.7226
Phosphorylcholine 0.0600 0.7091
3-Hydroxycapric acid 0.2915 0.7023
Pantothenate -0.0375 0.6280
L-Sorbose 0.8765 0.6103
Prostaglandin H2 0.7527 0.5996
Alpha-D-Glucose 0.0067 0.5657
D-galacturonic acid 0.1595 0.5494
Taurochenodeoxycholate 12.2075 0.5419
Xylitol -0.0245 0.5305
Nicotinamide -0.2203 0.5021
D-Glucuronate 0.1869 0.4959
Hippuric acid 0.2762 0.4874
N-Acetylglucosamine 1-phosphate 0.0691 0.4868
D-Maltose -0.8013 0.4844
Galactonic acid 0.1493 0.4764
N1-Methyl-2-pyridone-5-carboxamide 0.1818 0.4407
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
21
L-Leucine 0.0886 0.4392
Dihydrothymine 0.0696 0.4015
L-Methionine 0.0203 0.3983
Glucosamine -0.0168 0.3964
N-Acetyl-L-alanine -0.0106 0.3813
Capric acid 0.0590 0.3753
D-Xylulose 0.1148 0.3722
Deoxyinosine -0.5810 0.3708
Alpha-N-Phenylacetyl-L-glutamine 0.0222 0.3597
Dodecanoic acid 0.1601 0.3570
Hydroxyphenyllactic acid -0.0310 0.3515
Biliverdin -0.2165 0.3489
Indoleacrylic acid 0.1305 0.3458
D-Mannose -0.0403 0.3417
DL-3-Phenyllactic acid 0.1022 0.3147
3-Hydroxydodecanoic acid 0.1606 0.3047
D-Allose 0.3856 0.3043
D-(+)-Melibiose 0.6791 0.3014
3-Phenylpropanoic acid 2.9134 0.3001
Stavudine -0.0056 0.2980
L-Threonine 0.0145 0.2897
Inosine 0.1066 0.2864
L-Rhamnose -0.2380 0.2831
myo-Inositol -0.1179 0.2805
D-Lyxose -0.0634 0.2717
3-Hydorxy-3-methylglutaric acid -0.0049 0.2620
Anthranilic acid (Vitamin L1) 0.1419 0.2561
Mevalonic acid 0.2937 0.2506
Salicylic acid -0.1118 0.2474
Glyceric acid 0.1155 0.2437
L-Asparagine -0.0154 0.2345
Pseudouridine -0.0091 0.2322
pregnenolone sulfate -0.1086 0.2305
Azelaic acid -0.0239 0.2293
D-Proline 0.0437 0.2209
Pelargonic acid -0.1507 0.2198
L-Histidine -0.0426 0.2106
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
22
Cysteine-S-sulfate 0.1237 0.2099
cis-9-Palmitoleic acid 0.2582 0.2094
all cis-(6,9,12)-Linolenic acid 0.5030 0.2019
DL-Serine -0.0724 0.1953
L-Iditol -0.3103 0.1883
L-Threonate -0.0630 0.1841
Taurine -0.0204 0.1816
Hydroxyisocaproic acid 0.0150 0.1733
L-Tryptophan -0.2276 0.1708
1-methylguanosine 0.1781 0.1560
L-Glutamate -0.1167 0.1542
Myristic acid 0.2453 0.1540
D-glucosamine 6-phosphate 0.0939 0.1538
Linoleic acid 0.5141 0.1533
L-Cystine 0.0878 0.1457
4-Pyridoxic acid -0.0888 0.1392
3-Hydroxyisovaleric acid -0.0630 0.1340
Oleic acid 0.7902 0.1253
Allantoin 0.2822 0.1171
Succinate -0.0108 0.1167
Palmitic acid 0.1393 0.1162
L-Phenylalanine -0.0803 0.1097
ketoisocaproic acid -0.0490 0.1097
L-Pipecolic acid -0.0701 0.1090
Uracil -0.1341 0.1042
Acetylcarnitine -0.0486 0.0945
DL-O-tyrosine -0.0441 0.0944
Stearic acid 0.0026 0.0924
Heptadecanoic acid 0.0508 0.0850
D-Quinovose -0.0459 0.0849
2-Oxoadipic acid 0.0731 0.0829
Pentadecanoic Acid -0.0931 0.0721
D-Sorbitol -0.0484 0.0707
(4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,1 6,19-
Docosahexaenoic acid -0.0994 0.0683
4-Methoxyphenylacetic acid -0.0935 0.0656
Uric acid -0.0412 0.0651
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
23
L-Pyroglutamic acid 0.0464 0.0588
Ribothymidine -0.0110 0.0528
Dihydroxyacetone -0.0181 0.0493
O-Succinyl-L-homoserine -0.0107 0.0465
DL-lactate -0.0198 0.0445
D-Threitol -0.1441 0.0412
Salicyluric acid 0.0372 0.0368
L-Carnitine -0.1119 0.0356
Glycyl-L-leucine -0.2446 0.0174
1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphate -0.0608 0.0168
2-Hydroxy-3-methylbutyric acid -0.0245 0.0155
11-Keto-.beta.-boswellic acid 0.2580 0.0117
Barbituric acid -0.0584 0.0095
Bilirubin -0.1615 0.0017
Chenodeoxycholate -0.1014 0.0015
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate -0.0297 -0.0036
Hypoxanthine -0.0118 -0.0039
2'-Deoxy-D-ribose -0.0826 -0.0083
L-Glutamine -0.0453 -0.0181
Deoxycholic acid -0.1090 -0.0184
Taurolithocholic acid -0.2713 -0.0204
Thymidine -0.1538 -0.0290
Propionic acid -0.0007 -0.0414
Tetrahydrocorticosterone -0.2240 -0.0476
2-Deoxyribose 5-phosphate -0.0988 -0.0489
D-Galactarate 0.1024 -0.0496
Caprylic acid -0.0244 -0.0544
Acadesine (Drug) -0.1202 -0.0603
Thymine 0.0858 -0.0645
Enterostatin human -0.3029 -0.0679
1-Palmitoyl-2-hydroxy-sn-glycero-3-
phosphoethanolamine -0.3321 -0.0681
Acetylcholine -0.0324 -0.0725
Protoporphyrin IX 0.4796 -0.0751
L-Valine -0.0459 -0.0777
2-Oleoyl-1-stearoyl-sn-glycero-3-phosphoserine -0.1096 -0.0787
Cholic acid -0.0983 -0.1218
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
24
Oxypurinol -0.1057 -0.1228
1-Oleoyl-L-.alpha.-lysophosphatidic acid -0.3378 -0.1436
Benzoic acid -0.1799 -0.1698
Cholesteryl sulfate -0.0612 -0.1756
3,4-Dihydroxybenzoate (Protocatechuic acid) -0.3007 -0.1834
1-Stearoyl-sn-glycerol 3-phosphocholine -0.1496 -0.1904
1-Oleoyl-sn-glycerol 3-phosphate -0.0613 -0.1972
Phenol -0.0711 -0.2080
Indoxyl sulfate -0.3378 -0.2228
2'-O-Methyluridine -0.2198 -0.2248
5-Hydroxyindoleacetate -0.3856 -0.2699
Indolelactic acid -0.2507 -0.2792
D-Glucosamine 1-phosphate (Glucosamine-1P) -0.6699 -0.2870
Urocanic acid -0.0708 -0.3207
gamma-L-Glutamyl-L-valine -0.5672 -0.3979
Estrone 3-sulfate -0.6798 -0.8164
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
25
S Table 4B. In ESI+ model, the mean relative change values for all identified metabolites
in portal vein and central vein serum of HCC patients and normal controls
Name Mean of abundance
((portal vein-central vein)/central vein)
Normal HCC
Taurocholate 0.7245 6.0560
Pro-Thr -0.6825 5.1939
Cholic acid 0.5748 3.7968
Abacavir 2.1891 3.4566
Glycocholic acid 41.0489 3.3600
EDTA -0.2572 3.1757
Synephrine 4.9471 2.7561
2'-Deoxyadenosine 5'-monophosphate (dAMP) -0.1100 2.6493
Met-Tyr 0.2464 2.2836
Taurochenodeoxycholate 1.6744 2.2721
Glycochenodeoxycholate 11.0974 2.0125
Ile-Tyr 0.3730 1.9349
L-Abrine 0.1149 1.7305
sn-Glycerol 3-phosphoethanolamine 1.9113 1.5057
beta-Octylglucoside 0.3841 1.4473
Val-Met 0.4856 1.4369
Glu-Ser -0.0706 1.4048
Tyr-Pro 1.4455 1.3088
Chenodeoxycholate 0.6841 1.2674
Lys-Ser 0.0585 1.0750
(-)-Ephedrine -0.7994 1.0727
N-Phenylacetamide -0.2492 1.0174
Gln-Asn 0.3569 0.9955
Pro-Arg -0.3588 0.9826
Sphinganine 0.0837 0.9800
Argininosuccinic acid 0.5964 0.9596
Guanosine 0.2036 0.9538
Gly-Arg 0.4373 0.9178
1-Methylnicotinamide 0.2384 0.8758
L-Saccharopine 0.4772 0.8559
His-Tyr 0.2730 0.8463
5-Methyltetrahydrofolate (5-Methyl-THF) 3.3359 0.8432
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
26
N-(omega)-Hydroxyarginine 0.0767 0.8185
Val-Tyr 0.2307 0.7901
Lys-Pro 0.0265 0.7839
Pseudouridine 0.1163 0.6949
Monomethyl glutaric acid -0.4939 0.6721
D-gluconate 0.3356 0.6605
Isomaltose 1.4611 0.6403
Thr-Tyr 0.3022 0.6015
Lys-Asn 0.4297 0.5992
Nicotinamide 0.0528 0.5857
7-Keto-8-aminopelargonic acid 0.0719 0.5191
trans-Vaccenic acid -0.1235 0.4851
1,7-Dimethylxanthine -0.1624 0.4694
L-Citrulline 0.9665 0.4678
N-Acetylmannosamine 0.0275 0.4498
Adenosine -0.0002 0.4463
Stearamide 0.0748 0.4431
Methyldopa -0.5538 0.4369
2-Phenylbutyric acid 0.2728 0.4310
2-Furancarboxylic acid 0.7540 0.4038
1-Stearoyl-rac-glycerol -0.3648 0.3973
Arg-Ser -0.1586 0.3959
Pyridoxal (Vitamin B6) 0.1336 0.3954
Ile-Trp 0.2294 0.3907
2-Methyl-3-hydroxybutyric acid 0.9068 0.3776
Erucamide 0.1218 0.3745
1-methylguanosine 0.2769 0.3742
L-Threonate 0.3873 0.3739
L-Tyrosine -0.0167 0.3538
trans-3-Coumaric acid -0.0286 0.3523
1-Palmitoyl-sn-glycero-3-phosphocholine 0.2244 0.3499
Glutaraldehyde -0.0213 0.3481
Ile-Phe 0.2630 0.3445
3.alpha.-Mannobiose 0.5830 0.3423
Dopamine -0.0240 0.3398
3-(Methylthio)-1-propanol -0.1698 0.3362
Triethanolamine 0.1769 0.3315
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
27
Inosine 0.1196 0.3314
Ergothioneine 0.1116 0.3242
Pro-Tyr 0.3738 0.3109
Pimelic acid -0.6798 0.3090
2-Methylguanosine 0.1931 0.3014
Lisinopril 0.4556 0.3004
4-Imidazoleacetic acid 0.2406 0.2975
Phe-Tyr 0.0871 0.2939
N1-Methyl-2-pyridone-5-carboxamide 0.2662 0.2902
Coumarin 0.1919 0.2824
N-Acetylglutamine 0.1193 0.2717
Diethylcarbamazine 0.0907 0.2661
Pro-Met 0.0709 0.2609
Ala-Leu 0.1263 0.2599
5-Methoxyindoleacetate 0.0359 0.2473
Promethazine 0.0192 0.2424
2'-O-methylcytidine 0.0129 0.2417
Ser-Leu 0.2168 0.2392
DL-Methionine sulfoxide 0.1593 0.2273
Glycerophosphocholine -0.0001 0.2256
Hydroxyproline 0.0175 0.2251
Ala-Ala 0.0901 0.2178
Alpha-N-Phenylacetyl-L-glutamine 0.2192 0.2123
3-Furancarboxylic acid -0.0156 0.2065
Pentadecanoic Acid 0.0788 0.2065
L-Carnitine -0.0101 0.2060
L-Leucine 0.0814 0.2057
Biliverdin -0.0909 0.2050
Urocanic acid -0.0063 0.2013
1-Stearoyl-sn-glycerol 3-phosphocholine 0.3076 0.1982
5-Aminopentanoic acid 0.1858 0.1957
L-Palmitoylcarnitine -0.1457 0.1945
N-Acetyl-L-tyrosine 0.1791 0.1811
D-Ornithine 0.0013 0.1806
L-Methionine 0.0127 0.1664
Pro-Val 0.2528 0.1651
DL-Phenylalanine 0.0632 0.1604
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
28
Pro-Gly -0.3462 0.1603
trans-cinnamate 0.0452 0.1593
L-Kynurenine 0.0493 0.1582
DL-3-Phenyllactic acid 0.0422 0.1578
N-Benzyloxycarbonylglycine 0.0177 0.1575
1-Stearoyl-2-hydroxy-sn-glycero-3-
phosphocholine 0.2782 0.1552
2'-O-methylinosine -0.0088 0.1527
Phosphorylcholine 0.0063 0.1522
Tyramine 0.0453 0.1496
L-Arabinose 0.0558 0.1432
Nortriptyline 0.4086 0.1412
Diaminopimelic acid -0.0645 0.1283
D-Lyxose 0.0513 0.1280
N6-methyladenosine 0.0132 0.1253
N-Acetylaspartylglutamate (NAAG) -0.2536 0.1251
D-Mannose 0.0716 0.1249
Acetoacetic acid -0.0226 0.1226
Lys-Cys -0.0311 0.1212
2-Amino-1-phenylethanol 0.1892 0.1147
(R)-3-Hydroxybutyric acid 0.1651 0.1145
Trimethylamine N-oxide -0.0899 0.1133
L-Phenylalanine 0.0438 0.1130
3,3-Dimethylglutaric acid -0.1522 0.1087
Pyruvaldehyde 0.0691 0.1063
Ser-Val -0.3474 0.1060
Taurodeoxycholic acid -0.0038 0.1018
Stearoylcarnitine -0.0853 0.1012
DL-2-Aminoadipic acid 0.0285 0.0978
cis-9-Palmitoleic acid -0.2065 0.0955
6-Hydroxydopamine -0.2825 0.0929
Cyclohexylamine -0.1282 0.0900
Guanidine 0.0855 0.0891
N-Acetyl-L-glutamate 0.0127 0.0886
Diacetyl 0.0264 0.0872
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine 0.0265 0.0832
DL-O-tyrosine 0.0002 0.0817
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
29
5-L-Glutamyl-L-alanine 0.0932 0.0775
Indoleacrylic acid -0.2084 0.0761
N,N-Dimethylaniline 0.0326 0.0735
Cytidine 0.1298 0.0686
Thr-Trp 2.4761 0.0673
Glutathione disulfide 0.6441 0.0660
Uridine -0.0962 0.0654
Ile-Met 0.3355 0.0652
3-(2-Hydroxyethyl)indole -0.1746 0.0649
Taurine -0.0092 0.0630
Indole-3-pyruvic acid -0.1659 0.0624
Pyridoxamine (PM) -0.0032 0.0622
Betaine 0.1613 0.0574
L-Proline 0.0747 0.0541
2(1H)-Pyridinone -0.2434 0.0539
Cytosine 0.0807 0.0539
Imidazole 0.0365 0.0529
Sarcosine 0.1230 0.0523
Anthranilic acid (Vitamin L1) -0.0046 0.0522
6-Aminocaproic acid -0.1975 0.0509
Uracil -0.0837 0.0431
L-Valine -0.0850 0.0369
DL-Indole-3-lactic acid 0.0055 0.0353
5-Methyl-5,6-Dihydrouracil 0.1069 0.0350
Linoleic acid -0.1769 0.0305
Lys-Gly -0.3048 0.0303
Formylanthranilic acid -0.1987 0.0303
3-Methylhistamine -0.2721 0.0301
L-Tryptophan -0.1984 0.0280
Acetylcarnitine -0.0130 0.0229
N6-Acetyl-L-lysine -0.1972 0.0218
Ala-Gly -0.0821 0.0191
2'-Deoxyguanosine 5'-diphosphate (dGDP) -0.3828 0.0186
Pyrocatechol 0.0333 0.0170
L-Asparagine 0.1048 0.0156
PC(16:0/16:0) -0.1798 0.0089
L-Histidine -0.0570 0.0073
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
30
3-Methylhistidine -0.1498 -0.0008
His-Ser -0.2994 -0.0105
N2-Acetyl-L-ornithine 0.0462 -0.0124
Ile-Lys -0.0544 -0.0134
Glycodeoxycholic acid -0.1480 -0.0179
Urea 0.0239 -0.0223
L-Pipecolic acid -0.0535 -0.0228
Myristic acid 0.0933 -0.0230
.gamma.-L-Glu-.epsilon.-L-Lys -0.0377 -0.0247
DL-Arginine 0.1043 -0.0270
Pro-Asn 0.0379 -0.0290
3-Methoxytyramine -0.0038 -0.0330
Nicotinuric acid -0.0834 -0.0334
Arg-Ala -0.3098 -0.0335
Pro-Phe -0.1255 -0.0354
Histamine -0.0162 -0.0375
trans-2-Octenoic acid -0.1316 -0.0394
6-Benzylaminopurine -0.0739 -0.0411
Betaine aldehyde -0.0229 -0.0411
DL-Vanillylmandelic acid 0.0159 -0.0418
L-Serine 0.1815 -0.0419
Succinic semialdehyde -0.0221 -0.0483
Hypoxanthine -0.0285 -0.0542
Diltiazem -0.4040 -0.0543
Dihydroxyfumarate 0.0019 -0.0550
L-Pyroglutamic acid 0.0403 -0.0551
trans-2-Hydroxycinnamic acid -0.0048 -0.0553
3-Hydroxycapric acid 0.8663 -0.0557
Pyrrolidine 0.0043 -0.0571
Sphingomyelin (d18:1/18:0) -0.0404 -0.0589
Dimethyl sulfone 0.0294 -0.0596
DL-2,4-Diaminobutyric acid 0.0308 -0.0604
Leu-Thr -0.1360 -0.0628
1-Eicosatrienoyl-sn-glycero-3-
phosphoethanolamine 0.0608 -0.0635
Pantothenate -0.1680 -0.0635
1-O-Octadecyl-sn-glyceryl-3-phosphorylcholine -0.0615 -0.0644
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
31
L-Histidinol -0.0325 -0.0681
Pro-Asp -0.1839 -0.0725
1-Palmitoyl-2-hydroxy-sn-glycero-3-
phosphoethanolamine -0.2277 -0.0765
1-Oleoyl-sn-glycero-3-phosphocholine 0.0972 -0.0780
D-Proline -0.1032 -0.0800
.beta.-Cyano-L-alanine -0.0189 -0.0808
1-Stearoyl-2-arachidonoyl-sn-glycerol 0.1083 -0.0880
1-Aminocyclopropanecarboxylic acid -0.1178 -0.0892
Creatine -0.0478 -0.0922
Nadolol 0.0411 -0.0972
Ile-Arg 0.0311 -0.0985
Creatinine -0.1818 -0.0988
N6,N6,N6-Trimethyl-L-lysine -0.2434 -0.1016
3-Methylphenylacetic acid -0.2171 -0.1138
Diethanolamine 0.2561 -0.1139
Lys-Val -0.2602 -0.1165
His-Gly -0.1333 -0.1213
.beta.-Homoproline -0.0538 -0.1226
gamma-L-Glutamyl-L-glutamic acid -0.2937 -0.1249
Quinaldic acid -0.3320 -0.1259
N-.alpha.-Acetyl-L-arginine -0.0667 -0.1263
D-erythro-Sphingosine-1-phosphate -0.1813 -0.1297
Gly-Val -0.2074 -0.1301
Gly-Glu 0.0916 -0.1345
Ornithine 0.0133 -0.1411
His-Glu -0.1630 -0.1432
beta-Hydroxybutyrate -0.1564 -0.1450
1-Myristoyl-sn-glycero-3-phosphocholine -0.1249 -0.1464
Arg-Val -0.3430 -0.1531
1-Stearoyl-2-oleoyl-sn-glycerol 3-phosphocholine
(SOPC) -0.0950 -0.1609
N-Docosanoyl-4-sphingenyl-1-O-
phosphorylcholine -0.0214 -0.1694
Raffinose -0.4984 -0.1709
L-Arginine -0.1383 -0.1749
20-Hydroxyarachidonic acid -0.3727 -0.1824
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
32
N-Methyl-1-deoxynojirimycin -0.0130 -0.1844
Stearic acid 0.2808 -0.1852
Phe-Ser -0.1921 -0.1912
gamma-L-Glutamyl-L-valine -0.2224 -0.1954
N6-Methyl-L-lysine -0.1702 -0.1985
NG,NG-dimethyl-L-arginine(ADMA) -0.1155 -0.2020
Butyl lactate -0.3074 -0.2091
Bilirubin -0.0833 -0.2399
DL-.alpha.-Phenylglycine -0.2894 -0.2418
Pro-Glu -0.2081 -0.2470
Dimethylglycine 0.0586 -0.2533
Hexanoic acid -0.7300 -0.2699
His-Ala -0.2629 -0.2802
Palmitic acid -0.5243 -0.2922
Altretamine 0.6852 -0.2933
Phenylethylamine 0.1412 -0.2984
4-Guanidinobutyric acid -0.0842 -0.3055
His-Ile -0.3377 -0.3077
Arecoline 0.5126 -0.3245
2-Ethoxyethanol -0.4809 -0.3524
Arg-Ile -0.1206 -0.3541
Phe-Asp -0.2907 -0.3548
His-Gln -0.3134 -0.4033
Azelaic acid -0.2214 -0.4704
Pro-Ala -0.0921 -0.4838
DL-a-Hydroxybutyric acid -0.1431 -0.4863
Phe-Ile -0.4950 -0.5108
Ser-Ala -0.3948 -0.5408
Val-Arg -0.2294 -0.5817
Cellobiose 0.1352 -0.5882
Pro-Ser -0.3132 -0.5950
Asp-Arg -0.5008 -0.6372
Phe-Trp -0.3061 -0.7082
Ala-Ile -0.5180 -0.7131
Phe-Phe -0.6178 -0.7171
Ile-Val -0.8929 -0.8642
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
33
S Table 5A. Mean relative abundance of 28 elevated metabolites in portal vein in HCC
patients compared to healthy controls in ESI- model
Name C_CV C_PV N_CV N_PV QC
Dihydrothymine 0.166612612 0.233506392 0.130120635 0.139138824 0.163885451
N-Acetylneuraminic acid 0.016669161 0.029770496 0.010459836 0.009383379 0.016824213
D-Lyxose 0.224908061 0.286023248 0.173942623 0.161358308 0.220140426
Glycerol 3-phosphate 0.007346036 0.025578197 0.01420329 0.013876529 0.017625273
D-Allose 0.261940317 0.341645257 0.1312095 0.177136257 0.214146602
L-Sorbose 0.049839066 0.080254507 0.020197924 0.036012712 0.050491243
DL-3-Phenyllactic acid 0.574581481 0.755426107 0.409108444 0.451668127 0.528422697
Hydroxyphenyllactic acid 1.014656298 1.371335081 0.822433339 0.787622876 1.072640796
3-Hydroxycapric acid 0.09528992 0.162212234 0.079801267 0.102165977 0.098933203
Galactonic acid 1.095379698 1.617267542 0.832832092 0.948815708 1.14594237
Cysteine-S-sulfate 0.101490191 0.122791205 0.068773641 0.075656609 0.122238509
Sebacic acid 0.115753878 0.763681095 0.200359314 0.248349774 0.309374583
L-Tryptophan 1.582292049 1.852482857 1.477081072 1.142587312 1.532317115
Pantothenate 0.439174953 0.714968571 0.465992433 0.444777155 0.593334501
Traumatic Acid 0.039509437 0.104216086 0.022703307 0.022430092 0.053563103
Pseudouridine 1.438679313 1.772684277 1.191265283 1.167566318 1.502641667
Stavudine 0.016889042 0.021921501 0.013631582 0.013304787 0.019016384
Phenylacetylglycine 0.008280838 0.018814963 0.005890866 0.007165869 0.013110972
3-Methoxy-4-
Hydroxyphenylglycol Sulfate
0.132859585 0.228867243 0.112249481 0.135374611 0.172637801
Xanthosine 0.173440414 0.319099678 0.08902597 0.075936448 0.335394887
D-Xylulose 0.014930492 0.020487597 0.0109489 0.011992995 0.014818931
D-(+)-Melibiose 0.085835295 0.111707882 0.033352377 0.054176101 0.054530402
Glycolithocholic acid 0.061892891 0.116174297 0.004729991 0.03741698 0.034313867
Glycochenodeoxycholate 3.626417055 11.69800975 0.50653251 3.859160287 8.255234083
Glycocholic acid 0.777282162 4.730249771 0.146672823 1.908934514 4.209213502
Taurochenodeoxycholate 5.599891612 8.634513197 0.183319281 1.382515197 3.980621591
Taurocholate 0.509911191 2.945064936 0.078757047 0.74293256 3.081538833
Biliverdin 0.059773257 0.080629909 0.054686007 0.04200109 0.075946748
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
34
S Table 5B. Mean relative abundance of 28 elevated metabolites in portal vein in HCC
patients compared to healthy controls in ESI+ model
Name C_CV C_PV N_CV N_PV QC
Glutaraldehyde 0.042439137 0.057210374 0.031091238 0.030315044 0.042262656
3-Furancarboxylic acid 0.019152667 0.023107639 0.015195935 0.015127274 0.015223176
Dopamine 0.134836139 0.180648995 0.097883762 0.095210856 0.13221767
1-Methylnicotinamide 0.48173952 0.903651789 0.422061319 0.516899125 0.559372455
trans-3-Coumaric acid 0.020335801 0.027499438 0.014897073 0.014403136 0.020434167
3-(Methylthio)-1-
propanol
0.067382835 0.090034512 0.048030953 0.040293754 0.055628858
L-Tyrosine 0.602131788 0.815164257 0.457680197 0.448616627 0.595145054
Glu-Ser 0.002034475 0.004892556 0.001748574 0.001595073 0.002104305
D-gluconate 0.000567582 0.000942494 0.000261805 0.000310832 0.001510908
N-Acetylglutamine 0.001759667 0.002237717 0.000992385 0.001074841 0.001661875
Methyldopa 0.022266837 0.031994495 0.000709735 0.000249623 0.007570188
Pro-Thr 0.034204643 0.211860269 0.041114243 0.011666971 0.220710643
Gln-Asn 0.002073425 0.004137592 0.001224746 0.001608517 0.002021843
Lys-Pro 0.055724877 0.09940581 0.047360113 0.048136437 0.056947876
Pseudouridine 0.038171613 0.064695925 0.037215404 0.04145042 0.047228491
L-Abrine 0.001307932 0.003571316 0.001652894 0.001771558 0.003248889
EDTA 0.070405354 0.293988876 0.023328214 0.012682637 0.321169964
Ile-Tyr 0.006529253 0.019162615 0.005643015 0.006946276 0.031269583
Tyr-Pro 0.019019089 0.043911042 0.008564974 0.017133948 0.080247126
Adenosine 0.006992035 0.010112637 0.005861011 0.005618835 0.015178508
Met-Tyr 0.000763965 0.002508573 0.00077935 0.000825773 0.005898772
His-Tyr 0.005951819 0.010988992 0.005315242 0.00643902 0.015002467
Isomaltose 0.001400867 0.002297881 0.000356579 0.000758784 0.002470289
3.alpha.-Mannobiose 0.002546664 0.003418371 0.001080094 0.001651011 0.001913551
7-Keto-8-
aminopelargonic acid
0.008861363 0.013461156 0.003835445 0.004064539 0.00572942
Glycochenodeoxycholate 0.184697245 0.556400414 0.017445085 0.153157962 0.252618963
Taurocholate 0.006942138 0.048983956 0.003908711 0.006206794 0.018192958
Biliverdin 0.062388884 0.075177406 0.046834582 0.042914464 0.032494318
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
35
S Table 6. The mean value of elevated metabolites in the discovery cohort was also
increased in portal vein of HCC patients in the independent validation cohort compared
with healthy controls
Name FC log2FC Mean value
in HCC
Mean value in
healthy control
3-Methoxy-4-
Hydroxyphenylglycol
Sulfate
1.1221527 0.166269008 0.0072219 0.006435755
3-Hydroxycapric acid 1.223765856 0.291327553 0.005031094 0.004111158
Dihydrothymine 1.405544895 0.491129536 0.084997133 0.060472727
Sebacic acid 1.455343765 0.541359971 0.005980904 0.004109616
D-Lyxose 1.528222139 0.611854265 0.004574658 0.002993451
Hydroxyphenyllactic acid 1.657853543 0.729316562 0.010832151 0.00653384
N-Acetylneuraminic acid 2.320493115 1.214431417 0.000623088 0.000268515
Glycolithocholic acid 3.097834121 1.631259895 0.00086187 0.000278217
Taurochenodeoxycholate 4.903964215 2.293948451 0.168391586 0.03433785
L-Tryptophan 56.06040729 5.808910321 0.003144127 5.60846E-05
3-Furancarboxylic acid 1.006397521 0.009200274 0.021545384 0.021408424
Pantothenate 1.516958502 0.60118162 0.03002918 0.01979565
Phenyllactic acid 1.597961604 0.676232743 0.013299051 0.00832251
Biliverdin 1.632891683 0.707429094 0.004040243 0.002474288
Pseudouridine 1.771171923 0.824704258 0.00289172 0.001632659
Adenosine 1.82009062 0.864010282 0.043172656 0.023720058
Glutaraldehyde 1.839075309 0.878980558 0.076462041 0.041576352
Dopamine 1.850221465 0.887697967 0.239738338 0.12957278
1-Methylnicotinamide 1.900382528 0.926289848 0.735676418 0.387120176
L-Tyrosine 1.915918525 0.938036211 1.05941896 0.552956165
Lys-Pro 3.669011965 1.875391609 0.11702441 0.031895347
Glycocholic acid 5.819015229 2.540775022 0.202302175 0.034765706
Taurocholate 6.051827319 2.597370823 0.082855829 0.013691043
Ile-Tyr 6.92552537 2.791923518 0.009428392 0.001361397
L-Abrine 14.51204826 3.859179253 0.039626408 0.002730587
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
36
S Table 7. The follow-up information for 52 HCC patients
Participants Death/Alive Surgical option Date of
operation Date of death
Survival
time
(month)
HCC1 Alive Hepatectomy 2018/12/11 2020/10/25 22
HCC2 Alive Hepatectomy 2019/1/24 2020/10/25 21
HCC3 Alive Liver transplantation 2019/2/15 2020/10/25 20
HCC4 Death Liver transplantation 2019/2/20 2020/1/21 11
HCC5 Alive Hepatectomy 2019/2/19 2020/10/25 20
HCC6 Alive Hepatectomy 2019/2/22 2020/10/25 20
HCC7 Alive Hepatectomy 2019/3/1 2020/10/25 19
HCC8 Death Hepatectomy 2019/3/6 2019/5/8 2
HCC9 Alive Hepatectomy 2019/3/1 2020/10/25 19
HCC10 Alive Hepatectomy 2019/3/21 2020/10/25 19
HCC11 Alive Liver transplantation 2019/3/21 2020/10/25 19
HCC12 Alive Hepatectomy 2019/3/27 2020/10/25 18
HCC13 Alive Hepatectomy 2019/4/2 2020/10/25 18
HCC14 Alive Liver transplantation 2019/4/2 2020/10/25 18
HCC15 Alive Hepatectomy 2019/4/12 2020/10/25 18
HCC16 Death Hepatectomy 2019/4/18 2020/10/2 17
HCC17 Alive Hepatectomy 2019/4/18 2020/10/25 18
HCC18 Death Hepatectomy 2019/4/17 2019/12/15 7
HCC19 Death Hepatectomy 2019/5/29 2020/6/10 12
HCC20 Alive Hepatectomy 2019/5/7 2020/10/25 17
HCC21 Alive Hepatectomy 2019/5/9 2020/10/25 17
HCC22 Alive Hepatectomy 2019/5/10 2020/10/25 17
HCC23 Alive Liver transplantation 2019/5/17 2020/10/25 17
HCC24 Alive Liver transplantation 2019/5/17 2020/10/25 17
HCC25 Alive Liver transplantation 2019/5/20 2020/10/25 17
HCC26 Death Hepatectomy 2019/5/22 2020/8/12 14
HCC27 Alive Hepatectomy 2019/6/4 2020/10/25 16
HCC28 Alive Hepatectomy 2019/6/6 2020/10/25 16
HCC29 Alive Hepatectomy 2019/5/5 2020/10/25 17
HCC31 Alive Hepatectomy 2019/1/17 2020/10/25 21
HCC34 Alive Hepatectomy 2019/3/22 2020/10/25 19
HCC35 Alive Hepatectomy 2019/4/24 2020/10/25 18
HCC37 Alive Hepatectomy 2019/2/28 2020/10/25 19
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
37
HCC39 Death Hepatectomy 2019/1/21 2020/7/15 17
HCC40 Alive Hepatectomy 2018/12/12 2020/10/25 22
HCC41 Alive Hepatectomy 2019/4/3 2020/10/25 18
HCC42 Alive Hepatectomy 2019/4/29 2020/10/25 17
HCC43 Alive Hepatectomy 2019/4/2 2020/10/25 18
HCC44 Alive Hepatectomy 2019/3/4 2020/10/25 19
HCC45 Alive Hepatectomy 2019/4/16 2020/10/25 18
HCC46 Alive Hepatectomy 2019/1/17 2020/10/25 21
HCC47 Alive Hepatectomy 2019/1/23 2020/10/25 21
HCC48 Alive Hepatectomy 2019/6/20 2020/10/25 16
HCC49 Alive Hepatectomy 2019/6/18 2020/10/25 16
HCC50 Alive Hepatectomy 2019/7/9 2020/10/25 15
HCC51 Alive Hepatectomy 2019/6/21 2020/10/25 16
HCC52 Death Hepatectomy 2019/7/19 2020/5/17 9
HCC53 Alive Hepatectomy 2019/7/2 2020/10/25 15
HCC54 Alive Hepatectomy 2019/7/8 2020/10/25 15
HCC55 Alive Hepatectomy 2019/7/17 2020/10/25 15
HCC56 Alive Hepatectomy 2019/7/8 2020/10/25 15
HCC57 Death Liver transplantation 2019/5/30 2019/10/15 4
Notes: The follow-up period was from date of operation to 10/25/2020. Survival time was
calculated in months.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
38
S Table 8A. In the discovery cohort, the relative concentration of linoleic acid and
phenol in portal vein serum of HCC patients and healthy controls
Name Linoleic acid Phenol
type MS2 forward MS2 forward
FC (fold change) 0.701194 0.725678
log2FC -0.512115 -0.462598
P value 0.000123 0.001826
ROC (receiver-operating
characteristic) 0.706923 0.679231
VIP (variable importance in
the projection) 1.251515 0.9979
C_PV1 12.5363551 0.16700268
C_PV2 21.3311681 0.32111229
C_PV3 18.7832351 0.08723051
C_PV4 7.96548473 0.68266988
C_PV5 9.53611807 0.14348187
C_PV6 9.9659174 0.91561916
C_PV7 5.48859468 0.33174328
C_PV8 8.80354456 0.41771869
C_PV9 23.1892218 0.10870076
C_PV10 10.9117413 0.8319994
C_PV11 14.0595486 0.47995609
C_PV12 10.7167926 0.1172332
C_PV13 9.62803313 0.24898527
C_PV14 11.6826897 0.15623619
C_PV15 13.1086602 0.23066627
C_PV16 18.829648 0.12728651
C_PV17 10.6664024 0.13419757
C_PV18 12.0348535 1.03048715
C_PV19 20.333153 0.02322651
C_PV20 14.0657853 0.04115915
C_PV21 7.05244886 0.43029847
C_PV22 14.2869089 0.70779292
C_PV23 10.4309958 0.10432627
C_PV24 5.97619373 0.44936777
C_PV25 11.7725996 0.1136337
C_PV26 18.3921202 2.09935939
C_PV27 13.3471604 0.10958645
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
39
C_PV28 17.2116263 0.71159666
C_PV29 15.6359018 0.27403812
C_PV31 25.6054048 0.19485169
C_PV34 10.6092633 0.10640185
C_PV35 13.2084915 0.16852495
C_PV37 10.6096206 0.16183229
C_PV39 26.1833326 0.29235477
C_PV40 5.23947388 0.33299133
C_PV41 14.3565679 0.28135485
C_PV42 9.59601916 0.2521919
C_PV43 9.42924934 0.11650804
C_PV44 6.39743111 0.17809473
C_PV45 14.8783855 0.19354798
C_PV46 8.76048177 0.67616118
C_PV47 9.55905432 0.10915239
C_PV48 16.960719 0.25439181
C_PV49 15.823751 0.83592285
C_PV50 9.97209349 0.27741214
C_PV51 6.53231936 0.13671384
C_PV52 14.2930979 0.80699019
C_PV53 23.835348 0.16730378
C_PV54 12.5441681 0.39549744
C_PV55 19.5467446 0.21298706
C_PV56 15.3817979 1.05784812
C_PV57 11.2078196 0.15770251
N_PV1 12.005697 0.85017089
N_PV2 12.3123946 0.38673953
N_PV3 15.1241546 1.37907001
N_PV4 19.9601844 0.10453
N_PV5 9.38908819 0.17656085
N_PV6 22.0878683 1.00404139
N_PV7 8.85694959 0.45685879
N_PV8 12.2197694 0.32993979
N_PV9 33.5250118 0.65316168
N_PV10 14.5546697 0.60241163
N_PV11 35.002819 0.24827302
N_PV12 27.7027103 1.15767177
N_PV15 11.0422526 0.2158617
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
40
N_PV16 15.110998 0.48108168
N_PV17 20.9418884 0.23217262
N_PV18 33.6215417 0.47478508
N_PV19 8.98740219 1.1838042
N_PV20 15.833174 0.84947361
N_PV21 13.7820978 0.17849349
N_PV22 9.93242225 0.51906405
N_PV24 5.18699545 0.34246032
N_PV25 14.8314644 0.20873346
N_PV26 19.5837488 0.41908966
N_PV27 16.949168 0.56224544
N_PV28 24.9335363 0.56548988
N_PV29 12.4344138 0.63824718
N_PV30 16.6413871 0.35130583
N_PV31 9.87883803 0.12951703
N_PV32 25.7316633 0.3444258
N_PV33 13.1910548 0.60932883
N_PV34 14.4023863 0.33168275
N_PV35 25.1046092 0.60378772
N_PV36 23.023461 0.18087269
N_PV37 12.3384046 0.2017758
N_PV38 19.815242 0.26860166
N_PV39 9.32992957 0.34112282
N_PV40 19.8679582 0.16314164
N_PV41 34.5254807 0.48410661
N_PV42 9.32727193 1.43254492
N_PV43 35.9007057 0.2405316
N_PV44 23.06326 0.13814074
N_PV45 21.8368647 0.64296498
N_PV46 16.1811776 0.25488756
N_PV47 15.9056168 0.83422227
N_PV48 16.5714949 0.71300661
N_PV49 20.5086886 0.4220145
N_PV50 20.7131564 0.84493569
N_PV51 39.4889937 0.89981337
N_PV52 32.9257385 0.23743933
N_PV53 21.6351111 0.23634645
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
41
S Table 8B. In the discovery cohort, the relative concentration of linoleic acid and
phenol in stool samples of HCC patients and healthy controls.
Name Linoleic acid Phenol
type MS2 forward MS2 forward
FC (fold change) 0.748661 0.880705
log2FC -0.417616 -0.183269
P value 0.003197 0.048335
ROC (receiver-operating
characteristic) 0.657692 0.649231
VIP (variable importance in
the projection) 1.710916 1.167809
C_S1 0.09599996 1.32E-05
C_S2 0.05713232 1.09E-05
C_S3 0.10509806 1.32E-05
C_S4 0.12063126 2.95E-06
C_S5 0.03663404 1.01E-05
C_S6 0.0485574 9.66E-06
C_S7 0.04603684 1.85E-05
C_S8 0.07710917 1.35E-05
C_S9 0.04140628 1.52E-05
C_S10 0.04948928 6.79E-06
C_S11 0.03055135 3.01E-05
C_S12 0.06256747 0.00010376
C_S13 0.10402999 1.21E-05
C_S14 0.10925143 1.01E-05
C_S15 0.05392193 1.78E-05
C_S16 0.04175076 0.00054226
C_S17 0.07689715 1.93E-05
C_S18 0.02071969 2.66E-05
C_S19 0.05207227 2.99E-05
C_S20 0.05751238 8.88E-06
C_S21 0.07290445 5.61E-06
C_S22 0.02634574 2.50E-05
C_S23 0.01350831 1.14E-05
C_S24 0.07404078 1.27E-05
C_S25 0.16640281 5.01E-05
C_S26 0.1069527 9.33E-05
C_S27 0.04004673 1.87E-05
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
42
C_S28 0.04288252 1.90E-05
C_S29 0.05148245 2.10E-05
C_S31 0.01056399 1.90E-05
C_S34 0.10817619 1.29E-05
C_S35 0.03131126 1.17E-05
C_S37 0.06756829 2.81E-05
C_S39 0.09956897 2.57E-05
C_S40 0.04701921 1.95E-05
C_S41 0.08580379 2.13E-05
C_S42 0.01847928 6.69E-06
C_S43 0.06964614 1.40E-05
C_S44 0.10218387 6.79E-05
C_S45 0.07125654 5.72E-05
C_S46 0.15404118 3.98E-05
C_S47 0.04761525 0.0001015
C_S48 0.02568422 2.69E-05
C_S49 0.04890937 4.27E-05
C_S50 0.11952682 8.72E-06
C_S51 0.1425128 1.65E-05
C_S52 0.06980333 3.85E-05
C_S53 0.05446272 2.07E-05
C_S54 0.09851855 1.64E-05
C_S55 0.08831718 1.33E-05
C_S56 0.05136656 1.81E-05
C_S57 0.01940944 1.02E-05
N_S1 0.08127647 2.58E-05
N_S2 0.05757304 2.40E-05
N_S3 0.094744 0.00013462
N_S4 0.1862758 5.32E-05
N_S5 0.03732016 2.15E-05
N_S6 0.08274913 2.72E-05
N_S7 0.11845468 2.55E-05
N_S8 0.15114932 6.66E-06
N_S9 0.04649447 2.59E-05
N_S10 0.09718301 6.91E-05
N_S11 0.1120922 1.94E-05
N_S12 0.15310203 2.92E-05
N_S15 0.15774338 8.05E-05
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
43
N_S16 0.03318744 1.66E-05
N_S17 0.03996298 0.00030834
N_S18 0.07935281 2.23E-05
N_S19 0.03929316 3.10E-05
N_S20 0.11391847 2.55E-05
N_S21 0.05131924 6.08E-05
N_S22 0.13399539 4.50E-05
N_S24 0.11125631 8.45E-05
N_S25 0.0265275 3.21E-05
N_S26 0.11162019 1.30E-05
N_S27 0.05211083 1.39E-05
N_S28 0.03636114 8.57E-05
N_S29 0.10740166 2.76E-05
N_S30 0.06957671 3.85E-06
N_S31 0.0467139 5.15E-05
N_S32 0.18126406 1.68E-05
N_S33 0.03024387 2.53E-05
N_S34 0.12757413 2.36E-05
N_S35 0.12380176 2.38E-05
N_S36 0.12438481 3.33E-05
N_S37 0.12084733 1.28E-05
N_S38 0.03547538 4.98E-05
N_S39 0.06962933 1.38E-05
N_S40 0.08499203 7.17E-05
N_S41 0.07662653 3.44E-05
N_S42 0.06745412 1.47E-05
N_S43 0.09836867 3.81E-05
N_S44 0.06518169 1.76E-05
N_S45 0.08689088 1.34E-05
N_S46 0.07345377 1.30E-05
N_S47 0.10711888 1.11E-05
N_S48 0.13222245 2.35E-05
N_S49 0.1363112 8.13E-05
N_S50 0.0662135 1.66E-05
N_S51 0.12851564 4.64E-05
N_S52 0.05236571 2.45E-05
N_S53 0.09508451 5.18E-06
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
44
S Table 8C. In the validation cohort, the relative concentration of linoleic acid and
phenol in portal vein serum of HCC patients and healthy controls
Name Linoleic acid Phenol
type MS2 forward MS2 reverse
FC (fold change) 0.899156 0.75218
log2FC -0.153357 -0.410851
P value 0.151755 0.002582
ROC (receiver-operating
characteristic) 0.5656 0.6596
VIP (variable importance in
the projection) 0.478015 1.200111
C_PV1 0.00822635 0.00191993
C_PV2 0.00527742 0.00046185
C_PV3 0.00528516 0.00130636
C_PV4 0.00454204 0.00364151
C_PV5 0.00354828 0.00953456
C_PV6 0.00638638 0.00131668
C_PV7 0.005341 0.00036536
C_PV8 0.00419606 0.00018564
C_PV9 0.0055938 0.00599952
C_PV10 0.00629904 0.00070624
C_PV11 0.00340525 0.00324568
C_PV12 0.00635891 0.00595834
C_PV13 0.00989447 0.00644957
C_PV14 0.00169381 0.0038065
C_PV15 0.00574636 0.0031121
C_PV16 0.00256311 0.00324315
C_PV17 0.00228421 0.00169848
C_PV18 0.00526093 0.00132863
C_PV19 0.0041745 0.00245611
C_PV20 0.00464078 0.01063984
C_PV21 0.00513509 0.00138597
C_PV22 0.00413627 0.00713669
C_PV23 0.00990958 0.00092818
C_PV24 0.00331768 0.00252817
C_PV25 0.00247479 0.00053125
C_PV26 0.00711941 0.00147478
C_PV27 0.00312012 0.00401627
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
45
C_PV28 0.00241028 0.00260374
C_PV29 0.00658847 0.00431618
C_PV30 0.00240451 0.00372806
C_PV31 0.00708228 0.0007207
C_PV32 0.00584383 0.00654245
C_PV33 0.00892246 0.00171079
C_PV34 0.00760649 0.0121006
C_PV35 0.00247363 0.00267267
C_PV36 0.00471119 0.00329276
C_PV37 0.00688856 0.00289066
C_PV38 0.00379301 0.00524177
C_PV39 0.00558988 0.00317972
C_PV40 0.00508077 0.00523344
C_PV41 0.00422011 0.00800291
C_PV42 0.00399457 0.00493011
C_PV43 0.00868543 0.00081282
C_PV44 0.00654323 0.00137222
C_PV45 0.00254832 0.02408888
C_PV46 0.00466861 0.00266437
C_PV47 0.00289143 0.02108126
C_PV48 0.00202597 0.00375627
C_PV49 0.00329288 0.00494327
C_PV50 0.00267132 0.00387102
N_PV1 0.0034794 0.00919237
N_PV2 0.00325307 0.00129249
N_PV3 0.00448553 0.00657155
N_PV4 0.0052568 0.00547358
N_PV5 0.00429192 0.01081295
N_PV6 0.00489265 0.00790858
N_PV7 0.00649654 0.00580272
N_PV8 0.00202729 0.00362013
N_PV9 0.00256857 0.00624849
N_PV10 0.00929496 0.00569707
N_PV11 0.00693711 0.00247754
N_PV12 0.00241729 0.01373775
N_PV13 0.00635021 0.01129053
N_PV14 0.00450577 0.00121014
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J
46
N_PV15 0.00468362 0.00230291
N_PV16 0.0075272 0.00610962
N_PV17 0.00820661 0.0026536
N_PV18 0.00773941 0.00579429
N_PV19 0.00675496 0.00345854
N_PV20 0.00518767 0.00251057
N_PV21 0.00340046 0.00213207
N_PV22 0.006052 0.00745737
N_PV23 0.00300825 0.01497979
N_PV24 0.0062068 0.0075382
N_PV25 0.00542492 0.00325258
N_PV26 0.00592457 0.00785059
N_PV27 0.00772863 0.0029052
N_PV28 0.00429514 0.00360775
N_PV29 0.00462485 0.00168399
N_PV30 0.00342184 0.00440886
N_PV31 0.01428984 0.00522155
N_PV32 0.00545394 0.00277737
N_PV33 0.00419869 0.00135902
N_PV34 0.00383563 0.00428277
N_PV35 0.01060003 0.00593306
N_PV36 0.00757625 0.00917979
N_PV37 0.00524534 0.00338706
N_PV38 0.00424806 0.01047224
N_PV39 0.00368757 0.00396407
N_PV40 0.00965844 0.00349979
N_PV41 0.00382187 0.01194259
N_PV42 0.00750138 0.01508509
N_PV43 0.00559493 0.00811842
N_PV44 0.00377138 0.00225896
N_PV45 0.00343512 0.00913842
N_PV46 0.00630462 0.0031932
N_PV47 0.00407322 0.00427576
N_PV48 0.00627863 0.00322665
N_PV49 0.00356373 0.00697122
N_PV50 0.00497255 0.00174526
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceSupplemental material placed on this supplemental material which has been supplied by the author(s) Gut
doi: 10.1136/gutjnl-2021-325189–11.:10 2021;Gut, et al. Liu J