Clin Exp HEPATOL 2019; 5, 4: 317–326

DOI: https://doi.org/10.5114/ceh.2019.89478
Received: 26.05.2019, Accepted: 10.08.2019, Published: 08.11.2019

Original paper

Profiling of plasma metabolomics in patients with

hepatitis C-related liver cirrhosis and hepatocellular
carcinoma
Azhar Mohamed Nomair1, Marwa Ahmed Madkour2, Mohammed Mohammed Shamseya2, Heba Gaber Elsheredy3,
Ahmed Shokr4
1
Department of Chemical Pathology, Medical Research Institute, Alexandria University, Egypt
2
Department of Experimental and Clinical Internal Medicine, Medical Research Institute, Alexandria University, Egypt
3
Department of Cancer Management and Research, Medical Research Institute, Alexandria University, Egypt
4
City of Scientific Research and Technology Applications, Egypt

Abstract

Aim of the study: The diagnosis of hepatocellular carcinoma (HCC) is usually late, due to the lack of early de-
tection of biomarkers for HCC. Metabolomics analysis has emerged as a useful tool for studying human diseases.
The objective of the study was to investigate the differences in plasma metabolites between hepatitis C virus
(HCV)-induced cirrhosis and HCC.
Material and methods: 22 subjects with HCV-related liver cirrhosis and 22 subjects with HCC were enrolled.
Clinical, routine laboratory and imaging studies were done. Gas chromatography/mass spectrometry (GC/MS)
was used for metabolomics analysis of patients’ plasma samples.
Results: 34 known metabolites were detected, of which five metabolites were identified to have the strongest
discriminatory power for separation between HCC and cirrhosis groups: octanoic acid (caprylic acid), decanoic
(capric acid), oleic acid, oxalic acid and glycine. These are 3 fatty acids (FA), a dicarboxylic acid and a glucogenic
amino acid, respectively. No significant correlation was found between the relative intensities of the five me-
tabolites and any of the patient or tumor characteristics (Child-Turcotte-Pugh (CTP) score, Barcelona Clinic Liver
Cancer (BCLC) stage, number of focal lesions and size of largest focal lesion). ROC curve analysis was performed
and area under the curve (AUC) was calculated, revealing that oleic acid, octanoic (caprylic) acid and glycine had
higher positive predictive value than α-fetoprotein.
Conclusions: The study of metabolomics (particularly involving FA) may help define distinct metabolic patterns
to distinguish HCV-induced liver cirrhosis from HCC patients. Future research in this field is still needed, particu-
larly concerning HCC treatment strategies which target fatty acid-related metabolic pathways.
Key words: liver cirrhosis, fatty acids, hepatocellular carcinoma, metabolomics, hepatitis C virus (HCV).

Address for correspondence

Dr. Azhar Mohamed Nomair, Department of Chemical Pathology, Medical Research Institute, Alexandria University,
165 El-Horreya St., El-Hadara, PB: 21561, Alexandria, Egypt, e-mail: azhar.mohmd@yahoo.com

Introduction more than half a million new cases diagnosed world-

wide every year. Cirrhosis due to chronic viral hepa-
Hepatocellular carcinoma (HCC) is increasing in titis B or C is now considered the main risk factor for
incidence, representing the fifth and ninth most fre- HCC worldwide [2]. Clinically, most patients rarely
quently occurring cancer in men and women, respec- have symptoms until the later stages of the disease.
tively [1]. It is considered to be the second leading Thus, the diagnosis is usually late, which is – in turn –
cause of cancer related mortality in the world, with responsible for the high morbidity and mortality rates

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) 317

Azhar Mohamed Nomair, Marwa Ahmed Madkour, Mohammed Mohammed Shamseya, Heba Gaber Elsheredy, Ahmed Shokr

associated with HCC [3]. The only chance of long- Hepatology Department of the Medical Research Insti-
term disease-free survival in asymptomatic patients tute Hospital, Alexandria University, Egypt during the
depends on early diagnosis of HCC [4]. Ultrasound period from December 2017 to April 2018. Informed
imaging and serum α-fetoprotein (AFP) have long consent was obtained from all participants before the
been considered to be the classic screening methods study and it was approved by the local Ethics Com-
for early detection of primary liver cancer. However, mittee of the institute in accordance with the Code of
around 30% of HCC patients are AFP-negative. There- Ethics of the World Medical Association (Declaration
fore, new screening methods for primary liver cancer of Helsinki) for experiments involving humans.
are increasingly needed [5, 6]. A complete history and physical examination were
Metabolomics analysis is a new technology which performed for all patients, followed by analysis of the
refers to the scientific study of the small-molecular in- biochemical and radiological profile. The subjects were
termediates and products of metabolism. It is a quan- divided into two groups: 22 patients with liver cirrho-
titative measurement of endogenous low molecules, sis due to HCV infection and 22 patients with HCC
with a relative molecular mass of less than 1000 Dal- complicating HCV-related cirrhosis. All patients had
tons, hence identifying the unique chemical patterns no prior history of receiving antiviral therapy. HCV
produced by specific cellular processes [7, 8]. It is infection was identified by positive serum anti-HCV
a powerful tool in exploring mechanisms of different antibodies, which was confirmed by a polymerase
diseases, including minimal changes in genes and ex- chain reaction (PCR) test. Patients had negative se-
pression of proteins, which provides ample informa- rum markers of active infection with hepatitis B virus
tion on discovery of new biomarkers, disease patho- (HBV), human immunodeficiency virus (HIV) and
genesis, diagnosis and personalized treatment [9, 10]. schistosomiasis. Also, patients with a history of alcohol
Techniques based on mass spectrometry (MS) such consumption > 30 g/day, autoimmune diseases, malig-
as gas chromatography/mass spectrometry (GC/MS) nancies, diabetes mellitus and non-HCV related liver
and liquid chromatography/mass spectrometry (LC/ cirrhosis were excluded from the study.
MS) are the most widely used and effective technologies
in metabolomics analysis. The former is suitable for an-
Clinical and radiological evaluation
alyzing the thermally stable, volatile and gaseous com-
pounds of small molecular mass, while the latter can Liver cirrhosis was diagnosed based on clinical,
analyze the more polar compounds with higher relative laboratory and imaging criteria (coarse echo pattern
molecular mass and lower thermal stability [11]. GC/ of the liver on ultrasound), with reporting of the pres-
MS is considered to be the gold standard technique in ence/absence of portal hypertension and splenomeg-
metabolomics [8]. It is a collective system where the vol- aly. Ascites was graded as none, mild/moderate or se-
atile and thermally stable compounds are first separated vere. Child-Turcotte-Pugh (CTP) score and class were
by GC, followed by detection of the eluting compounds used for assessing the severity of liver disease [15].
by electron-impact mass spectrometers. Human blood Hepatocellular carcinoma cases were diagnosed ac-
is a good source for metabolomics research, as there cording to the guidelines of the American Association
are large amounts of metabolites in blood. Studies have for the Study of Liver Disease (AASLD) published in
succeeded in extracting and detecting metabolites from 2011, which comprised the presence of a hepatic focal
human blood by applying GC/MS [12-14]. lesion on ultrasound, verified by either a contrast-en-
The analysis of specific patterns of metabolic alter- hanced triphasic CT-scan study or dynamic con-
ations associated with HCC can help in providing in- trast-enhanced MRI that showed characteristic criteria
sight into its etiology and mechanisms. This study aims for HCC diagnosis (arterial uptake of contrast material
to compare between the plasma metabolite levels in followed by washout) [16]. The Barcelona Clinic Liver
hepatitis C virus (HCV)-related HCC cases and cirrhot- Cancer (BCLC) system was applied for staging of HCC
ic patients, and to evaluate the capability of candidate cases [17].
metabolites in distinguishing between the two groups.
Biochemical analysis
Material and methods
After an overnight eight-hour fasting period, 10 ml
of whole venous blood samples were withdrawn from
Study population
each subject. One ml was collected in EDTA tubes for
Forty-four subjects with HCV-related liver cir- complete blood picture and two ml were collected in
rhosis with or without HCC were recruited from the citrated plasma tubes for prothrombin time and INR

318 Clinical and Experimental Hepatology 4/2019

 Metabolomics in hepatocellular carcinoma

determination. Four ml serum samples were prepared added to the residue, vortex-mixed for 60 seconds and
for routine clinical chemistry (using an Olympus heated in a water bath at 70°C for one hour with a glass
AU400 clinical chemistry analyzer; Beckman Coulter, plug. The final solution was taken for GC/MS analysis.
Inc.), according to the methods recommended by the All the samples were analyzed by GC/MS at random
International Federation of Clinical Chemistry and after being preprocessed [12].
Laboratory Medicine [18]. Serum levels of alanine GC/MS analysis: Analysis was performed in the
aminotransferase (ALT), aspartate aminotransferase research laboratories of City of Scientific Research
(AST), bilirubin (total and direct), albumin, alkaline and Technology Applications, New Borg El-Arab city,
phosphatase, gamma-glutamyl transferase (GGT), Alexandria, Egypt using a Shimadzu GC-2010 gas
blood urea nitrogen (BUN) and creatinine were as- chromatography instrument coupled with a Shimad-
sessed. In addition, serum AFP level was measured zu QP2010 mass spectrometer (Shimadzu Co., Kyoto,
for all patients with cirrhosis and HCC (using the Japan). The capillary column used for all analyses was
automated IMMULITE 1000 immunoassay analyzer; an Agilent DB-5MS with a deactivated fused silica col-
Siemens Medical Solutions Diagnostics Corporation, umn (inner diameter: 30 m × 0.25 mm, film thickness:
Erlangen, Germany). A serum cut-off value equal to 0.25 µm). The column temperature was initially main-
or more than 200 ng/ml was considered diagnostic tained at 80°C for one minute, programmed to 300°C
for HCC [19]. Anti-HCV-antibodies were measured at a rate of 15°C/min, and then held for one min. Ultra
by immunoassay technique, and HCV RNA load was high purity helium (99.9%) was used as a carrier gas
quantitatively determined using a real-time PCR sys- with a constant flow rate of 1.45 ml/min. The septum
tem. purge was turned on with a flow rate of one ml/min all
The remaining three ml of whole blood were col- the time. The injector temperature, the interface tem-
lected in separate EDTA tubes and centrifuged at 2000 perature and the ion source temperature were set at
rpm for 10 min at 4°C. The plasma was aliquoted into 250°C, 150°C and 230°C, respectively. Ionization was
Eppendorf tubes and stored at –80°C for metabolom- achieved by a 70 eV electron beam. The mass spec-
ics measurement. trometer was operated under electron impact (EI) in
a full-scan mode over the range from m/z 50 to 800
with a 0.5 second scan velocity, and the detector volt-
Metabolomics analysis
age was 0.96 kV [20].
Chemicals and reagents: N-methyl-N-(trimethyl-
silyl) trifluoroacetamide (MSTFA) with 1% trimethyl-
Data processing and statistical analysis
chlorosilane (TMCS) of > 99.0% purity, methoxyamine
hydrochloride (> 98.0% purity) and pyridine (> 99.8% The identification of compounds from the peaks
purity) were commercially obtained from Sigma-Al- was based on the interpreted tables of m/z values and
drich (St. Louis, MO, USA). High performance liquid normalized migration times, by comparing the mass
chromatography (HPLC) grade methanol was pur- spectrum with Library Spectra v. 2.0 of the National
chased from the Tedia Company (Inc., Fairfield, USA). Institutes of Standards and Technology, Gaithersburg,
Sample preparation: Each 100 μl plasma-sample MD (NIST). The identification of metabolites was
was thawed at 37°C for 10 min, vortexed and mixed established by matching masses (m/z) between the
for 15 seconds. 800 μl of methanol, 100 μl of distilled peak’s fragmentation pattern and the standard data-
water and 10 μl of “heptadecanoic acid in methanol” base. Peaks with more than 80% similarity were allo-
(1 mg heptadecanoic acid in 1 ml methanol) were add- cated compound names, while those having less than
ed and mixed. Ten μl per sample were vortex-mixed 80% similarity were listed as unknown metabolites.
for one minute, kept on ice for 10 minutes, ultrason- The chromatograms were subjected to noise reduc-
icated at room temperature for 5 minutes, then put tion before peak area integration. Peaks due to noise,
again on ice for 10 minutes and centrifuged for 10 min column bleed and MSTFA derivatization procedures
(1200 rpm). The supernatant (200 μl) was transferred were excluded from the data set. Integrated peak ar-
into a 5 ml glass centrifugation tube and evaporated eas of multiple derivative peaks which belonged to the
to dryness by N2 gas. Next, 50 μl of methoxyamine/ same compound were added together and considered
pyridine were taken per sample (15 mg/ml = 0.0015 g as a single compound [21].
methoxyamine hydrochloride in 100 μl of pyridine) SPSS version 20.0 software was used in statistical
to the dry tube, vortexed for 30 seconds and left for analysis. The discriminant function analysis test was
16 hours at room temperature with a glass plug. Finally, used to determine which metabolites discriminate
50 μl of MSTFA + 1% TMCS derivatization agent were between the two groups of the study (HCV cirrho-

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Azhar Mohamed Nomair, Marwa Ahmed Madkour, Mohammed Mohammed Shamseya, Heba Gaber Elsheredy, Ahmed Shokr

sis versus HCC). The discriminant function analysis (SD) or proportions. Comparison between two means
model was created by a retrospective stepwise discrim- was performed using the non-parametric Mann-Whit-
inant approach using the data set of the patients. Ini- ney U-test for abnormally distributed quantitative
tial classification functions were applied to determine variables. Comparison between proportions was de-
to which group each case most likely belongs. After termined by the chi square (χ2) test or Fisher’s exact
alignment and normalization of significant GC data, test (FET). Spearman’s correlation coefficient (r) was
multivariate statistical analyses were conducted to applied to our results. A p-value equal to or less than
estimate the sensitivity and specificity of each signif- 0.05 was considered to be statistically significant. ROC
icant variable in predicting HCC at a defined cut-off curve analysis was performed using the relative inten-
value. Clinical, routine laboratory and imaging data of sity values of the identified plasma metabolites and the
patients were expressed as mean ± standard deviation area under the curve (AUC) was calculated to deter-
mine their individual ability in predicting HCC cases
Table 1. Clinical and radiological data of the studied groups
among cirrhotic subjects.

Variable HCC (n = 22) Cirrhosis (n = 22) P-value Results

n (%) n (%)
Age (years) (X ±SD) 60.09 ±5.06 58.59 ±7.83 0.455
Clinical, radiological and routine laboratory
Male sex 9 (40.9) 14 (63.6) 0.131 evaluation
Splenomegaly by US 12 (54.5) 20 (90.9) 0.007*
Males represented 40.9% of patients in the HCC
Ascites grade by US 0.060
group with a mean age of 60.1 years, versus 63.6% of
None 1 (4.5) 7 (31.8) patients in the cirrhosis group with a mean age of 58.6
Mild-moderate 2 (9.1) 2 (9.1) years (p > 0.05). There was no statistically significant
Severe 19 (86.4) 13 (59.1) difference between the two groups regarding CTP score
CTP score (X ±SD) 11.18 ±2.04 9.91 ±2.91 0.100 and class (p > 0.05). Triphasic CT evaluation of HCC
CTP class 0.338
patients showed that the majority of patients had 2-3 or
> 3 focal lesions on presentation (72.8%), the tumor in-
Class A 0 (0.0) 2 (9.1)
volved both lobes of the liver in 54.5% of patients, with
Class B 9 (40.9) 9 (40.9) malignant portal vein thrombosis detected in 31.8%,
Class C 13 (59.1) 11 (50.0) lymph node involvement in only one patient and ex-
PV thrombosis by CT 7 (31.8) 0 (0.0) 0.004* trahepatic spread in none. The mean size of the largest
Number of FL by CT – – focal lesion was 4.43 ±2.01 cm. BCLC staging of HCC
Single 6 (27.3)
patients revealed that more than half of them were at
the end stage of the disease (59.1%), while nearly one
Two/three 8 (36.4)
third of them (31.8%) were at the early stage (Table 1).
More than three 8 (36.4) The biochemical analysis revealed that there was
Size of largest FL (cm) 4.43 ±2.01 – – a statistically significant increase in the levels of AST,
(X ±SD) ALT, GGT, INR, AFP and white blood cell count
Liver lobes involved – – (WBC) in the HCC group compared to the cirrhosis
One lobe 10 (45.5) group, while there was no statistically significant dif-
Both lobes 12 (54.5) ference between the two studied groups regarding oth-
er parameters (Table 2). Also, there was no statistically
LN involvement by CT 1 (4.5) – –
significant correlation between serum level of AFP and
Extrahepatic spread 0 (0.0) – –
any of the patient or tumor characteristics (CTP score,
BCLC stage of HCC – – BCLC stage, number of focal lesions and size of largest
Very early stage 0 (0.0) focal lesion (p > 0.05).
Early stage 7 (31.8)
Intermediate stage 2 (9.1) Metabolomics analysis
End-stage 13 (59.1)
Examples of GC-MS total ion chromatograms
Data are expressed as mean (X) ± standard deviation (SD) or as number (n) and percent
(TIC) of plasma samples derived from the studied
(%), *Statistically significant at p < 0.05, HCC – hepatocellular carcinoma, CTP – Child-
Turcotte-Pugh, US – ultrasound, CT – triphasic CT-scan, PV – portal vein, FL – focal groups are shown in Fig. 1. Around 61 signals were
lesions, LN – lymph nodes, BCLC – Barcelona Clinic Liver Cancer. detected in the samples using mass spectral decon-

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 Metabolomics in hepatocellular carcinoma

Table 2. Routine laboratory parameters of studied groups

Parameter HCC (n = 22) Cirrhosis (n = 22) P-value

ALT (U/l) 89.14 ±86.36 27.59 ±12.71 0.002*
AST (U/l) 154.14 ±112.44 55.59 ±26.66 0.001*
Albumin (g/dl) 2.07 ±0.48 2.32 ±0.43 0.076
Total bilirubin (mg/dl) 6.17 ±5.29 3.80 ±6.44 0.189
Direct bilirubin (mg/dl) 4.02 ±3.98 2.32 ±4.63 0.199
Alkaline phosphatase (U/l) 130.95 ±66.30 93.95 ±72.56 0.085
GGT (U/l) 80.64 ±50.73 49.27 ±47.75 0.041*
BUN (mg/dl) 72.41 ±39.15 56.91 ±35.50 0.176
Creatinine (mg/dl) 1.20 ±0.38 1.33 ±0.54 0.357
AFP 276.21 ±252.92 69.93 ±43.82 0.002*
PT (s) 19.01 ±3.14 17.31 ±4.21 0.137
INR 1.64 ±0.23 1.45 ±0.35 0.038*
Hb (g/dl) 10.49 ±1.43 10.46 ±1.50 0.951
RBC (× 10 cells/mm )
6 3
3.12 ±0.60 3.26 ±0.58 0.416
WBC (× 10 cells/mm )
3 3
12.21 ±8.19 8.18 ±3.59 0.041*
Platelets (× 10 cells/mm )
3 3
117.41 ±46.16 118.50 ±62.83 0.948
Data are expressed as mean ± standard deviation, *statistically significant at p < 0.05, n – number, HCC – hepatocellular carcinoma, ALT – alanine transaminase, AST – aspartate
transaminase, GGT – gamma-glutamyl transpeptidase, BUN – blood urea nitrogen, AFP – alpha-fetoprotein, PT – prothrombin time, INR – international normalized ratio,
Hb – hemoglobin, RBC – red blood cell count, WBC – white blood cell count.

volution software for peak detection. However, many the patient or tumor characteristics (CTP score, BCLC
of them were not consistently found in other samples stage, number of focal lesions and size of largest focal
or presented too low abundance or too poor spectral lesion), as shown in Table 5.
quality to be accurately assigned to specific metabo- ROC curve analysis was performed using the rel-
lites. A total of 34 peaks could be auto-identified by ative intensity values of the identified plasma metab-
the NIST library through comparing the fragmenta- olites in comparison to serum concentration of AFP.
tion patterns composed of all fragment ions, as shown The area under the curve (AUC) was calculated to de-
in Table 3. The remaining peaks which could not be termine their individual ability in predicting HCC cas-
identified were not reported. These metabolites are es among cirrhotic subjects, revealing that oleic acid,
suggested to be involved in energy metabolism, lipid octanoic (caprylic) acid and glycine had higher posi-
metabolism, protein metabolism, and amino acid me- tive predictive value than AFP (Table 6, Fig. 2).
tabolism.
Five metabolites were finally selected among all
variables with the strongest discriminatory power for Discussion
separation between the HCC group and the liver cir-
rhosis group. The five peaks were identified as octanoic Hepatocellular carcinoma is often advanced and
acid (caprylic acid), decanoic (capric acid), oleic acid, incurable at presentation, which is partially attribut-
oxalic acid and glycine, which were all significantly ed to the absence of appropriate biomarkers for early
higher in the HCC patients compared to the HCV-cir- diagnosis [22]. The technology of metabolomics has
rhotic group. emerged as a useful analytical tool for human disease
When statistically comparing the mean values of study, because of its high sensitivity and capability to
the relative intensities of the five plasma metabolites simultaneously measure many metabolites [23].
between the two studied groups, they were found to be The objective of the present study was to investigate
significantly higher among the HCC group compared the unique differences in plasma metabolites between
to the cirrhosis group (Table 4). However, no statis- HCV-cirrhotic and HCC patients. Based on non-tar-
tically significant correlation was found between the geted metabolomic analysis, the present work detected
relative intensities of the five metabolites and any of 61 metabolites, of which 34 metabolites were known

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Azhar Mohamed Nomair, Marwa Ahmed Madkour, Mohammed Mohammed Shamseya, Heba Gaber Elsheredy, Ahmed Shokr

TIC*1.00

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0
min
B

TIC*1.00

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0

min
Fig. 1. Example of GC-MS total ion chromatogram (TIC) of plasma sample of HCC versus cirrhosis patient. A) HCC patient chromatogram demonstrating high
peaks of decanoic acid (blue arrow), oleic acid (green arrow), and glycine (red arrow); B) Cirrhosis patient chromatogram demonstrating low peaks of glycine (red
arrow) and oleic acid (green arrow), while decanoic acid was undetectable

compounds. Five metabolites were successfully identi- the body. The liver, being the principal metabolic hub
fied with the strongest discriminatory power for distin- of fats, carbohydrates and proteins, undergoes major
guishing between the HCC group and the liver cirrhosis metabolic alterations under these circumstances. HCC
group, namely octanoic acid (caprylic acid), decanoic malignant cells require building blocks to provide
(capric acid), oleic acid, oxalic acid and glycine. The material for cellular membranes, signaling molecules
first two of the identified metabolites are medium-chain and energy as they proliferate and spread. Glycolysis,
saturated FA, oleic acid is a monounsaturated FA, ox- of course, acts as the primary source of energy [24].
alic acid is a saturated dicarboxylic acid, while glycine In addition, gluconeogenesis from lipids and proteins
is a simple glucogenic amino acid. ROC curve analysis also plays a key role, which consequently increases the
demonstrated that oleic acid, octanoic (caprylic) acid turnover of glucogenic amino acids (e.g. glycine, ala-
and glycine had higher ability than AFP in predicting nine) and free FA [25, 26]. This might be one explana-
HCC cases among HCV-cirrhotic patients. tion for the findings of our metabolomics analysis. In
Hepatocellular carcinoma, as well as other malig- fact, a metabolomics study by Di Poto et al. identified
nant tumors, is known to generate a catabolic state in glycine as having better performance than AFP, and

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 Metabolomics in hepatocellular carcinoma

Table 3. Discriminant analysis of different plasma metabolites in the studied groups

No. Identified metabolites Retention time m/z ratio HCC Cirrhosis Wilks’ lambda P-value
(min) n = 22 n = 22
1 Dihydroxyacetophenone 5.793 281.00 5.132 ±3.905 5.475 ±1.021 0.971 0.078
2 Trisiloxane 6.136 73.00 8.220 ±4.657 7.545 ±4.232 0.949 0.084
3 Benzenedicarboxaldehyde 6.958 133.00 2.733 ±1.942 1.043 ±0.986 0.983 0.400
4 Carbamic acid 7.304 149.00 7.645 ±7.578 2.817 ±1.910 0.917 0.058
5 3-ethyl 2-methylhexane 7.465 84.00 5.857 ±1.443 4.675 ±0.007 0.940 0.069
6 Silane 7.585 207.00 2.860 ±0.000 1.193 ±0.543 0.995 0.635
7 Pyridinecarbonitrile 7.811 221.00 5.66 ±0.000 14.120 ±0.000 0.994 0.605
8 Caprylic acid 8.106 57.00 7.334 ±2.623 2.711 ±1.802 0.985 0.023*
9 Oxomalonic acid 8.087 57.00 3.770 ±0.000 2.736 ±2.006 0.988 0.480
10 Oxalic acid 8.110 57.00 3.223 ±1.001 2.112 ±0.701 0.934 0.001*
11 Neohexane 9.050 57.00 3.950 ±0.000 1.090 ±0.000 0.952 0.155
12 Enanthic acid 9.078 57.00 2.945 ±0.306 3.983 ±2.448 1.000 0.985
13 Caproic acid 9.084 57.00 2.945 ±0.306 3.983 ±2.448 1.000 0.985
14 Butane 9.315 57.00 2.731 ±1.228 4.770 ±0.000 0.976 0.319
15 Iodododecane 9.386 57.00 8.570 ±0.000 2.060 ±0.000 0.949 0.141
16 Valeric acid 9.629 191.00 3.3120 ±2.000 3.146 ±2.153 0.997 0.704
17 Glutaric acid 9.697 191.00 3.312 ±2.000 3.146 ±2.153 0.997 0.704
18 Methoxy benzoic acid 10.617 135.00 3.801 ±2.025 2.564 ±1.593 0.961 0.199
19 Ethanol 12.668 179.00 5.025 ±2.147 3.634 ±2.023 0.973 0.290
20 Hypoxanthine 13.115 55.00 1.800 ±0.000 0.650 ±0.000 0.982 0.389
21 Arachidic acid 13.133 73.00 3.129 ±2.491 3.424 ±5.271 0.991 0.535
22 Palmitic acid 13.221 73.00 3.129 ±2.491 3.424 ±5.271 0.991 0.535
23 Pentadecylic acid 13.234 73.00 3.129 ±2.491 3.424 ±5.271 0.991 0.535
24 Heptadecanoic acid 13.893 57.00 1.920 ±1.392 1.983 ±1.352 0.988 0.484
25 Propionic acid 14.547 57.00 1.382 ±1.268 0.616 ±0.220 0.929 0.081
26 Capric acid 14.601 73.00 0.713 ±0.210 0.343 ±0.208 0.923 0.009*
27 Oleic acid 14.621 55.00 4.743 ±1.442 1.602 ±0.333 0.929 0.041*
28 Stearic acid 14.747 60.00 4.460 ±0.057 2.440 ±2.258 0.997 0.721
29 Glycine 14.774 223.00 55.245 ±1.734 50.611 ±2.341 0.939 0.015*
30 Methionine 15.153 223.00 1.071 ±0.663 0.947 ±0.658 0.995 0.639
31 L-Leucine 15.193 223.00 1.071 ±0.663 0.947 ±0.658 0.995 0.639
32 Butylhydroquinone 15.273 295.00 1.737 ±0.313 1.525 ±0.335 0.994 0.102
33 Acrylic acid 16.164 311.00 14.200 ±0.000 6.070 ±6.772 0.996 0.674
34 Isophthalic acid 16.904 267.00 57.710 ±0.000 50.360 ±19.638 0.976 0.318
Relative intensities of plasma metabolites in the two study groups (HCC vs. cirrhosis) are given as their peak areas, and expressed as mean ± standard deviation, *Statistically significant
at p ≤ 0.05, n – number, HCC – hepatocellular carcinoma.

oxalic acid as clearly distinguishing HCC cases from Qiu et al. used chromatography–mass spectrometry
HCV-cirrhotic controls [27]. Another metabolom- to prove that linoleic acid, oleic acid, arachidonic acid
ics study by Muir et al. demonstrated elevated oleic, and palmitic acid were potential fatty acid biomarkers
adrenic, and osbond acids in the plasma of patients of HCC patients [29].
with nonalcoholic steatohepatitis-associated hepato- Cancer-induced dysregulation of FA metabolism
cellular carcinoma [28]. Furthermore, a third study by has been receiving particular attention in recent years.

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Table 4. Comparison between the two studied groups according to the relative It was suggested that cancer cells fulfill their require-
intensities of the five identified plasma metabolites ment for energy and building materials either by up-
HCC group Cirrhosis group Test P-value regulating de novo FA synthesis, or by altering FA
(n = 22) (n = 22) of sig. oxidation [30]. At this point, studies seem to disagree
Octanoic (caprylic)
on how FA regulation is involved in tumorigenesis.
acid While some studies linked the downregulation of FA
Range
oxidation with HCC [31], others associated increased
3.5-11.2 0.9-6.4 U = 9.0* < 0.001*
catabolism of certain saturated lipids with high AFP
Mean ±SD 7.3 ±2.6 2.7 ±1.8 levels in the serum of HCC patients, concluding that
Oxalic acid lipidomics analysis may provide new biomarkers for
Range 1.6-4.7 1-3.1 t = 2.774* 0.011* HCC [32-34]. Li et al. also demonstrated that aberrant
Mean ±SD 3.2 ±1.1 2.1 ±0.7 lipid metabolism was an evident feature of HCC, and
Decanoic (capric)
that the severity of the condition correlated with high-
acid er tissue concentrations of saturated triglycerides (TG)
and lower concentrations of polyunsaturated TG [35].
Range 0.3-1.1 0-0.7 U = 22.0* 0.003*
Lin et al. revealed similar outcomes and concluded
Mean ±SD 0.7 ±0.2 0.3 ±0.2
that their findings offer the biomedical potential to use
Oleic acid the altered lipid metabolism as a diagnostic marker for
Range 2.6-6.9 1-2.1 t = 7.053* < 0.001* cancer cells, which – in turn – opens the opportunity
Mean ±SD 4.7 ±1.4 1.6 ±0.3 for treating aggressive HCC by targeting altered lipid
Glycine metabolism pathways [36].
Nevertheless, our results showed no correlation be-
Range 52.1-57.3 45.8-53.6 t = 5.369* < 0.001*
tween patient/tumor characteristics (CTP score, BCLC
Mean ±SD 55.2 ±1.7 50.6 ±2.3 stage, number of focal lesions and size of largest fo-
U – Mann-Whitney test, t – Student’s t-test, p – p value for comparison between the two cal lesion) and the relative intensities of the identified
studied groups, *statistically significant at p < 0.05

Table 5. Relation between relative intensities of the five plasma metabolites and different patient and tumor characteristics

Octanoic (caprylic) acid Decanoic (capric) acid Oleic acid Oxalic acid Glycine
CTP-score r = –0.131 r = 0.171 r = 0.244 r = 0.246 r = 0.182
(n = 44) p = 0.542 p = 0.426 p = 0.251 p = 0.256 p = 0.395
BCLC stage* U = 9.0 U = 8.0 t = 1.351 t = 0.389 t = 0.572
(n = 20) p = 0.833 p = 0.667 p = 0.214 p = 0.707 p = 0.583
Number of FL H = 0.932 H = 1.682 F = 0.332 F = 1.223 F = 1.448
(n = 22) p = 0.628 p = 0.431 p = 0.727 p = 0.344 p = 0.291
Size of largest FL r = 0.124 r = 0.417 r = 0.096 r = 0.272 r = 0.453
(n = 22) p = 0.717 p = 0.202 p = 0.778 p = 0.418 p = 0.161
CTP – Child-Turcotte-Pugh, BCLC – Barcelona Clinic Liver Cancer (*Intermediate stage patients were excluded from analysis due to small sample size, n = 2), FL – focal lesion,
n = number of patients, r – Pearson coefficient, U – Mann-Whitney test, t – Student’s t-test, F – ANOVA test, H – Kruskal Wallis test, p – level of significance between the different
categories (statistically significant at p ≤ 0.05)

Table 6. Sensitivity and specificity of AFP versus plasma metabolites in predicting HCC cases among cirrhotic patients

AUC P 95% CI Cut-off Sensitivity Specificity PPV NPV

AFP 0.867* 0.002* 0.725-1.009 > 95 72.73 84.62 80.0 78.6
Octanoic (caprylic) acid 0.937* < 0.001* 0.847-1.028 > 5.003 81.82 92.31 90.0 85.7
Oxalic acid 0.762* 0.030* 0.552-0.973 > 3.142 63.64 100.0 100.0 76.5
Decanoic (capric) acid 0.846* 0.004* 0.691-1.001 > 0.512 72.73 84.62 80.0 78.6
Oleic acid 1.000* < 0.001* 1.000-1.000 > 2.087 100.0 100.0 100.0 100.0
Glycine 0.951* < 0.001* 0.871-1.031 > 53.57 81.82 100.0 100.0 86.7
AUC – area under curve, p – value: probability value, CI – confidence interval, NPV – negative predictive value, PPV – positive predictive value, AFP – alpha-fetoprotein, *statistically
significant at p ≤ 0.05

324 Clinical and Experimental Hepatology 4/2019

 Metabolomics in hepatocellular carcinoma

plasma metabolites. In comparison, Muir et al. found 100

a great discrepancy among the different identified fatty
90
acids and their relation to HCC tumor size/burden in
mice and humans. While some of them showed a pos- 80
itive correlation (e.g. oleic and adrenic acids), others 70
showed negative (e.g. margaric and linoleic acids) or
60
no correlation with tumor size/burden [28].

Sensitivity
In Egypt, cases of hepatocellular carcinoma are 50
mostly secondary to HCV-induced liver cirrhosis 40
[37]. HCV infection seems to have a synergistic effect
on lipid turnover, namely by encouraging lipogenesis 30

and steatosis to provide a lipid-rich environment for 20

viral replication [38]. This is achieved by augmenting
10
the expression and activation of specific transcription
factors that activate the synthesis of FA, triglycerides 0
and cholesterol, causing their accumulation in the liv- 0 10 20 30 40 50 60 70 80 90 100
er [39]. Added to the previously described HCC-re- 100-Specificity

lated alteration of lipid metabolism, these findings AFP Octanoic caprylic acid Decanoic capric acid
emphasize the role of dysregulated FA particularly in Oleic acid Oxalic acid Glycine

HCV-induced HCC carcinogenesis, and indicates that Fig. 2. ROC curves of AFP and plasma metabolites demonstrating their
interfering with lipogenesis may represent a potential different abilities to predict HCC cases among cirrhotic patients
therapeutic strategy for these cases.
Numerous therapeutic agents are already targeting Disclosure
key and/or lipogenic enzymes and pathways in lip-
id metabolism and have shown good efficacy against The authors report no conflict of interest.
several cancers. However, this progress in therapeutic
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