Serum metabolomic profile and potential biomarkers for severity of fibrosis in nonalcoholic fatty liver disease
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- Tokushige, K., Hashimoto, E., Kodama, K. et al. J Gastroenterol (2013) 48: 1392. doi:10.1007/s00535-013-0766-5
Biomarker for usefulness in diagnosing advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is expected. In order to discover novel biomarkers for NAFLD and its pathogenesis, we performed matabolomics screening.
(1) The initial cohort was 44 NAFLD patients. (2) This validation cohort was 105 NAFLD patients, 26 primary biliary cirrhosis (PBC) patients, and 48 healthy controls. Using capillary electrophoresis and liquid chromatography with mass spectrometry, we analyzed low molecular weight metabolites in these groups.
1. In the initial cohort, we found 28 metabolites associated with advanced fibrosis. Among them, 4 sulfated steroids showed the greatest difference. A decrease of dehydroepiandrosterone sulfate (DHEA-S) and 5α-androstan-3β ol-17-one sulfate (etiocholanolone-S) was observed with the progression of fibrosis. Furthermore, 16 hydroxydehydroepiandrosterone sulfate (16-OH-DHEA-S) increased with the progression of fibrosis. 2. In the validation cohort, the decrease of DHEA-S and etiocholanolone-S, as well as the increase of 16-OH-DHEA-S, with the progression of fibrosis was confirmed. The 16-OH-DHEA-S/DHEA-S ratio and 16-OH-DHEA-S/etiocholanolone-S ratio were even more strongly associated with the grade of fibrosis. Among PBC patients, 16-OH-DHEA-S tended to be higher in stages 3 and 4 than in stages 1 and 2. However, levels of DHEA-S, etiocholanolone-S, and the two ratios were not associated with the stage of PBC.
Several metabolic products were found to be biomarkers of fibrosis in NAFLD and could also be useful for diagnosis of this condition. Our findings suggested disturbance of hormone metabolism in NAFLD and might lead to the development of new therapy.
Nonalcoholic fatty liver disease
Primary biliary cirrhosis
5α-androstan-3β ol-17-one sulfate
16 hydroxydehydroepiandrosterone sulfate
Nonalcoholic fatty liver disease (NAFLD) has become the most common type of liver disease in developed countries. NAFLD covers a wide spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC) [1, 2, 3]. NASH was initially identified as a clinicopathological entity, and biopsy is still considered to be the “gold standard” for definitive diagnosis. However, liver biopsy has several drawbacks because it is an invasive, painful, and costly procedure that is associated with the possibility of sampling error and variability in interpretation. Moreover, the extremely high prevalence of NAFLD in the general population makes liver biopsy unsuitable as a diagnostic procedure for NASH. These shortcomings and drawbacks of liver biopsy highlight the urgent need to find noninvasive markers, such as biochemical markers, or imaging techniques for the assessment of NASH. So far, biomarkers have been evaluated for usefulness in distinguishing NASH from NAFLD and/or diagnosing advanced fibrosis or cirrhosis .
Recently, Younossi et al.  reported that a number of pathologic features were associated with hepatic mortality on univariate analysis, but fibrosis was the only independent predictor according to multivariate analysis, so the severity of fibrosis rather than a diagnosis of NASH determined hepatic mortality. In other words, liver fibrosis is the most important index of the severity of NAFLD.
Metabolomics involves the measurement of a large number of low molecular weight metabolites, including sugars, amino acids, and hormones, and it has emerged as a powerful tool for discovering novel biomarkers. Metabolomics has already identified new biomarkers for prostate cancer , Parkinson’s disease , type 2 diabetes , acute myocardial infarction , and preeclampsia . This promising area of research has also recently provided some important insight into the pathogenesis of human NAFLD and NASH [11, 12, 13]. However, sample size was small or their method was not in detail. We have attempted to discover novel fibrosis markers by detail method and their pathological role in the progression of NAFLD.
The present study was performed to investigate the differences of serum metabolomics between NAFLD patients with or without advanced fibrosis and to validate any novel biomarkers identified by the initial metabolomic analysis in another cohort of subjects.
Materials and methods
A total of 149 patients with a clinical and/or histological diagnosis of NAFLD made at Tokyo Women’s Medical University between 2008 and 2011 were enrolled in this study. Forty-four patients formed the in initial cohort and 105 patients were used as the validation cohort. Diagnosis of NAFLD was based on the following criteria: (1) macrovesicular steatosis affecting at least 5 % of hepatocytes, (2) intake of less than 20 g of ethanol per day (confirmed by the attending physician and family members residing with the patient), and (3) appropriate exclusion of other liver diseases such as alcoholic liver disease, viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis [2, 14].
A complete history was obtained and physical examination was performed in all patients, followed by measurement of the following laboratory parameters: aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count, hepatitis B serology (hepatitis B surface antigen, antibody to hepatitis B surface antigen, and hepatitis B core antigen antibody), hepatitis C serology (hepatitis C virus antibody), and autoantibodies (antinuclear antibody and antimitochondrial antibody). All liver biopsy specimens were examined by using hematoxylin-eosin, Mallory, and silver reticulin staining. Fibrosis was scored on a 5-grade scale: F0, normal connective tissue; F1, focal perivenular or pericellular fibrosis in zone 3; F2, perivenular or pericellular fibrosis confined to zones 3 and 2 with portal/periportal fibrosis; F3, bridging or septal fibrosis; and F4, cirrhosis [14, 15]. Steatosis was graded on a scale of 1 to 3:1, mild (affecting 5–33 % of hepatocytes); 2, moderate (33–66 % of hepatocytes); 3, severe (>66 % of hepatocytes). Inflammation was graded as mild, moderate or severe based on the pathologist’s overall impression.
The healthy control subjects (HC) were volunteers. They were all Japanese and were matched for age with the NAFLD patients. All control subjects were confirmed to have normal liver function and no viral hepatitis.
Twenty-five PBC patients were used as the disease control group. The diagnosis of PBC was based on the following three criteria: (1) detection of serum anti-mitochondrial antibody, (2) elevated liver enzymes at the initial diagnosis, and (3) histological features compatible with PBC on liver biopsy . Fibrosis was classified according to Scheuer’s classification  and the PBC patients were divided into 2 groups (stage 1–2 or stage 3–4). PBC patients had no other chronic liver diseases, including viral hepatitis.
Informed consent was obtained from all subjects before enrollment in the study. The study protocol conformed to the ethical guidelines of the 2008 Declaration of Helsinki and was approved by our institution’s research committee.
Sample collection and metabolomics analysis
Serum was obtained by centrifugation of blood samples for 10 min at 1500×g and 4 °C, and was stored at −80 °C. Preparation of serum for capillary electrophoresis (CE)-TOF mass spectrometry (MS) or liquid chromatography (LC)-TOFMS and measurement were performed as described previously . With each system, two mode of the measurement were employed to detect both of cationic and anionic metabolites. Identification of compounds from the peaks was based on the annotated tables of m/z values and normalized migration times.
Important metabolites were confirmed by measurement of standard compounds. As chemical standards, dehydroepiandrosterone sulfate (DHEA-S) and 5β-androstan-3α ol-17-one sulfate (etiocholanolone-S) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), 16 hydroxydehydroepiandrosterone sulfate (16-OH-DHEA-S) was purchased from Steraloids Inc. (Newport, RI, USA), and dehydroepiandrosterone disulfate (DHEA-2S) was obtained by the sulfation of dehydroepiandrosterone as reported previously .
Identification of candidate biomarkers
The initial cohort was studied to seek candidate biomarkers for detection of advanced fibrosis. The 44 NAFLD patients in initial cohort were divided into 2 groups based on the severity of fibrosis (F0-2 vs. F3-4). We compared the two groups with respect to the results of metabolomics analysis. Peaks showing significant differences between the two groups were selected as potential predictors of advanced fibrosis.
Several important biomarkers and their ratios were validated in another 105 NAFLD patients, 48 control subjects, and 25 PBC patients (disease control group). Further analysis was done by comparing NAFLD patients with control subjects or stage 1–2 versus stage 3–4 PBC patients.
Diagnosis of fibrosis
We investigated the diagnosis of fibrosis ≥F3. All NAFLD patients from the combined initial and validation cohort were used for this study. Diagnostic performance was assessed by ROC analysis, with calculation of the area under the ROC curve (AUC).
Peak areas were normalized against those of the internal standards in each measurement mode and the resulting values (relative areas) were used for calculation. For assessment of the selected biomarkers, the absolute serum concentration was determined from the calibration curve obtained with standard chemicals added to serum. For the assessment of ratios between two markers, ratio values were changed into log-values. For comparison between groups of different stages, Steel–Dwass test for NAFLD or Welch’s t test for PBC was employed. The association between age and several metabolomic parameters was determined by Pearson’s correlation coefficient analysis and correlation coefficients (R values) were calculated.
Profile of the subjects
A. Initial cohort
55.8 ± 14.3 (29–82)
B. Validation cohort
51.5 ± 15.4 (18–84)
26 (Stage 1–2, 20; Stage 3–4, 6)
64.2 ± 12.8 (47–90)
55.7 ± 3.4 (50–60)
Potential biomarkers found by screening
F < 3
F ≥ 3
0.1022 ± 0.0882
0.0269 ± 0.0269
0.0083 ± 0.0027
0.0144 ± 0.0144
0.0182 ± 0.0147
0.0402 ± 0.0402
4.0088 ± 1.3949
5.0026 ± 5.0026
0.0036 ± 0.0021
0.0068 ± 0.0068
0.0035 ± 0.0017
0.0024 ± 0.0024
0.0207 ± 0.0049
0.0254 ± 0.0254
0.0363 ± 0.0364
0.0150 ± 0.0150
cis-11-Eicosenoic acid or cis-13-Eicosenoic acid
0.0371 ± 0.0228
0.0585 ± 0.0585
0.0144 ± 0.0053
0.0107 ± 0.0107
0.0512 ± 0.0089
0.0450 ± 0.0450
0.3946 ± 0.0806
0.4802 ± 0.4802
0.0053 ± 0.0025
0.0037 ± 0.0037
0.0538 ± 0.0391
0.0352 ± 0.0352
0.0465 ± 0.0108
0.0403 ± 0.0403
0.0122 ± 0.0080
0.0207 ± 0.0207
0.0121 ± 0.0095
0.0213 ± 0.0213
0.0260 ± 0.0064
0.0226 ± 0.0226
2.3337 ± 1.5646
1.6048 ± 1.6048
0.0087 ± 0.0016
0.0098 ± 0.0098
0.0037 ± 0.0024
0.0074 ± 0.0074
Butyric acid (or) isobutyric acid
0.0341 ± 0.0135
0.0275 ± 0.0275
0.0200 ± 0.0032
0.0183 ± 0.0183
0.0064 ± 0.0027
0.0047 ± 0.0047
0.0128 ± 0.0155
0.0057 ± 0.0057
0.2776 ± 0.2319
0.1424 ± 0.1424
0.0497 ± 0.0582
0.0258 ± 0.0258
0.0188 ± 0.0058
0.0163 ± 0.0163
The ratio of 16-OH-DHEA-S to DHEA-S (16/D) and the ratio of 16-OH-DHEA-S to etiocholanolone-S (16/E) were more clearly associated with the fibrosis grade (Fig. 1d–e) (16/D ratio: HC, −1.31 ± 0.18; NAFLD F0-1, −1.15 ± 0.47/F2, −0.86 ± 0.50/F3, −0.43 ± 0.48/F4, −0.056 ± 0.47; 16/E ratio: HC, −0.76 ± 0.25; NAFLD F0-1, −0.46 ± 0.76/F2, −0.12 ± 0.68/F3, 0.57 ± 0.64/F4, 0.96 ± 0.65).
NAFLD patients were divided by gender and separately compared the markers for mild fibrosis (F0-2) and severe fibrosis (F3-4) (supplementary Fig. 1A and B). In male cases (n = 57), all markers and both ratios were significantly different between mild fibrosis and severe fibrosis. In female cases (n = 48), etiocholanolone-S and both ratios were significantly different. DHEA-S and 16-OH-DHEA-S were not significantly different, but showed similar tendencies.
Diagnosis of fibrosis
By metabolomics analysis, we found several biomarkers that were associated with progression of fibrosis in NAFLD, and our findings suggested disturbance of hormone metabolism in this disease.
In the present study, we investigated metabolites related to the severity of fibrosis in NAFLD. We identified 28 peaks with significant differences between patients with and without advanced fibrosis, which were candidate biomarkers. Most of these candidates are well known to have a role in the pathogenesis of NAFLD/NASH, including fatty acids, ADMA, DHEA-S, glutamate, carnosine, hypotaurine, serotonin, and pipecolic acid [11, 20, 21, 22, 23, 24, 25, 26]. However, other candidates have not been associated with the pathogenesis or progression of NAFLD.
We focused on sex hormone metabolites because of these marked differences. DHEA is a potential mediator of reactive oxygen species scavenger synthesis and has also been reported to augment insulin sensitivity . DHEA-S is the most abundant circulating androgen, and it has already been reported to be useful for diagnosis and monitoring of fibrosis in NAFLD .
Supplementary Tables 1A and B show candidates for biomarkers of inflammation (activity) and steatosis. Steatosis grade and activity grade were influenced by fibrosis. Therefore, we need further analysis to discover real novel biomarkers for inflammation and steatosis in NAFLD.
We compared these metabolites between simple steatosis and NASH. However, as there were only 8 simple steatosis cases in the initial and validation studies, we could not find novel markers for the discrimination between simple steatosis and NASH.
2.085 ± 2.020
3.400 ± 2.609
0.361 ± 0.417
0.866 ± 0.966
0.342 ± 0.324
0.442 ± 0.437
−0.736 ± 0.546
−0.893 ± 0.625
0.156 ± 0.835
−0.201 ± 0.820
By using metabolomic screening, we found some novel biomarkers that suggested disturbance of hormone metabolism is associated with progression of fibrosis in NAFLD. It may be important to investigate the mechanisms of these changes and their pathological significance in NAFLD, since the results could lead to the development of new therapies. In conclusion, we hope that our findings contribute to understanding both the clinical and pathological aspects of NAFLD.
This work was supported in part by a grant-in aid from the Ministry of Health, Labor and Welfare of Japan.
Conflict of interest
The authors declare that they have no conflict of interest.
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