gutjnl-2021-325189-r1 supplemental materials

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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

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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



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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



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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



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(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



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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.



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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.

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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.

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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


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



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

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●

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●●●●●

−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

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●

●

●

●

●

●

●

●

●

●

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●

−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

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●

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●

●

●●●●●●●●

●

●●●●●●●●●●●●●

−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

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●●●●●●●●●●●●●●●●●●●●

−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

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●

●

●

●

●

●

●

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●

●

●●

●

●

●●●●●●●●●●●●●●●●●●●●

−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

●

●●

●

●

●

●

●

●

●

●

●

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●

●

●

●

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●

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●

●

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●●

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●●

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●

●

●

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●●

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●

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●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

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●

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●

●

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●

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●●●●●●●●●●●●

−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



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



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



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%) ——



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



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.



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.



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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


HCC3 Alive Liver transplantation 2019/2/15 2020/10/25 20

HCC4 Death Liver transplantation 2019/2/20 2020/1/21 11




HCC8 Death Hepatectomy 2019/3/6 2019/5/8 2




























37



















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.



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



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



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



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.


type MS2 forward MS2 forward

FC (fold change) 0.748661 0.880705

log2FC -0.417616 -0.183269

P value 0.003197 0.048335




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



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



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



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


type MS2 forward MS2 reverse

FC (fold change) 0.899156 0.75218

log2FC -0.153357 -0.410851

P value 0.151755 0.002582




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



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



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



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