Table S1. Clinical characteristics of 24 HCC patients and 24 healthy individuals(Post-hoc analysis)

Characteristicsa / HCC patients / Healthy individuals / p valueb
No. of subjects / 24 / 24
Gender (M/F) / 17/7 / 19/5 / 0.740
Age (year) / 53 (38~70) / 55 (28~64) / 0.370
AFP (ng/mL) / 23.8 (3.7~24200) / -
ALT (U/L) / 63.5 (22~695) / -
AST (U/L) / 78.0 (21~542) / -
GGT (U/L) / 109.5 (25~469) / -
HBsAg (positive/negative) / 23/1 / 0/24
HCV Ab (positive/negative) / 1/23 / -
Cirrhosis/no cirrhosis / 16/8 / 0/24
TNM stage
T1N0M0 / 6
T2N0M0 / 6
T3N0M0 / 6
T4N0M0 / 6

a Age, AFP, ALT, AST, and GGT were expressed as median (range).

b Chi-squared test was conducted for gender differences, and independent-samples T test was conducted for agedifferences.

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Table S2. Clinical characteristics of 18 HCC patients, 20 liver cirrhosis patients, and 20 healthy individuals(Validation set)

Characteristicsa / HCC patients / Liver cirrhosis patients / Healthy individuals / p valueb
No. of subjects / 18 / 20 / 20
Gender (M/F) / 14/4 / 13/7 / 16/4 / 0.480 *
Age (year) / 60 (43~79) / 59 (38~78) / 61 (28~69) / 0.770
AFP (ng/mL) / 7.7 (1.6~1210) / 3.0 (1.4~147) / - / 0.028
ALT (U/L) / 43.5 (29~171) / 42.5 (18~495) / - / 0.851
AST (U/L) / 51.5 (24~181) / 57.0 (14~221) / - / 0.942
GGT (U/L) / 111.9(24.4~336) / 105.9 (15.8~221) / - / 0.919
HBsAg (positive/negative) / 15/3 / 14/6 / 0/24 / 0.454#
HCV Ab (positive/negative) / 3/15 / 6/14 / - / 0.454#
Cirrhosis/no cirrhosis / 11/7 / 20/0 / 0/24
TNM stage
T1N0M0 / 12
T2N0M0 / 1
T3N0M0 / 4
T4N0M0 / 1

a Age, AFP, ALT, AST, and GGT were expressed as median (range).

bChi-squared test was conducted for gender, HBsAg and HCV Abdifferences, one-way ANOVA was for age differences, and independent-samples T test was conducted for AFP, ALT, AST, and GGT differences.

* The pvaluesare 0.485 (HCC vs Cirrhosis), 1.000 (HCC vs Healthy) and 0.480 (Cirrhosis vs Healthy) within every two groups’ comparison.

#Thepvaluesare obtained by comparison between HCC and liver cirrhosis patients.

Table S3. Reproducibility evaluation of LC-MS and GC-MS based on selected peaks in quality control samples (Discovery set).

Peak
No. / LC-MS, positive ion mode / Peak
No. / LC-MS, negative ion mode / Peak
No. / GC-MS
m/z / R.T.(min) / RSD a(100%) / m/z / R.T.(min) / RSD a(100%) / m/z / R.T.(min) / RSD a(100%)
1 / 104.11 / 0.4 / 7.8 / 1 / 151.03 / 0.5 / 9.4 / 1 / 159.12 / 4.4 / 5.7
2 / 258.11 / 0.7 / 5.3 / 2 / 119.04 / 1.2 / 6.5 / 2 / 207.03 / 5.1 / 4.2
3 / 384.05 / 1.7 / 6.0 / 3 / 593.20 / 4.4 / 7.5 / 3 / 147.07 / 6.4 / 5.1
4 / 260.19 / 2.7 / 9.0 / 4 / 498.29 / 5.8 / 7.0 / 4 / 179.05 / 8.1 / 8.3
5 / 529.40 / 3.2 / 8.7 / 5 / 448.31 / 6.2 / 5.4 / 5 / 299.07 / 8.6 / 7.0
6 / 286.20 / 4.3 / 4.8 / 6 / 507.28 / 8.9 / 20.9 / 6 / 228.07 / 10.6 / 8.4
7 / 310.20 / 5.3 / 9.5 / 7 / 253.22 / 12.2 / 10.6 / 7 / 223.15 / 12.4 / 4.9
8 / 466.32 / 7.3 / 8.2 / 8 / 283.27 / 13.6 / 11.0 / 8 / 217.11 / 15.0 / 6.1
9 / 414.30 / 8.6 / 11.7 / 9 / 204.10 / 16.1 / 9.8
10 / 616.18 / 10.0 / 10.1 / 10 / 313.25 / 17.2 / 7.6
11 / 520.35 / 10.9 / 8.5 / 11 / 117.04 / 19.0 / 5.1
12 / 424.35 / 11.7 / 6.4 / 12 / 207.03 / 24.9 / 8.9
13 / 426.36 / 12.6 / 8.0

a RSD: relative standard deviation of peak areas.

Table S4. MS and MS/MS information of 36 metabolites identified by LC-MS system

Metabolites / R.T.(min) / m/z / Fragments / Formula / Exact mass / ppm
[M+H]+ / [M+Na]+ / [M-H]-
N-Acetylneuraminic Acid / 0.30 / 308.0941 / 87, 98, 128, 170 / C11H19NO9 / 309.1060 / 14.93
Glycerol 3-phosphate / 0.31 / 171.0094 / 63, 79 / C3H9O6P / 172.0137 / -17.54
Glycerophosphocholine / 0.34 / 280.0908 / 86, 104, 214, 221 / C8H20NO6P / 257.1028 / 4.28
Acetylcarnitine / 0.39 / 204.1227 / 85, 204 / C9H17NO4 / 203.1158 / 1.47
Phosphoethanolamine / 0.39 / 140.0090 / 63, 79 / C2H8NO4P / 141.0191 / 20.00
Hydroxybutyrylcarnitine / 0.40 / 248.1497 / 85, 103 / C11H21NO5 / 247.1420 / -2.01
Malate / 0.40 / 133.0131 / 71, 92, 115 / C4H6O5 / 134.0215 / 8.27
Uric acid / 0.44 / 167.0248 / 69, 96, 124 / C5H4N4O3 / 168.0283 / -22.15
Niacinamide / 0.45 / 123.0552 / 80, 95 / C6H6N2O / 122.0480 / 0.81
Propionylcarnitine / 0.51 / 218.1383 / 85, 204 / C10H19NO4 / 217.1314 / 1.83
Cytidine monophosphate / 0.58 / 324.0547 / 70, 112, 176, 282, 306 / C9H14N3O8P / 323.0519 / 13.58
Methylglutarylcarnitine / 0.65 / 290.1610 / 85, 129, 213 / C13H23NO6 / 289.1525 / -4.14
Tiglylcarnitine / 1.02 / 244.1553 / 60, 85, 146, 188 / C12H21NO4 / 243.1471 / -4.10
Purine / 1.20 / 119.0361 / 65, 92 / C5H4N4 / 120.0436 / 1.68
Decanoylcarnitine / 1.49 / 316.2576 / 85, 109, 137, 211 / C17H33NO4 / 315.2410 / -29.72
Pimelylcarnitine / 1.56 / 304.1766 / 85, 125, 227 / C14H25NO6 / 303.1682 / -3.62
Suberylcarnitine / 1.93 / 318.1927 / 85, 157 / C15H27NO6 / 317.1838 / -5.03
Riboflavin / 2.26 / 377.1443 / 99, 172, 243, 319 / C17H20N4O6 / 376.1383 / 3.45
Hexanoylcarnitine / 2.69 / 260.1871 / 85 / C13H25NO4 / 259.1784 / -5.77
Taurocholic acid / 2.85 / 516.3052 / 86, 247, 417 / C26H45NO7S / 515.2917 / -12.20
Dodecanoylcarnitine / 3.09 / 344.2880 / 85, 119, 147 / C19H37NO4 / 343.2723 / -24.69
Sebacylcarnitine / 3.22 / 346.2218 / 85, 139, 241, 269 / C17H31NO6 / 345.2151 / 1.73
Xanthurenic acid / 3.74 / 206.0484 / 145, 190 / C10H7NO4 / 205.0375 / -17.47
Cortisone / 4.17 / 361.1911 / 199, 273 / C21H28O5 / 360.1937 / 27.41
Glycocholic Acid / 5.05 / 464.3031 / 74 / C26H43NO6 / 465.3090 / -2.80
Glycodeoxycholic acid / 6.17 / 448.3085 / 74 / C26H43NO5 / 449.3141 / -3.79
LysoPC(14:0) / 9.84 / 468.3106 / 86, 104, 184, 398 / C22H46NO7P / 467.3012 / -4.48
Tetradecenoylcarnitine / 10.31 / 370.2969 / 85, 144 / C21H39NO4 / 369.2879 / -4.59
LysoPC(16:1) / 10.37 / 494.3270 / 86, 104, 184 / C24H48NO7P / 493.3168 / -5.87
Tetradecanoylcarnitine / 11.00 / 372.3144 / 85 / C21H41NO4 / 371.3036 / -9.67
Hexadecenoylcarnitine / 11.42 / 398.3293 / 85 / C23H43NO4 / 397.3192 / -7.03
LysoPC(16:0) / 11.46 / 496.3399 / 104, 184, 313 / C24H50NO7P / 495.3325 / -0.20
LysoPC(18:0) / 11.79 / 524.3681 / 86, 104, 184, 341, 506 / C26H54NO7P / 523.3638 / 5.72
Myristate / 11.95 / 227.2024 / 86, 126 / C14H28O2 / 228.2089 / -3.08
Palmitoylcarnitine / 12.13 / 400.3449 / 85, 283 / C23H45NO4 / 399.3349 / -6.99
Stearoylcarnitine / 13.34 / 428.3786 / 85, 369 / C25H49NO4 / 427.3662 / -12.14

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Table S5. Pearson’s correlation analysis in 50 HCC patients (Discovery set)

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

Table S6. Pearson’s correlation analysis in 24 HCC patients (Post-hoc analysis)

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

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Table S7. Acetylcarnitine levels (Mean ± SD) in 18 HCC patients, 20 liver cirrhosis patients, and 20 healthy individuals (Validation set)

HCC
patients / Liver Cirrhosis
patients / Healthy
individuals / Fold change / p valuea
HCC/
Cirrhosis / HCC/
Healthy / Cirrhosis/
Healthy / HCCvs.
Cirrhosis / HCCvs.
Healthy / Cirrhosisvs.
Healthy
No. of subjects / 18 / 20 / 20
Acetylcarnitine (μM/L) / 4.25 ± 0.45 / 6.83 ± 0.87 / 5.94 ± 0.73 / 0.62 / 0.72 / 1.15 / 0.010 / 0.035 / 0.398

aIndependent-samples T test was conducted for thedifferences.

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Figure S1.

Figure S2.

Figure S3.

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Figure S4.

Figure S5.

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