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, 20–26]. 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 .
Etiocholanolone-S and 16-OH-DHEA-S were novel biomarkers detected in this study. Etiocholanolone-S is one of the end products of androgen metabolism derived from testosterone (Fig. 4). Its biological significance in relation to liver disease is not clear. In the initial cohort, we also observed that DHEA-2S, which is another end product of androgen metabolism derived from DHEA-S, decreased along with the progression of fibrosis. In contrast, the serum level of 16-OH-DHEA-S, an intermediate of estrogen biosynthesis, was higher in patients with advanced fibrosis. This metabolite is DHEA-S with a hydroxyl group at position 16 and is formed during biosynthesis of estriol from DHEA in the fetal liver . Although it has been reported that the serum level of 16-OH-DHEA-S was increased in patients with breast cancer and endometrial cancer , the biological role of this intermediate in liver disease remains unclear. Our validation analysis confirmed that DHEA-S, etiocholanolone-S, and 16-OH-DHEA-S are useful serum markers for detecting fibrosis in NAFLD. In addition, the 16/D and 16/E ratios were even more clearly associated with the grade of fibrosis.
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.
However, there are limitations to the use of these novel fibrosis markers in NAFLD. When we investigated the influence of age and gender on these markers, we found that DHEA-S and etiocholanolone-S both decreased with increasing age (Fig. 5), although 16-OH-DHEA-S was not influenced by age. Regarding the ratios, the R values of the 16/D and 16/E ratios were decreased compared with those of DHEA-S and etiocholanolone-S (R values; DHEA-S, R = 0.589; etiocholanolone-S, R = 0.460; 16-OH DHEA-S, R = −0.165; 16/D ratio, R = 0.334; 16/E ratio, R = 0.294).
Concerning the influence of gender, we found that DHEA-S, etiocholanolone-S, and the 16/E ratio were significantly different between male and female patients, although 16-OH-DHEA-S and the 16/D ratio showed no influence of gender (Table 3). To investigate the effect of gender and age in our results, we performed 5 multivariate analyses including for each marker, age and gender. In the results, etiocholanolone-S, 16-OH-DHEA-S and both ratios were significant independent markers of comparison between mild fibrosis and severe fibrosis (supplementary Table 2). Both rations only showed small effects of age and gender. Because these compounds are precursors and intermediates of estrogen biosynthesis, their ratios represent the flux of this metabolic pathway. Therefore, our findings suggest that a disturbance of hormone metabolism, which cannot be detected by classical target analysis, is associated with the progression of fibrosis in NAFLD and that these indicators are useful for assessment of the pathology of NAFLD. Recently, adrenal dysfunction in liver diseases became a target of attention . In addition, Lonardo et al. reported that many endocrine derangements were associated with NAFLD through changes in energy and glycolipid homeostasis and/or a central shift in body fat distribution . Naugler et al.  reported that estrogen-mediated inhibition of IL-6 production reduced the liver cancer risk in females. Our data suggest that androgen metabolism might be decreased and estrogen metabolism might be increased. It remains a future issue whether sex hormone metabolism and IL-6 are associated with progression and carcinogenesis in NAFLD. In PBC patients, DHEA-S and etiocholanolone-S were not associated with the stage of fibrosis. Thus, these changes were only seen in NAFLD and may have an important role in its pathogenesis.
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.