Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases
Patients with chronic liver diseases (CLD) including chronic hepatitis B and hepatic cirrhosis (CIR) are the major high-risk population of hepatocellular carcinoma (HCC). The differential diagnosis between CLD and HCC is a challenge. This work aims to study the related metabolic deregulations in HCC and CLD to promote the discovery of the differential metabolites for distinguishing the different liver diseases. Serum metabolic profiling analysis from patients with CLD and HCC was performed using a liquid chromatography–mass spectrometry system. The acquired large amount of metabolic information was processed with the random forest–recursive feature elimination method to discover important metabolic changes. It was found that long-chain acylcarnitines accumulated, whereas free carnitine, medium and short-chain acylcarnitines decreased with the severity of the non-malignant liver diseases, accompanied with corresponding alterations of enzyme activities. However, the general changing extent was smaller in HCC than in CIR, possibly due to the special energy-consumption mechanism of tumor cells. These observations may help to understand the mechanism of HCC occurrence and progression on the metabolic level and provide information for the identification of early and differential metabolic markers for HCC.
KeywordsHepatocellular carcinoma Metabolomic profiling Random forest–recursive feature elimination Acylcarnitine Fatty acid oxidation
Carcinoma embryonic antigen
Chronic hepatitis B
Chronic liver diseases
- CPT 1
Carnitine palmitoyl transferase 1
- CPT 2
Carnitine palmitoyl transferase 2
Fatty acid oxidation
Chenodeoxycholic acid glycine conjugate
Hepatitis B surface antigen
Hepatitis B virus
Liquid chromatography–mass spectrometry system
Recursive feature elimination
Random forest–recursive feature elimination
Rapid-resolution liquid chromatography
Type 2 diabetes
Hepatocellular carcinoma (HCC) has been demonstrated to be strongly related to chronic hepatitis virus infection  and is a major cause of death among patients with cirrhosis (CIR) . Especially in China, most of HCC patients have the disease background of chronic hepatitis B (CHB) or further CIR. The pathological changes of liver caused by hepatitis virus are complicated. It begins with the cycle of hepatic injury and regeneration, and then to more severe abnormal liver nodule formation (cirrhosis), and finally to the precancerous lesions of HCC . And in the early stage of HCC, there are large scale of hepatic injury and cirrhosis around the malignant lesions. This makes it difficult to identify early HCC from chronic liver diseases (CLD).
Despite increasing studies of genes and proteins involved in the occurrence and development of HBV infection-related HCC [4, 5, 6, 7, 8, 9], the knowledge on pathogenesis and diagnosis of this malignant tumor remains scarce. As a major organ of metabolism in the body, liver takes part in many metabolic pathways and also has a great impact on the systemic state of the organism. And metabolic dysregulation in liver may influence the incidence and development of HCC . For example, the activation of lipogenic pathway was found contributing to cell proliferation and prognosis  in HCC patients.
Through screening as many metabolites as possible, metabolic profiling or metabolomics can provide a large amount of endogenous metabolite information, facilitating a systematic view of metabolic deregulations [11, 12]. Metabolomics has already been used in the study of pathogenesis of liver diseases or malignant hepatic tumors and the discovery of novel diagnostic markers based on the comprehensive analysis of differentially expressed metabolites. It has been reported that there is an increase of total lipid profile and a decrease of lipid polyunsaturation in liver biopsies with increasing hepatic fibrosis in patients with chronic hepatitis C . Differentially expressed metabolites were found in urine of hepatitis C virus infected Egyptian patients with HCC. Among the differential metabolites, glycine, citrate, carnitine, and trimethylamine-N-oxide are related to energy production and aberrant chromosomal methylation . Recenly, γ-glutamyl dipeptides have also been reported as biomarkers to discriminate six different CLD and HCC . And also, some important serum metabolites have been found altered in HCC and CIR involved in lipid metabolism, increased protein degradation, perturbations of the tricarboxylic acid (TCA) cycle . Importantly, when comparing the serum metabolic changes of CIR and HCC in our previous study, we found not only several differential metabolites specific to CIR and HCC but also different metabolic correlation networks .
Generally, rich metabolome data can be collected by the metabolic profiling analysis based on liquid chromatography-mass spectrometry [18, 19, 20], gas chromatography–mass spectrometry  or NMR . In order to mine important deregulation information and find out metabolic features, an effective multivariate analysis method is necessary. Random forest (RF)  is a very efficient multivariate analysis technique, and has been successfully applied in many fields such as text mining, genomics, and metabolomics [23, 24, 25]. It can efficiently process large number of variables and small samples, such as metabolome data, due to its two randomness . Furthermore, it can deal with classification problems as well as feature selection problems. RF could measure the variable importance through “Gini importance”  or “permutation importance” . Random forest–recursive feature elimination (RF-RFE) which combines RF with RFE  is a recursive backward feature elimination procedure. It begins with all the features. In each iteration, a random forest is constructed to measure the features’ importance and the feature which is the least important is removed. This procedure is repeated until there is no feature left. Finally, the features are ranked according to the deleted sequence, the top ranked feature is the last deleted and the most important.
In this study, the samples collected from CHB, CIR, and HCC patients and healthy controls were analyzed on a LC-MS. RF-RFE was adopted to select the informative variables from the serum metabolome data. The study aimed at the exploration of important metabolic deregulations related to the CLD and HCC in order to provide useful pathogenesis information and facilitate the discovery of differential metabolites for different liver diseases.
Reagents and chemicals
Acetonitrile and formic acid (HPLC grade) was purchased from Merck (USA), and Tedia (USA), respectively. And home filtered Milli-Q water was used as solvent or for preparing mobile phases. The standard compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Baseline characteristics of patients with liver diseases
N (n = 30)
CHB (n = 30)
CIR (n = 30)
HCC (n = 30)
40 ± 2
46 ± 2
55 ± 2
59 ± 2
2.4 ± 0.16
42.2 ± 17.91
15.5 ± 6.21
246.9 ± 77.94
1.8 ± 0.22
31.3 ± 29.16
3.4 ± 0.39
8.4 ± 3.2
31.6 ± 4.91
167.3 ± 32.1
44.3 ± 4.9
56.7 ± 7.67
25.6 ± 1.83
113.4 ± 22.88
59 ± 6.45
86.2 ± 11.16
90.2 ± 4.68
101 ± 6.15
128 ± 12.89
125.6 ± 13.69
54 ± 16.15
93.1 ± 10.68
113.7 ± 36.88
107.7 ± 17.4
45.1 ± 1.19
39.9 ± 1.16
31.7 ± 1.03
34.6 ± 1.79
27.5 ± 0.66
31.6 ± 1.26
31.7 ± 1.28
32.3 ± 2.14
73.7 ± 0.68
68.9 ± 2.99
63.3 ± 1.65
60.3 ± 3.4
1.7 ± 0.05
1.9 ± 0.63
1.1 ± 0.06
8.6 ± 4.36
17.1 ± 1.23
27.9 ± 7.9
52.1 ± 10.7
134.4 ± 77.44
318.1 ± 283.92
63.7 ± 8.79
112.5 ± 44.82
8 ± 0.39
6,505.4 ± 579.74
2,334.7 ± 204.15
3,357.4 ± 411.24
Sample preparation for analysis
Four hundred-microliter acetonitrile was added to 100-μl serum after thawing at room temperature. Each mixture was votexed vigorously and then centrifuged at 15,000×g for 10 min at 4 °C. About 100-μl supernatant was moved to autosampler vial for analysis. Ten-microliter supernatant from each sample was mixed as the quality control (QC) sample . The QC was run ten times to equilibrate the liquid chromatographic system before the batch analysis and was run every ten samples to monitor the stability of instrument during the batch analysis .
Liquid chromatographic separation
The supernatant from each sample was injected into an Agilent 1200 Rapid Resolution Liquid Chromatography (RRLC) system (Agilent, USA) for the metabolic profiling and fatty acid analysis. A reversed-phase HSS T3 column (100 × 2.1 mm, 1.7 μm; Waters, USA) was used for chromatographic separation. The mobile phase A was of 0.1% formic acid in water (v/v) and mobile phase B was of 0.1% formic acid in acetonitrile (v/v). The gradient started with 2% mobile phase B, increasing to 30% B at 3 min, then to 80% B at 22 min, and then to 100% B at 24 min. The total run time was 35 min including equilibration of 5 min. The flow rate was 0.3 ml/min, and the injection volume was 4 μl.
Mass spectrometry analysis
Data acquisition was performed on an Agilent 6510 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent, USA) equipped with dual-electrospray source which was operated in the positive ion mode for the metabolic profiling and in the negative ion mode for the fatty acid analysis, separately. The ion source temperature was set at 300 °C and other parameters were set as our previous protocol . The mass accuracy was calibrated before analysis and a real-time calibration of mass accuracy was implemented to ensure the stability of mass accuracy. Mass spectra data were collected in the centroid mode with acquisition rate of 2 spectra/second. The scan range was from m/z 100 to 1,000. And at the end of a sequence in each ion mode, data-dependent information acquisition was performed to acquire MS/MS fragments with the collision energies set at 10 and 30 V, respectively.
The raw total ion current spectra acquired from RRLC/Q-TOF MS were directly analyzed by the Molecular Features Extraction from Agilent to pick compounds. Then, the peak alignment was done on the export files by GeneSpring MS 1.2 (Agilent, USA). After these steps, an excel table including retention time, m/z, peak intensity was given. The ions in the table were kept if they exist in 80% samples of at least one group . With this method, most of the missing values can be removed. And then the remaining variables were imported to SIMCA-P 11.0 (Umetrics AB, Umea, Sweden) for principal component analysis (PCA) to evaluate the reliability of the obtained profiling data.
Multivariate analysis and feature selection
RF-RFE was performed on the constricted excel table to select the informative ions. The algorithm was written in C++. Each decision tree in the random forest is constructed as a binary tree, its training set is produced by the bootstrapping technique and one sample can be repeated at most two times in each training set. To value the variable’s splitting effect, we adopt distinct class based splitting measure , which consists of two parts: one is related to the number of sample classes in each child partition and the other is related to each class’s proportion in the partition. To find meaningful metabolites which can discriminate liver diseases from the control, discriminate among HCC, CHB and CIR, further discriminate between each two different liver diseases, RF-RFE was conducted on five cases, labeled with RF-RFEi (i = 1, 2, 3, 4, and 5) in sequence. RF-RFE1 considers CHB, CIR, and HCC as one liver disease group, trying to select variables which have a significant difference between the disease group and the control group. RF-RFE2 compares three liver diseases, aiming at finding discriminative variables for three different liver diseases. And the remaining three cases were conducted as the binary classification problems among three liver diseases.
Each RF-RFEi (i = 1, 2, 3, 4, and 5) was run 50 times. For each run, one third of the data was randomly selected as an independent test set and the remaining two thirds was used as the training set to conduct the feature selection. The procedure starts from all the input variables on the training data. In each loop, a random forest is constructed based on the training data, the features’ scores are calculated by the permutation importance , and the feature having the lowest score is deleted. The procedure is repeated until there is no feature left. In each iteration, the performance of the random forest is tested by the out-of-bag (OOB) data, the maximal OOB accuracy rate and its corresponding random forest model are kept. When the iteration terminates, a feature rank is produced according to the deleting sequence of the features, the top-ranked features are the last deleted and the most important ones. The occurrence frequency of each variable in the top 50 ranked features of each run is calculated. The variables whose frequencies are bigger than 0.5 are selected. The feature subsets selected by RF-RFEi are denoted as FSSi (i = 1, 2, 3, 4, and 5), respectively. The final feature subset is a union of FSSi (i = 1, 2, 3, 4, and 5).
Univariate analysis: the informative ions were imported into SPSS 11.5 for Mann–Whitney U test analysis. A significant level of p < 0.05 was chosen.
Feature ion identification
Identification of the remaining differential expression ions was performed according to our former work  with the small modification. Firstly, quasi-molecular ions were picked out from their product and adduct ions. And then online MS/MS spectra databases such as Metlin (http://metlin.scripps.edu/) or HMDB (http://www.hmdb.ca/) were searched using the exact mass with a mass deviation of 0.1 Da. If an ion was not in the databases, accurate mass with mass deviation smaller than 3 ppm was measured to calculate its element composition, and multi-stage MS fragmentation was obtained to deduce the possible chemical structures. Also, the fragments of possible endogenous compounds searched from Chemspider (http://www.chemspider.com/) and KEGG (http://www.genome.jp/kegg/kegg2.html) were analyzed with the MassFrontier 6.0 software (HighChem, Slovakia). And then retention time and MS/MS spectra of authentic standards were checked to confirm the identification results.
Results and discussion
Serum metabolic profiling of HCC and CLD patients
Sera from patients with HCC and CLD including CHB and CIR, and the healthy controls were profiled to investigate metabolic variations among liver diseases. In order to evaluate the injury extent of liver mitochondria, the ratio of AST to ALT was calculated. It was found that the level of liver mitochondria injury increased with the liver disease severity from 0.8 ± 0.08 in CHB patients, 1.42 ± 0.08 in CIR to 1.63 ± 0.18 in HCC patients.
Significantly changed metabolites among liver diseases defined by RF-RFE
In order to explore more valuable information from the massive metabolic data, RF-RFE was applied. Since RF is not sensitive to data standardization and data normalization , the data pretreatment is not necessary except for the removal of the missing values with the 80% rule .
In RF, the size of the candidate splitting feature subset was set as its default value sqrt (N), where N was the number of the features; the tree number of each random forest was set as 100. Each RF-RFEi (i = 1, 2, 3, 4, and 5) was run 50 times; the average accuracy rates of RF-RFEi (i = 1, 2, 3, 4, and 5) on the independent test sets were 96.55 ± 3.50%, 66.07 ± 7.64%, 71.60 ± 9.34%, 84.40 ± 6.36%, and 78.40 ± 8.89%, respectively. This implies that RF-RFE can measure the features properly and produce a good model performance.
In RF-RFEi (i = 1, 2, 3, 4, and 5), each run could generate a feature ranking sequence. The top-ranked features were the most informative. The occurrence frequency of each variable in the top 50 ranked features of each run was calculated. The variables with the frequency bigger than 0.5 were selected. The final feature subset including 96 feature ions was the union of all selected features from RF-RFEi (i = 1, 2, 3, 4, and 5).
Identified differential expression compounds compared with healthy controls (N) selected by RF-RFE
Mass error (ppm)
Mann–Whitney U test (p)
HCC vs. CIR
HCC vs. CHB
CIR vs. CHB
Systemic metabolic deregulation in the HCC and CLD patients
The related metabolic changes may help to interpret the biological events in the different liver diseases. From Table 2, we can see a decrease in tryptophan, several acylcarnitines (CN) with medium and short fatty acid chain including C10-acylcarnitine (C10-CN), C10:1-CN, C8-CN, C6-CN, and some lysophospholipid molecules and an increasing level of cortisol, GCA, GCDCA, C16:1-CN, and other lysophospholipid molecules in the liver diseases.
It was found that tryptophan decreased in the sera of CHB, CIR and HCC patients. Tryptophan can be metabolized by rate-limiting enzyme IDO [34, 35] induced by interferons  or other inflammatory cytokines after the system immune activation to resist viral infection [37, 38] or tumor.
Cortisol increased in both CHB and HCC groups compared with the healthy control. Most of CHB patients in this study were with active hepatitis virus B replication. It was reported that serum cortisol significantly increased in patients with the chronic active hepatitis B while increased but not statistically significant in cases with HBeAg (+) and chronic persistent hepatitis B . And elevated cortisol was also observed in plasma of 15 Southern African black patients with HCC in comparison to ten control subjects which was significantly correlated with alpha (2)-macroglobulin elastase binding capacity .
The formation of acylcarnitines is essential to the mitochondria shuttle of long-chain fatty acid for β-oxidation. And acylcarnitines with different acyl residues are usually used to screen or diagnose inborn fatty acid oxidation (FAO) errors . And also, to examine FAO alteration in obesity and type 2 diabetes (T2DM), acylcarnitines in human plasma from individuals with obesity and T2DM during fasting and insulin-stimulated conditions have been profiled. Higher level of long-chain acylcarnitines has been found both in obesity and T2DM with accumulated shorter-chain species in T2DM as well . In comparison, in this study of CLD and HCC cases, C16:1-CN as one of the long-chain acylcarnitines, increased with severity of chronic liver diseases, while acylcarnitines with medium and short-chain including C10-CN, C10:1-CN, C8-CN, and C6-CN decreased. So, it is important to further investigate serum free fatty acid level and the special alteration of FAO in different liver diseases.
Fatty acid profiling of HCC and CLD in the negative mode
Alteration trends of fatty acids in CHB, CIR, and HCC, compared with healthy controls (N)
Mann–Whitney U test (p))
HCC vs. CIR
HCC vs. CHB
CIR vs. CHB
Alterations of serum acylcarnitine level among HCC and CLD
Deregulation of fatty acid related metabolism in HCC and CLD
SCD, with its main isoform SCD1 expressed in liver , was activated in CHB, CIR and HCC, characterized by reversely decreasing ratio of saturated FFAs to monounsaturated FFAs (Fig. 3). The benefit of SCD1 deficiency to increase FAO and inhibit lipid synthesis has been reviewed , concluding that increased SCD activity in humans and animals is related to significantly accumulated lipids in liver . And the significantly decreased ratio of saturated FFAs to monounsaturated FFAs in CIR and HCC compared with CHB indicated that the SCD activity increased with the severity of liver diseases.
The combustion of FFAs in different liver diseases can be evaluated on the levels of acylcarnitines. Long-chain acylcarnitines which are responsible for shuttling into mitochondria of long-chain fatty acids, increased in liver diseases compared with the controls (Fig. 4a), suggesting that more fatty acids can enter mitochondria . Moreover, there was a trend that the more severe the liver injury was, the more long-chain acylcarnitines accumulated in plasma.
With the decline of free carnitine and increase of long-chain acylcarnitines in liver disease state, FFAs were readily translocated onto carnitines by carnitine palmitoyl transferase 1 (CPT 1), reflected by higher level of (C16 + C18)-CN/carnitine (Fig. 5a). CPT 1 has been reported to be regulated by long-chain FFAs, independently from peroxisome proliferator-activated receptor alpha (PPARα) , a sensor for FFAs. However, the lower level of carnitine and higher level of long-chain acylcarnitnes indicated a deficiency of carnitine palmitoyl transferase 2 (CPT 2), demonstrated by reversely increased (C16 + C18:1)-CN/C2-CN (Fig. 5b). CPT 2 locates at the inner side of inner mitochondria membrane, responsible for fatty acid conjugated back to CoA releasing carnitine to mitochondrial matrix. Also, the increasing ratio of C16-CN to C8-CN (Fig. 5c) and decreasing medium and short-chain acylcarnitines (Fig. 4b) in liver diseases, indicated that once the fatty acids were conjugated back to CoA in the mitochondria matrix, they may be easily oxidized into acetyl-CoA. That is to say, FAO in liver diseases is mainly limited by CPT 2. Seelaender et al. have also found that CPT 2 activity shrank in rats bearing the Walker 256 carcinosarcoma and the expression of CPT 2 may be affected by a prostaglandin, PGE(2) .
Free carnitine decreased progressively in non-malignant liver diseases with a little rebound in HCC compared with CIR (Fig. 4b). It has been reported that carnitine strikingly decreases in PPARα (−/−) mice, due to both direct and indirect effects of PPARα inactivity. And there are reduced tissue levels of short-chain acylcarnitine intermediates and tissue accumulation of long-chain acyl-CoAs, as well . So the expression level of PPARα in liver diseases needs to be investigated further. Free carnitine supplement has been proved to effectively prevent hepatitis and subsequent HCC in Long-Evans Cinnamon rats via protection of mitochondria from oxidative injury .
C2-CN is mainly from acetyl-CoA, the ultimate product of FAO. The significantly decreased plasma level of C2-CN in liver diseases (Fig. 4b) implies both the lack of long-chain acyl-CoA in mitochodria matrix for production of acetyl-CoA and the fast consumption of acetyl-CoA. As is mentioned above, cancer cell reprograms its energy metabolism by promoting glycolysis and suppressing the TCA cycle, which would inevitably causes the accumulation of carboxylic acid in the mitochondria. When these carboxylic acids are transported outside the mitochondria by carnitines, the relative accumulation of C2-CN and other medium and short-chain acylcarnitines would be detected in HCC compared with CIR (Fig. 4b). And these glycolytic intermediates may subsequently facilitate the proliferation of cancer cells . Moreover, their accumulation may reversely inhibit the formation of long-chain acyl-CoA, resulting a relative smaller accumulation of long-chain acylcarnitines in HCC compared with CIR (Fig. 4a), and lower ratios of (C16 + C18)-CN to carnitine, and C16-CN to C8-CN as well (Fig. 5a, c). Moreover, C2-CN/carnitine, an indicator for beta-oxidation of even-numbered fatty acids, rose in CIR and HCC (Fig. 5c), resulting from more severe liver mitochondria injury with corresponding higher AST/ALT.
Through combining serum metabolic profiling with the RF-RFE algorithm for feature selection, important metabolic changes among liver diseases were found out from a large amount of metabolome data. These metabolic deregulations are related to pathways of tryptophan, glucocorticoid, bile acids and FFAs among the liver diseases. The deregulation of FFA metabolism is a primary focus subsequently for it is closely related with the reprogramming of energy metabolism in cancer cell. It was found that a lowered level of free carnitine and a deficiency of CPT 2 are correlated with liver disease severity. Besides, the alterations of FFA (16:1), SCD activity, acylcarnitines and some corresponding enzymes also imply the possible mechanisms related to the development of HCC. These results provide an important insight into metabolic deregulations contributing to the development of liver diseases and may further help to explore new target for preventing deterioration of liver diseases and differential diagnosis of HCC.
The study has been supported by the State Key Science and Technology Project for Infectious Diseases (2008ZX10002-017 and 2008ZX10002-019) from State Ministry of Science and Technology of China, and the key foundation (no. 20835006) and the creative research group project (no. 21021004) from National Natural Science Foundation of China.
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