Equol Production Status is An Effective Predictor of Nonalcoholic Steatohepatitis in Women


 Equol is a metabolite of daidzein, a major soybean isoflavone with estrogenic and antioxidant activities. As the production of equol depends on the presence of certain members of the intestinal microflora, not all individuals can produce equol. The incidence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) increases in menopausal women, and estrogen is associated with the progression of NAFLD/NASH. Therefore, the status of equol production might also be related to the pathogenesis of NAFLD/NASH in women. We, thus, examined the relationship between NASH histological features and equol production. Thirty-eight NAFLD patients who underwent liver biopsy were included in this study. In women, the degree of fibrosis and ballooning in nonproducers was significantly higher than that in producers. The percentage of nonproducers with NAFLD activity score (NAS) ≥5 was significantly higher than that of producers. None of the histological features were significantly different between nonproducers and producers in men. Decision tree analysis identified the predictors of NAS ≥5 in women. The status of equol production was the strongest predictor, followed by fasting glucose. Equol can be noninvasively detected in urine, suggesting its application as a screening test for predicting the diagnosis and progression of NASH in women.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is globally the most common chronic liver disease, representing a signi cant health burden worldwide. Brie y, NAFLD includes a broad range of conditions, as among which simple steatosis and nonalcoholic steatohepatitis (NASH) 1 . The pathophysiology of NASH is multifactorial, involving genetic and epigenetic factors, insulin resistance, adipose-derived hormones, and nutritional factors 2 . The most potent driver of NASH is lipotoxicity-induced hepatocyte death, which triggers in ammation and brosis, leading to cirrhosis or liver cancer.
After menopause, women have an increased risk for insulin resistance, hyperlipidemia, and visceral fat accumulation, all of which are known risk factors for NAFLD 3 . A higher incidence of NAFLD is found in postmenopausal women than in premenopausal women, as estrogens inhibit stellate cell activation and brogenesis, suggesting their correlation with the progression of NAFLD/NASH 4 .
Soy-based foods, which are largely consumed in Asian countries, have become popular in non-Asian countries because of their bene cial effects on health. Equol, which is produced by the gut microbiota in the gastrointestinal tract, is a metabolite of daidzein, a major iso avone found in soybean 5 . Equol has been reported to exert estrogenic activity, with a nity for both ER alpha and ER beta estrogen receptors.
Equol is superior to all other iso avones in its antioxidant activity 6 . As the production of equol depends on the presence of certain intestinal micro ora, not all individuals have the ability to produce equol from the metabolism of iso avones 7 . Only 25-30 % of the adult population in Western countries have the ability to produce equol when fed soy-based foods containing iso avones [8][9][10] . This percentage is signi cantly lower than the reported 50-60 % frequency of equol-producers in adults from Japan, Korea, and China 11-13 , or Western adult vegetarians 14 . Usui et al. reported that the ratio of equol nonproducers in overweight or obese Japanese women was lower than the generally reported range 15 . Yoshikaka et al. reported that equol production was signi cantly associated with lower arterial stiffness and uric acid levels, and a high ratio of eicosapentaenoic acid to arachidonic acid in women in their 50s 16 . As NAFLD/NASH has been associated with both the visceral fat area and metabolic syndrome, and the incidence of NAFLD/NASH is known to increase after menopause in women, the ability to produce equol is expected to be related to the pathogenesis of NAFLD/NASH; however, to date, no relationship has been reported between them. Therefore, we aimed to examine the relationship between the pathogenesis of NASH and the status of equol production.

Study populations
Forty Japanese patients with clinically suspected NASH (13 men and 27 women) who visited the Department of Gastroenterology, Nara Medical University Hospital (Kashihara, Japan) between October 2017 and March 2019, were recruited for this study. All patients underwent a liver biopsy. The upper limit of alcohol consumption for NASH was de ned as less than 30 g/d for men and less than 20 g/d for women in terms of ethanol content. Exclusion criteria included: those positive for hepatitis B surface antigen or antihepatitis C virus antibodies, those with other hepatobiliary diseases, those on antibiotics or hormone therapy, those with diarrhea, and those unable to consume soy food products due to allergy or preference. Following the exclusion of 2 women who did not consume soy food products due to preference, a total of 38 (13 men and 25 women) patients were enrolled in this study.

Clinical and Laboratory assessments
The information collected using a questionnaire included alcohol consumption, medication history, and soy food consumption habits. In accordance with the de nition of the Japanese Society of Obstetrics and Gynecology, women without a menstrual period for 12 mo are considered menopausal. Hypertension was de ned as a systolic blood pressure ≥ 140 mmHg, a diastolic blood pressure ≥ 90 mmHg, or current use of antihypertensive medication. Diabetes mellitus was de ned as a fasting blood glucose level ≥ 126 mg/dL or current use of antidiabetic medication. Dyslipidemia was de ned as the current use of lipid-lowering medication. Insulin resistance was assessed using the homeostasis model assessment-insulin resistance (HOMA-IR), which was calculated as fasting insulin (µU/mL) × fasting glucose (mg/dL) / 405. The FIB-4 index for noninvasive markers of liver brosis was calculated as To determine the equal-producing status, participants consumed soy food products containing approximately 50 mg iso avones twice a day, and urine samples were collected the following morning. The levels of daidzein and equol in the urine of patients were measured in the Saga Nutraceuticals Research Institute (Otsuka Pharmaceutical Co., Ltd., Japan) by high-performance liquid chromatography (HPLC; Nexera X2; Shimadzu, Japan) using a type C18 column (Cortecs C18, 2.7 µm, 3.0 × 150 mm; Waters, USA) and an SPD-M30A PDA and RF-20Axs detection system (Shimadzu), according to a modi ed method by Lundh et al. 30 . This method has been used for the detection of metabolites in human urine in previous studies 31 . Brie y, 100 µL urine sample was deconjugated by incubating with 2 µL (ca. 300 U) β-glucuronidase (G0876, Sigma-Aldrich, USA) in sodium acetate buffer (pH 5.0) at 37°C for 30 min. Samples were then extracted using OASIS HLB microelution plates (Waters) prior to HPLC analysis. Quantitation was performed by means of UV response (254 nm and 280 nm) for daidzein and uorescence response (Ex: 255 nm, Em: 310 nm) for equol. The laboratory precision was assessed for each batch of samples through the analysis of the standard solution. The limits of quanti cation (LOQ) for daidzein and equol were 0.076 and 0.080 nmol/mL, respectively. The equol-producing status of each urine collection was de ned by using a log-transformed equol/daidzein ratio of − 1.75 or more 14 .

Pathology
The liver tissue biopsy specimens were stained with hematoxylin-eosin, silver, and Azan stain, and analyzed by experienced pathologists blinded to the clinical data. NASH was de ned as fat accumulation in more than 5 % of hepatocytes, in ammation, and hepatocellular ballooning on liver biopsies. Disease activity was scored using the NAFLD activity score (NAS) of the Nonalcoholic Steatohepatitis Clinical Research Network. The scoring system comprised 4 histological features: steatosis (0-3), lobular in ammation (0-2), hepatocellular ballooning (0-2), and brosis (0-4) 23,24 . Accordingly, NAS is the unweighted sum of steatosis, lobular in ammation, and hepatocellular ballooning scores. The substages (1a, 1b, and 1c) of brosis stage 1 were combined as a single stage.

Statistical analysis
All data were expressed as the mean ± SD. Continuous variables were compared between 2 groups using the Student's t-test or the Mann-Whitney U test, whereas categorical variables were compared using the chi-square test. Statistical signi cance was set at p < 0.05. A decision-tree algorithm was constructed to reveal the pro les associated with an NAS ≥ 5. The following factors were set as independent variables: age, menopause, hypertension, dyslipidemia, diabetes mellitus, BMI, platelet count, fasting glucose, HOMA-IR, ferritin, type 4 collagen 7S, P-III-P, FIB-4, APRI, and equol producers. All calculations were performed using the SPSS software (version 27, IBM Corp., Armonk, NY, USA).

Subject baseline characteristics
We found that the number of equol nonproducers was 23 out of 38. The prevalence of diabetes was shown to be signi cantly lower in nonproducers than in producers. We did not detect any signi cant differences in any of the other parameters tested between the 2 groups (Table 1). When subjects were strati ed by sex, 7 male (53 %) and 8 female (32 %) patients were identi ed as equol producers. No signi cant differences were detected in any of the tested parameters between nonproducers and producers in men. We observed, however, that in women the prevalence of diabetes was signi cantly lower, whereas the prevalence of dyslipidemia was higher, although not signi cantly different (p = 0.054), in nonproducers compared with those in producers ( Table 2). Values are presented as N (%) or mean ± standard deviation. BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HOMA-IR, homeostasis model assessment-insulin resistance; type III procollagen peptide; P-III-P, aspartate aminotransferase to platelet ratio index; APRI.

Comparison of pathological features between equol nonproducers and producers
We compared the 4 histological features of NAS (steatosis, lobular in ammation, hepatocellular ballooning, and brosis) between nonproducers and producers strati ed by sex (Table 3). We noticed that in men, none of the histological features were signi cantly different between nonproducers and producers. In contrast, we found that in women, the degree of brosis in nonproducers was signi cantly higher than that in producers. Although we did not observe any stage 4 brosis in producers, the percentage of stage 4 brosis in nonproducers was found to be 23.5 %. Likewise, the degree of ballooning in nonproducers was shown to be signi cantly higher than that in producers, with the percentage of ballooning score 2 in nonproducers being 58.8 %, whereas that in producers being 12.5 %.  We compared the percentage of nonproducers and producers with NAS ≥ 5. We accordingly found that in men, the percentage of nonproducers with NAS ≥ 5 was not signi cantly different from that in producers.
However, in women, the percentage of nonproducers with NAS ≥ 5 was demonstrated to be signi cantly higher than that in producers (Fig. 1).
Prediction model for NAS ≥ 5 using decision trees analysis To clarify the pro les associated with NAS ≥ 5, we created a decision-tree algorithm. We set the following factors as independent variables: age, menopause, hypertension, dyslipidemia, diabetes mellitus, BMI, platelet count, fasting glucose, HOMA-IR, ferritin, type 4 collagen 7S, P-III-P, FIB-4, APRI, and equol producers. Among the factors expected to predict NAS ≥ 5, we selected equol producers as the initial distinguishable factor for NAS ≥ 5 in women. We observed a NAS ≥ 5 in 25 % of equol producers. In contrast, a NAS ≥ 5 was identi ed in 82 % of equol nonproducers. Fasting glucose was demonstrated to be the second most distinguishable factor among equol nonproducers. We observed a NAS ≥ 5 in 93 % of nonproducers with fasting glucose levels > 84 mg/dL. In contrast, none of the nonproducer patients with fasting glucose ≤ 84 mg/dL was found to have a NAS ≥ 5 (Fig. 2).

Discussion
We showed in this study that in women with NASH, the degree of brosis and ballooning in equol nonproducers was signi cantly higher than that in producers. In addition, the percentage of nonproducers with NAS ≥ 5 was signi cantly higher than that in producers in women with NASH. In contrast, in men with NASH, no difference was identi ed between nonproducers and producers. As most women in this study were menopausal, it could be suggested that the status of equol production is associated with the pathogenesis of NASH in menopausal women.
The percentage of equol producers in Asia has been reported to be approximately 50-60 % 12,17 . In this study, 53 % of men with NASH were equol producers, similar to a previous report 17 . However, in women with NASH, the percentage of equol producers was 32 %, lower than previously reported 12 and similar to the percentage of equol producers in obese women reported by Usui et al. 15 . Equol is produced from the iso avone daidzein in the gut of humans and animals through the action of certain bacterial biotypes 18 . Gut dysbiosis has been signi cantly associated with obesity and the development and progression of NAFLD. The progression from NAFLD to NASH is known to largely occur due to bacterial dysbiosis, which activates in ammatory and pro brogenic intracellular pathways via Toll-like receptors (TLRs) and the activation of the in ammasome 19 . Bacterial dysbiosis might also be one of the causes of the inability of the host to produce equol, which in turn might be one of the reasons for the progression of NASH.
In women, the proportion of those with dyslipidemia was higher among equol nonproducers than producers, although the difference was not signi cant (p = 0.054 cholesterol than nonproducers 20 . Estrogen is known to increase the levels of HDL cholesterol and decrease those of LDL cholesterol, in uencing body fat deposition. In addition, equol has been reported to exert the strongest estrogenic activity among all known iso avones or iso avone-derived metabolites 21,22 . Both the central adiposity and levels of serum LDL cholesterol are known to increase in menopausal women. Therefore, the production of equol has been suggested to be associated with postmenopausal central adiposity and lipid metabolism.
In our study, the proportion of women affected with diabetes mellitus was higher in producers than nonproducers. Guo et al. reported that no signi cant difference was observed in the levels of fasting blood glucose between equol producers and nonproducers, neither in the overall group nor in patients strati ed by sex 20 , suggesting that equol production probably had no effect on glucose metabolism.
However, as diabetes is known to be closely related to the pathogenesis of NASH, it is likely that diabetes has a greater impact on NASH in equol producers.
The diagnosis of NASH is established by the presence of a characteristic pattern of steatosis, in ammation, and hepatocellular ballooning on liver biopsies. Accordingly, a NAS threshold value (NAS ≥ 5) has been used for the diagnosis of NASH 23,24 . Our decision-tree analysis results showed that in women, the ability for equol production was the strongest predictor of NAS ≥ 5, with fasting glucose being the second strongest predictor of NAS ≥ 5 in equol nonproducers. As equol is thought to affect lipid metabolism and oxidative stress, a combination of the inability to produce equol and glucose metabolism abnormalities might increase the risk of NASH progression. As equol can be noninvasively measured in urine samples, it could be used as a useful screening tool for the prediction of NASH in women.
Equol is known to be superior to all other iso avones in its antioxidant and estrogenic activities.
Moreover, equol has been reported to be effective in improving the ischemic cardiovascular risk pro le 25 , menopausal symptoms 26,27 , and suppression of decreased bone mineral density 28,29 . Although further research is needed, equol intake as a therapeutic strategy might lead to the improvement of NASH.
Our study had several limitations. The rst limitation was the small sample size. Second, this study had a cross-sectional design that did not identify whether equol nonproducers progressed to NASH. Third, this was a single-center study.

Conclusion
Among women with NASH, equol nonproducers, which were also associated with brosis progression, had a signi cantly higher percentage of NAS ≥ 5 than producers. The status of equol production was the strongest predictor of NASH in women. As equol can be noninvasively measured in urine, its detection could be employed as a simple screening test for predicting the diagnosis and progression of NASH in women.

Declarations
Ethics approval This study was approved by the Ethics Committee of Nara Medical University (approval no. 1607). The study was conducted in concordance with the Helsinki Declaration. All included patients have signed an informed consent form prior to their participation in the study. All research was performed in accordance with relevant guidelines and regulations.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request. 31. Ideno, Y. et al. Optimal cut-off value for equol-producing status in women: The Japan Nurses' Health Study urinary iso avone concentration survey. PLoS One 13, e0201318, doi:10.1371/journal.pone.0201318 (2018). Figure 1 Comparison of the percentage of individuals with NAS ≥5 between equol nonproducers and producers. A: Male. The percentage of nonproducers with NAS ≥5 was not signi cantly different from that in producers. B: Female. The percentage of nonproducers with NAS ≥5 was signi cantly higher than that in producers.