Shigeto Uchiyama

Possible role of equol status in the effects of isoflavone on bone and fat mass in postmenopausal Japanese women: a double-blind, randomized, controlled trial.

Objective:Equol is more biologically active than its precursor daidzein, which is the principal isoflavone found in soybean. There are interindividual differences in the ability to produce equol; these may lead to differences in the effects of isoflavone intervention on human health. This study aimed to investigate whether the effects of soy isoflavones on bone and fat mass are related to an individuals equol status. Design:We performed a 1-year double-blind, randomized trial to compare the effects of isoflavone (75 mg of isoflavone conjugates/day) with those of placebo on bone mineral density, fat mass, and serum isoflavone concentrations in early postmenopausal Japanese women who were classified based on their equol-producer phenotype. Results:After 1 year, the isoflavone intervention significantly increased the serum equol concentration in the equol producers but not in the nonproducers. In the isoflavone group, the annualized changes in the bone mineral density of the total hip and intertrochanteric regions were −0.46% and −0.04%, respectively, in the equol producers and −2.28% and −2.61%, respectively, in the nonproducers; these values were significantly different (P < 0.05 for both the regions). Significant differences were observed between the equol producers and nonproducers in the isoflavone group with regard to the annualized changes in the fat mass. No significant difference in the annualized changes in bone mineral density and fat mass was observed between the equol producers and nonproducers in the placebo group. Conclusions:Our data suggest that the preventive effects of isoflavones on bone loss and fat accumulation in early postmenopausal women depend on an individuals equol-producing capacity.

Effects of Isoflavone and Exercise on BMD and Fat Mass in Postmenopausal Japanese Women: A 1-Year Randomized Placebo-Controlled Trial†

The combined intervention of isoflavone intake and walking exercise over 1 year in postmenopausal Japanese women exhibited a trend for a greater effect on prevention of bone loss at the total hip and Wards triangle regions.

Effects of natural S‐equol supplements on overweight or obesity and metabolic syndrome in the Japanese, based on sex and equol status

Epidemiologic studies indicate that soy intake has an important role in the prevention of age‐related health problems. Daidzein, the principal isoflavone contained in soy, is converted to S‐equol by the intestinal bacteria. Not all individuals, however, can produce S‐equol, which is considered the most biologically active metabolite. We studied the effects of a natural S‐equol supplement on metabolic parameters associated with overweight or obesity and metabolic syndrome.

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

A Pilot Study on the Effects of S-Equol Compared to Soy Isoflavones on Menopausal Hot Flash Frequency

BACKGROUND S-equol, a metabolite of the soy isoflavone daidzein, has been proposed as having potential for relief of menopausal symptoms. This study compared the efficacy of the natural S-equol supplement, SE5-OH, with isoflavones for relieving hot flashes and other menopausal symptoms. METHODS An 8-week randomized, double-blind, active comparator trial with SE5-OH was conducted in postmenopausal women (aged 45-65 years), who experienced ≥5 hot flashes/day. Participants (n=102) were assigned to one of four treatment groups: 10 (n=24), 20 (n=27), or 40 (n=25) mg S-equol/day or soy isoflavones (n=26). Participants recorded their hot flash frequency and rated their menopause symptom severity. RESULTS Reductions in hot flash frequency at week 8 were similar for all treatment groups. However, based on analyses of the cumulative effect for the 8-week period, 40 mg/day S-equol had a greater reduction of hot flash frequency compared to isoflavones (p=0.021). A subgroup analysis further indicated that for subjects with >8 hot flashes/day at baseline, 20 and 40 mg/day S-equol were superior to isoflavones in reducing hot flash frequency (p=0.045 and p=0.001, respectively). In addition, 10 and 20 mg/day S-equol improved muscle and joint pain score compared with isoflavones (p=0.003 and p=0.005, respectively). CONCLUSIONS S-equol, 10 mg/day, appears to be as effective as soy isoflavones at reducing hot flash frequency and more effective for relieving muscle and joint pain in postmenopausal women. S-equol, ≥20 mg/day, alleviates hot flashes to a greater extent than soy isoflavones in those women who experience >8 hot flashes/day.

Identification of a Novel Dihydrodaidzein Racemase Essential for Biosynthesis of Equol from Daidzein in Lactococcus sp. Strain 20-92

ABSTRACT Equol is metabolized from daidzein, a soy isoflavone, by the gut microflora. In this study, we identified a novel dihydrodaidzein racemase (l-DDRC) that is involved in equol biosynthesis in a lactic acid bacterium, Lactococcus sp. strain 20-92, and confirmed that histidine-tagged recombinant l-DDRC (l-DDRC-His) was able to convert both the (R)- and (S)-enantiomers of dihydrodaidzein to the racemate. Moreover, we showed that recombinant l-DDRC-His was essential for in vitro equol production from daidzein by a recombinant enzyme mixture and that efficient in vitro equol production from daidzein was possible using at least four enzymes, including l-DDRC. We also proposed a model of the metabolic pathway from daidzein to equol in Lactococcus strain 20-92.

Dietary and lifestyle correlates of urinary excretion status of equol in Japanese women.

The isoflavone metabolite equol has been identified in urine or blood samples in some but not all humans. In this cross-sectional study, we examined the association between lifestyle, including diet, and the urinary excretion of equol. Study subjects were 419 Japanese women who were recruited from a breast cancer screening center. Each woman responded to a self-administered questionnaire seeking information about health and lifestyle factors. Diet was assessed by a validated semiquantitative food frequency questionnaire. Urinary isoflavones were measured using spot urine samples. Equol was detected in the urine of 84 (20.0%) women. After controlling for covariates, it was found that dairy product intake was significantly lower in those who excreted detectable equol levels in urine than in those who did not. Because equol is derived from daidzein, individuals with low intake of daidzein may produce undetectable levels of equol. To account for this, the study subjects were restricted to 163 women with urinary daidzein levels of 10 nmol/mg creatinine or higher. The association of equol excretion with dairy product intake remained significant. Demographic factors, smoking status, and menstrual and reproductive factors were unrelated to equol excretion. These data suggest that dairy product intake may be associated with the production of equol.

Effects of S-equol and natural S-equol supplement (SE5-OH) on the growth of MCF-7 in vitro and as tumors implanted into ovariectomized athymic mice.

Hormone replacement therapy (HRT) for treatment of menopausal symptoms is controversial because of reported breast cancer resulting from estrogen treatment and consequent estrogenic stimulation. S-equol, a natural metabolite of the soy isoflavone daidzein produced by intestinal bacteria, has been shown to ameliorate menopausal symptoms, with relatively low concomitant estrogenic receptor stimulation. Although synthesis of equol produces the racemate, the S-isomer may be produced in commercial amounts by bacterial fermentation of soy germ, during the production of the supplement SE5-OH. This study aims to investigate the effects of S-equol and SE5-OH on the growth of MCF-7 in vitro and in vivo. In vitro, purified S-equol, and the isoflavonoid mixture present in SE5-OH stimulated estrogenic transcriptional activity and proliferation of MCF-7-E10 cells, similar to that observed for genistein (another soy isoflavone), but at concentrations from 10(4)-fold to 10(6)-fold higher than seen with 17β-estradiol (E2). Ovariectomized (OVX) mice implanted with MCF-7-E10 cells were fed diets containing 250 or 500 ppm of purified S-equol, isoflavonoid mixture, or genistein. There were no significant differences in tumor growth between the treatment groups and control group. These results suggest that S-equol and natural S-equol in the supplement (SE5-OH), do not promote the progression of breast cancer.

Prevalence of the equol-producer phenotype and its relationship with dietary isoflavone and serum lipids in healthy Chinese adults.

Background Studies have suggested that daidzein-metabolizing phenotypes have beneficial effects on a range of health outcomes. We investigated the prevalence of equol producers and the relationship of equol phenotype with habitual isoflavone consumption and serum lipid concentrations in 200 Chinese adults in Beijing. Methods After the baseline survey and dietary records, 200 healthy adults in Beijing were challenged with a soy-isoflavone supplement for 3 days; 24-hour urine samples were collected before and after the challenge. Isoflavones and their metabolites in urine were measured to determine equol phenotype. Serum lipids, uric acid, and other biochemical markers were also measured. Results Only 26.8% of the participants excreted equol when on a regular diet, as compared with 60.4% after the challenge. After the challenge, urinary isoflavonoid excretion increased in all participants, while equol excretion increased only in equol producers. Isoflavone intake was correlated with urinary isoflavone (range r = 0.49–0.58, P < 0.01). As compared with nonproducers, equol producers were less likely to consume cereals (P < 0.001). There was no significant correlation between serum lipids and isoflavone intake. Serum lipids were not significantly affected by equol phenotype. Conclusions Urinary equol excretion was detected in about 25% of participants under their usual dietary conditions. Their potential to produce equol was increased after the challenge. Urinary isoflavone levels may serve as a useful biomarker for isoflavone intake in populations. We observed an association between equol phenotype and cereal intake. Our findings also suggest that dietary isoflavone intake has no significant effect on serum lipids in healthy participants, regardless of equol phenotype.

Identification of two novel reductases involved in equol biosynthesis in Lactococcus strain 20-92.

Lactococcus strain 20–92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20–92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20–92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20–92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.