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GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. While GmMYB176 is important for isoflavonoid synthesis, it is not sufficient for the function and requires additional cofactor(s). The aim of this study was to identify the GmMYB176 interactome for the regulation of isoflavonoid biosynthesis in soybean. Here, we demonstrate that a bZIP transcription factor GmbZIP5 co-immunoprecipitates with GmMYB176 and shows protein-protein interaction in planta. RNAi silencing of GmbZIP5 reduced the isoflavonoid level in soybean hairy roots. Furthermore, co-overexpression of GmMYB176 and GmbZIP5 enhanced the level of multiple isoflavonoid phytoallexins including glyceollin, isowighteone and a unique O-methylhydroxy isoflavone in soybean hairy roots. These findings could be utilized to develop biotechnological strategies to manipulate the metabolite levels either to enhance plant defense mechanisms or for human health benefits in soybean or other economically important crops.

We determined that consuming chungkookjang fermented by Bacillus subtilis (BS) or Bacillus amyloliquefaciens (BA) alleviated hyperglycemia in partially pancreatectomized (Px) rats, an Asian type 2 diabetic (T2D) animal model. Px rats had deteriorated glucose metabolism with decreased glucose-stimulated insulin secretion and insulin sensitivity. Insulin secretion capacity was improved in the ascending order of the Px-control, positive control (3 mg of metformin/kg of body weight), BS (4.5% BS diet), BA (4.5% BA diet), and normal-control (sham-operated rats). BA and BS increased β-cell mass and decreased malondialdehyde contents and tumor necrosis factor α expression in the islets. BA increased hepatic peroxisome proliferator-activated receptor (PPAR)-α and PPAR-β similar to the positive control. Bacillales , Lactobacillales , and Verrucomicrobiales ( Akkermensia muciniphila ) increased and Enterobacteriales decreased in the BA and BS compared to the Px-control. BA prevented the decrease in the villi area and the number of goblet cells in intestinal tissues. In conclusion, BA improved glucose regulation by potentiating insulin secretion and reducing insulin resistance while maintaining gut mucin contents by improving gut microbiota in lean T2D rats.

Equol improves menopausal symptoms and it is synthesized from daidzein, one of the isoflavonoids in soybeans, by the bacteria in the large intestines of some people. The purpose of this study was to isolate equol-producing bacteria using daidzein from the intestinal microflora and to produce equol-containing chungkookjang (short-term fermented soybean). Equol-producing bacteria from the feces of Sprague-Dawley female rats were isolated using media containing daidzein. The isolated bacteria were cultured in thioglycollate media and equol production was identified through thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry. The bacteria were identified by 16S rRNA sequencing. The rate of equol production in different concentrations of daidzein was assessed. The expression of genes that code for enzymes associated with the production of equol from daidzein was detected through reverse transcription quantitative PCR. The bacterium we isolated was Lactobacillus intestinalis (LC096206.1, 99%). L. intestinalis was found to express daidzein reductase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase, the enzymes involved in producing equol from daidzein. The conversion rate of equol from daidzein was highest (29.5%) using 200 μM daidzein for 48 h of incubation. When chungkookjang fermented with Bacillus amyloquencies SRCM100001 was incubated with L. intestinalis, 0.32 ± 0.04 mg equol/g chungkookjang was produced. In conclusion, L. intestinalis efficiently produces equol from not only daidzein but also in chungkookjang.

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC 50  = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

Estrogenic isoflavones were found, in the 1940s, to disrupt ewe reproduction and were identified in soy-consumers' urine in 1982. This led to controversy about their safety, often supported by current Asian diet measurements, but not by historical data. Traditional Asian recipes of soy were tested while assaying soy glycosilated isoflavones. As these compounds are water-soluble, their concentration is reduced by soaking. Pre-cooking or simmering time-dependently reduces the isoflavone:protein ratio in Tofu. Cooking soy-juice for 15 or 60min decreases the isoflavone:protein ratios in Tofu from 6.90 to 3.57 and 1.80, respectively (p<0.001). Traditional Tempeh contains only 18.07% of the original soybean isoflavones (p<0.001). Soy-juice isoflavones were reduced by ultra-filtration (6.54 vs 1.24 isoflavone:protein; p<0.001). Soy-protein and isoflavones are dissociated by water rinsing and prolonged cooking, but these have no equivalent in modern processes. As regards human health, a precise definition of the safety level of isoflavone intake requires additional studies., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Soy germ rich in isoflavones has attracted much attention for health-promoting characteristics. An effective approach via Monascus aged vinegar soaking was adopted to enhance the aglycone amount. The profiles and interconversion of soy germ isoflavones via Monascus aged vinegar soaking were investigated, and the distribution in vinegars were also explored. The aglycones were dramatically increased by 40.76 times. Concomitantly, β-glycosides and malonylglycosides were significantly decreased. The proportion of aglycones presented a sharp increase with the endogenous β-glucosidase activity at the initial 4h incubation. There appeared to be correlations between β-glucosidase activity and the hydrolysis of conjugated isoflavones. The results demonstrated that the reactions of decarboxylation, de-esterification and de-glycosylation were involved in the Monascus aged vinegar soaking, supporting synergistic effects of enzymolysis by endogenous β-glucosidase from soy germ and acid hydrolysis of vinegars. Soaking by vinegar is a promising pathway for preparing aglycone-rich soy germ., (Copyright © 2015. Published by Elsevier Ltd.)

The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α', α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

Traditionally fermented soybeans (chungkookjang; TFC) may have potent anti-diabetic activity, depending on the ambient microorganisms and conditions. We hypothesized that one of the major Bacillus species in TFC contributes to the anti-diabetic activity and could be used to standardize a highly functional TFC. We tested the hypothesis by using cell-based studies to evaluate insulin sensitizing and insulinotropic action of chungkookjangs fermented with various Bacillus spp. and fermentation periods. The 70% methanol and water extracts of chungkookjang fermented with Bacillus licheniformis (BL) for 48 h contained similar profiles of isoflavonoids and peptides to methanol and water extracts of TFC with potent anti-diabetic activity. Water extracts (mainly containing peptides) of TFC and BL fermented for 48 h and 72 h had a better insulin sensitizing action via activating peroxisome proliferator-activated receptor-γ (PPAR-γ) and increased the expression of PPAR-γ in 3T3-L1 adipocytes better than unfermented cooked soybeans (CSB). The 70% methanol extracts (predominantly isoflavone aglycones) of BL fermented for 48 h and 72 h improved glucose-stimulated insulin secretion and protected β-cell viability better than CSB in insulinoma cells, and the improvement by BL was similar to TFC. In conclusion, the BL water extract fermented for 48 h exhibited equal insulin sensitization as TFC and BL methanol extract exerted similar insulinotropic actions to those of TFC. B. licheniformis may be one of the major microorganisms responsible for anti-diabetic actions of chungkookjang. It is important to make chungkookjang that retains the anti-diabetic properties of the most efficacious traditional chungkookjang using a standardized method.

Background: Metabolic syndrome is a set of disorders that increases the risk of developing cardiovascular disease. The primary target of treatment of patients with metabolic syndrome is therapeutic lifestyle change. Numerous preclinical study have reported positive effects of chungkookjang in in vivo models of diabetes and obesity, but there is a paucity of controlled clinical trials on variables of metabolic syndrome in obese subjects. Thus, the objective of this trial is to examine the effect of chungkookjang compared to placebo on variables of metabolic syndrome in overweight/obese subjects., Methods: This double-blind randomized controlled crossover trial will be conducted on 120 overweight/obese subjects; aged 19-29 years. Subjects will be recruited from the Chonbuk National University, Jeonju, South Korea. Enrolled subjects will be randomly assigned to two groups of equal number; one group received 35 g of chungkookjang (n = 60) and the other group received placebo (n = 60) on a regular daily basis for 12 weeks. After a 12-week washout period, the groups will be crossed over. In addition to anthropometric measures and blood pressure, glucose parameter, lipid profile, adipocytokine, and carnitine assay will be determined at baseline and 12 week. Also, safety will be assessing by measuring total bilirubin, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, total protein, albumin, blood urea nitrogen, creatinine, and creatine kinase at baseline and 12 weeks. 24-hour dietary recalls were collected at the baseline and at the end of the trial., Discussion: This trial will evaluate the effects of chungkookjang on variables of metabolic syndrome in overweight/obese subjects. The results of this study may contribute to the reduction of risk factor for metabolic syndrome caused by obesity., Trial Registration: Clinical trials NCT01811511.

Unlabelled: The bacterial community of Chungkookjang and raw rice-straw collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods. Pure cultures were isolated from Chungkookjang and raw rice-straw on tryptic soy agar plates with 72 to 121 colonies and identified by 16S rDNA gene sequence analysis, respectively. The traditional culture-based method and denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rDNA confirmed that Pantoea agglomerans and B. subtilis were identified as predominant in the raw rice-straw and Chungkookjang, respectively, from Iljuk district of Gyeonggi province, P. ananatis and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Wonju district of Gangwon province, and Microbacterium sp. and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Sunchang district of Jeolla province. Other strains, such as Bacillus, Enterococcus, Pseudomonas, Rhodococcus, and uncultured bacteria were also present in raw rice-straw and Chungkookjang., Practical Application: A comprehensive analysis of these microorganisms would provide a more detailed understanding of the biologically active components of Chungkookjang and help improve its quality. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis can be successfully applied to a fermented food to detect unculturable or more species than the culture-dependent method. This technique is an effective and convenient culture-independent method for studying the bacterial community in Chungkookjang. In this study, the bacterial community of Chungkookjang collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods., (© 2012 Institute of Food Technologists®)

The role of food matrix and gender on soy isoflavone metabolism and biomarkers of activity were examined in twenty free-living adults (34.7 ± 11.5 years old) with hypercholesterolemia (221.9 ± 18.7mg dL(-1)). In a randomized crossover design study, participants consumed soy bread (3 wk) or a soy beverage (3 wk) containing 20 g soy protein with 99 and 93 mg isoflavones aglycone equivalents per day, respectively. During soy bread intervention, women had significantly greater microbial metabolite excretion (P = 0.05) of isoflavonoids than men. In men, isoflavone metabolite excretion was not discernibly different between the two matrices. Significant reductions (P≤ 0.05) in triglycerides (24.8%), LDL cholesterol (6.0%), apolipoprotein A-I (12.3%), and lipid oxidative stress capacity (25.5%), were observed after soy food intervention. Our findings suggest that the food matrix significantly impacts soy isoflavone metabolism, particularly microbial metabolites in women.

Background: Soy foods have been substituted for meat in recent years because of proposed health benefits. Research indicates, however, that soy protein and phytate in soy products inhibit the absorption of divalent cations., Methods: Our study was primarily designed to determine the effect of consuming two to three servings per day of soy foods, providing ∼19 g protein and ∼36 mg isoflavones, on iron and zinc status in premenopausal women during a 10-weeks period. As secondary outcomes, we also tested the effect of soy foods on biochemical markers of bone and thyroid hormones. Nonsmoking women (18-28 years) without chronic disease, anemia, pregnancy, or irregular menstrual cycles were randomly assigned to either the soy food (SF, n=31) or animal food (AF, n=32) group. Blood and urine samples and 3-day dietary records were collected at baseline and postintervention., Results: At baseline, iron and zinc status, bone markers, and thyroid hormones were not different between groups. After intervention, no significant changes were observed in hemoglobin, transferrin saturation, serum iron, ferritin, or transferrin receptor (TFR) concentrations. Plasma zinc, but not serum alkaline phosphatase, significantly decreased in both groups (-0.8 μmol/L). The change in bone-specific alkaline phosphatase was significant between SF (1.5 U/L) and AF (-0.7 U/L) groups. No significant changes were observed in bone resorption, thyroid-stimulating hormone (TSH), or free thyroxine after soy food intake., Conclusions: Incorporating ∼19 g soy protein from soy foods for 10 weeks had no significant effect on iron or zinc status, bone resorption or formation, or thyroid hormone status in premenopausal women.

Lactobacillus sp. FTDC 2113, L. acidophilus FTDC 8033, L. acidophilus ATCC 4356, L. casei ATCC 393, Bifidobacterium FTDC 8943 and B. longum FTDC 8643 were incorporated into soymilk supplemented with fructooligosaccharides (FOS), inulin, mannitol, maltodextrin and pectin. The objective of the present study was to evaluate the effects of prebiotics on the bioactivity of probiotic-fermented soymilk. Proteolytic activity was increased in the presence of FOS, while the supplementation of inulin and pectin increased the angiotensin I-converting enzyme inhibitory activity accompanied by lower IC(50) values. The beta-glucosidase activity was also enhanced in the presence of pectin. This led to higher bioconversion of glucosides to aglycones by probiotics, especially genistin and malonyl genistin to genistein. Results from this study indicated that the supplementation of prebiotics enhanced the in-vitro antihypertensive effect and production of bioactive aglycones in probiotic-fermented soymilk. Therefore, this soymilk could potentially be used as a dietary therapy to reduce the risks of hypertension and hormone-dependent diseases such as breast cancer, prostate cancer and osteoporosis.

Background: In vitro and animal studies suggested that soy protein and isoflavones promote weight and body fat loss. However, clinical trials in humans were few and the effects remained uncertain., Objectives: To examine whether soy protein with isoflavones and isoflavone extracts exert beneficial effects on body composition among postmenopausal women., Design: A double-blind randomized placebo-controlled trial was conducted among 180 postmenopausal Chinese women with mild hyperglycemia. After a 2-week adaptation, participants were randomly assigned to one of the three arms to receive 15 g soy protein and 100 mg isoflavones (Soy group), or 15 g milk protein and 100 mg isoflavones (Iso group), or 15 g milk protein (placebo group) on a daily basis for 6 months., Results: We observed a mild but significant favorable effect of soy protein with isoflavones on the changes of body weight (BW), body mass index, and body fat percentage relative to isoflavone extracts and milk protein after 6-month supplementation. The mean differences of BW between the Soy and the Iso groups were -0.75 kg (95% CI: -1.363 to -0.136, P=0.017), and between the Soy and the placebo groups were -0.60 kg (95% CI: -1.209 to -0.019, P=0.047). The mean difference of change percentage in body fat percentage between the Soy and the Iso groups was -3.74% (95% CI: -6.88 to -0.60%, P=0.02), and between the Soy and the placebo groups was -2.54% (95% CI: -5.69 to 0.12%. P=0.08)., Conclusions: Six-month supplementation of soy protein with isoflavones had a mild favorable effect on body composition in postmenopausal women.

Historically, the incidence of type 2 diabetes has been lower in Asian populations compared with those in Western countries. One possible reason for the lower incidence among Asians is that they consume fermented soybean products, which are unique to the traditional Asian diet. Some have hypothesized that dietary phytoestrogens and soy peptides in fermented soybean foods consumed in traditional Asian diets may help prevent and slow the progression of type 2 diabetes. This review evaluates the existing evidence from animal studies and clinical and epidemiologic investigations on fermented soybeans in the prevention and treatment of type 2 diabetes. Nutritional studies performed in animals and intervention studies with humans suggest that the ingestion of soy protein with isoflavones improves glucose control and reduces insulin resistance. Korean fermented soybean products such as doenjang, kochujang, and chungkookjang contain alterations in the structures and content of isoflavonoids and small bioactive peptides, which are produced during fermentation. Several studies revealed improvements in insulin resistance and insulin secretion with the consumption of these fermented products. Therefore, fermented soybean products may help prevent or attenuate the progression of type 2 diabetes. Although the lack of human intervention trials does not permit definitive conclusions, the evidence does suggest that fermented soy products may be better for preventing or delaying the progression of type 2 diabetes compared with nonfermented soybeans., (2010 Elsevier Inc. All rights reserved.)

Background: Health claims link soy protein (SP) consumption, through plasma cholesterol reduction, to a decreased risk of heart disease. Soy isoflavones (ISOs), particularly in individuals who produce equol, might also contribute to lipid lowering and thus reduce SP requirements., Objective: The objective was to examine the contributions of SP, ISOs, and equol to the hypocholesterolemic effects of soy foods., Design: Nonsoy consumers (33 men, 58 women) with a plasma total cholesterol (TChol) concentration >5.5 mmol/L participated in a double-blind, placebo-controlled, crossover intervention trial. The subjects consumed 3 diets for 6 wk each in random order, which consisted of foods providing a daily dose of 1) 24 g SP and 70-80 mg ISOs (diet S); 2) 12 g SP, 12 g dairy protein (DP), and 70-80 mg ISOs (diet SD); and 3) 24 g DP without ISOs (diet D). Fasting plasma TChol, LDL cholesterol, HDL cholesterol, and triglycerides (TGs) were measured after each diet., Results: TChol was 3% lower with the S diet (-0.17 +/- 0.06 mmol/L; P < 0.05) than with the D diet, and TGs were 4% lower with both the S (-0.14 +/- 0.05 mmol/L; P < 0.05) and SD (-0.12 +/- 0.05 mmol/L; P < 0.05) diets. There were no significant effects on LDL cholesterol, HDL cholesterol, or the TChol:HDL cholesterol ratio. On the basis of urinary ISOs, 30 subjects were equol producers. Lipids were not affected significantly by equol production., Conclusions: Regular consumption of foods providing 24 g SP/d from ISOs had no significant effect on plasma LDL cholesterol in mildly hypercholesterolemic subjects, regardless of equol-producing status.

Background: Evidence of the benefits of soy on cancer risk in Western populations is inconsistent, in part because of the low intake of soy in these groups., Objective: We assessed the validity of soy protein estimates from food-frequency questionnaires (FFQs) in a sample of Adventist Health Study-2 participants with a wide range of soy intakes., Design: We obtained dietary intake data from 100 men and women (43 blacks and 57 nonblacks). Soy protein estimates from FFQs were compared against repeated 24-h recalls and urinary excretion of daidzein, genistein, total isoflavonoids (TIFLs), and equol (measured by HPLC/photodiode array/mass spectrometry) as reference criteria. We calculated Pearson and Spearman correlation coefficients (with 95% CIs) for FFQ-24-h recall, 24 h-recall-urinary excretion, and FFQ-urinary excretion pairs., Results: Among soy users, mean (+/- SD) soy protein values were 12.12 +/- 10.80 g/d from 24-h recalls and 9.43 +/- 7.83 g/d from FFQs. The unattenuated correlation (95% CI) between soy protein estimates from 24-h recalls and FFQs was 0.57 (0.32, 0.75). Correlation coefficients between soy protein intake from 24-h recalls and urinary isoflavonoids were 0.72 (0.43, 0.96) for daidzein, 0.67 (0.43, 0.91) for genistein, and 0.72 (0.47, 0.98) for TIFLs. Between FFQs and urinary excretion, these were 0.50 (0.32, 0.65), 0.48 (0.29, 0.61), and 0.50 (0.32, 0.64) for daidzein, genistein, and TIFLs, respectively., Conclusions: Soy protein estimates from questionnaire were significantly correlated with soy protein from 24-h recalls and urinary excretion of daidzein, genistein, and TIFLs. The Adventist Health Study-2 FFQ is a valid instrument for assessing soy protein in a population with a wide range of soy intakes.

Background: The soy isoflavone daidzein is metabolized to equol and O-desmethylangolensin (ODMA) by intestinal bacteria in approximately 30-50% and 80-90% of persons, respectively. Studies suggest beneficial health effects associated with daidzein-metabolizing phenotypes; thus, assessing their determinants is an important goal., Objective: We evaluated relations between daidzein-metabolizing phenotypes and demographic, anthropometric, lifestyle, and dietary factors among premenopausal women in the United States., Design: Two hundred women provided a first-void urine sample after a 3-d soy challenge and completed a health and demographics questionnaire, physical activity questionnaire, food-frequency questionnaire, and 3-d food record. Urine samples were measured for isoflavones by gas chromatography-mass spectrometry to determine daidzein-metabolizing phenotypes., Results: Fifty-five (27.5%) and 182 (91%) women had detectable concentrations of urinary equol and ODMA (>87.5 ng/mL), respectively, and were classed as producers of these metabolites. Compared with nonproducers, equol producers were more likely (P < or = 0.05) to be Hispanic or Latino, to be highly educated, and to have frequent constipation, and ODMA producers were taller and less likely to be Asian than white. Equol and ODMA producers reported higher overall physical activity than did nonproducers., Conclusions: We observed associations between equol production and ethnicity, education, constipation, and physical activity and between ODMA production and race, height, and physical activity. Associations with race and ethnicity were based on small numbers of Asian and Hispanic or Latino women, and confirmation of these findings is needed. Few dietary factors, assessed with the use of either a food-frequency questionnaire or food record, were associated with daidzein-metabolizing phenotypes.

Equol, a metabolite of daidzein for some intestinal microflora, is known to retain highly estrogenic activity and is of wide interest in relation to human health. However, not all humans can produce equol. In this study, detection of urinary equol using thin-layer chromatography (TLC) was performed to distinguish between equol producers and non-producers. After 36 h of soy food intake, urine, collected from 7 volunteers, was hydrolyzed, purified by reverse phase silica gel column and applied to normal phase TLC. Consequently, equol was clearly separated from the urine samples and discriminated the equol producers in this system. The detection limit of equol was at least 20 ng.

High levels of insulin-like growth factor 1 (IGF-1) are associated with increased risk of prostate cancer, whereas increased levels of some of its binding proteins (IGFBPs) seem to be protective. High intakes of dietary protein, especially animal and soy protein, appear to increase IGF-1. However, soy isoflavones have demonstrated anti-proliferative and apoptotic effects both in vitro and in vivo. We evaluated dietary intakes of total protein and soy isoflavones in relation to the IGF axis in prostate cancer patients making comprehensive lifestyle changes including a very low-fat vegan diet supplemented with soy protein (58 g/day). After one year, intervention group patients reported significantly higher intakes of dietary protein and soy isoflavones compared to usual-care controls (P < 0.001). IGF-1 increased significantly in both groups, whereas IGFBP-1 rose in the experimental group only (P < 0.01). Increases in vegetable protein over one year were associated with increases in IGFBP-1 among intervention group patients (P < 0.05). These results suggest that dietary protein and soy isoflavones, in the context of comprehensive lifestyle changes, may not significantly alter IGF-1. However, given the recent literature indicating that high intake of protein rich in essential amino acids (animal or soy protein) may increase IGF-1, it may be prudent for men with early stage prostate cancer not to exceed dietary protein recommendations.

7-Hydroxy-3-(4'-hydroxyphenyl)-chroman (S-equol) is a specific end-metabolite formed in the biotransformation of the dietary soy isoflavones daidzin and daidzein by intestinal bacteria. The frequency of equol production varies among individuals and populations, and it is suggested that the efficacy of soy foods differs depending on the ability of an individual to produce equol. To develop a standardized approach to define equol-producer status that can be universally adopted to differentiate these 2 distinct populations, we measured isoflavones in serum and urine collected from a cohort of 41 healthy adults, comprising 29 vegetarians and 12 nonvegetarians, after consuming 2 x 250 mL/d soy milk on 3 consecutive days. Serum and urinary daidzein and S-equol concentrations were analyzed by MS. Serum S-equol and daidzein concentrations ranged from 10.3-139 nmol/L (2.5-33.6 microg/L) and 16-1401 nmol/L (4.0-356 microg/L), respectively, whereas in urine the corresponding concentrations ranged from 16-12,574 nmol/L (4-3043 microg/L) and 539-26,834 nmol/L (137-6816 microg/L), respectively. The log10-transformed urinary S-equol:daidzein ratio provided a clearer distinction of equol-producer status than the absolute serum or urinary S-equol concentrations because it is independent of isoflavone intake and minimizes interindividual variation in isoflavone pharmacokinetics or differences in analytical methodologies. A threshold value for the log10-transformed urinary S-equol:daidzein ratio of -1.75 provided a demarcation to define equol-producer status. The frequency of equol producers in the vegetarians was 59%, similar to the reported frequency in Japanese adults consuming soy, and much higher than for nonvegetarian adults (25%), suggesting that dietary components other than soy influence S-equol synthesis by intestinal bacteria.

Objectives: Intestinal bacterial metabolize the soy isoflavone daidzein to O-desmethylangolensin (O-DMA) or equol. Some individuals do not excrete O-DMA or equol after soy consumption, suggesting they do not harbor bacteria capable of producing these metabolites. The aim of this study was to evaluate bone mineral density (BMD) in relation to presence of these urinary metabolites., Methods: BMD, determined by whole-body dual X-ray absorptiometry scan, was age-adjusted and evaluated in relation to O-DMA-producer and equol-producer phenotypes in 92 postmenopausal women, aged 50-75 years. Women consumed supplemental soy foods (daidzein source) for 3 days and collected a first-void urine sample on the fourth day in order to determine metabolic phenotypes., Results: In O-DMA producers (n=76) compared to O-DMA non-producers (n=16), greater total, leg and head BMD (p<0.05) were observed. Total BMD among the O-DMA producers (geometric mean=1.04 g/cm2) was 6% greater than total BMD among the O-DMA non-producers (geometric mean=0.98 g/cm2). Total and site-specific BMD did not differ between equol producers (n=24) and non-producers (n=68) (p>0.05). In exploratory analyses, among regular soy consumers, spinal BMD was 20% lower among the equol producers than non-producers, whereas, among soy non-consumers, no such difference was observed (p-interaction<0.05). Among equol producers, circulating estrone and free estradiol concentrations were inversely or not associated with total BMD, whereas, among equol non-producers, these hormones were positively associated (p-interaction<0.05)., Conclusions: Our results provide evidence that intestinal bacterial composition may influence BMD in postmenopausal women. Further studies characterizing associations of intestinal bacterial profiles with BMD are warranted.

Postmenopausal women are at increased risk for cardiovascular disease because many risk factors are aggravated by menopause. Phytoestrogens may modulate risk factors favorably, involving mechanisms similar to estrogen. The effect of phytoestrogens on the atherogenic amino acids homocysteine and asymmetric dimethylarginine (ADMA) was investigated in a controlled intervention study in healthy postmenopausal women. A multicenter, double-blind, crossover intervention trial in 89 postmenopausal women from Denmark, Germany, and the UK was performed. Subjects consumed fruit cereal bars with or without soy isoflavones (50 mg/d) for 8 wk each with an 8-wk washout period in between. Urinary phytoestrogens increased significantly after isoflavone intervention (P < 0.001). Isoflavone supplementation did not affect plasma total homocysteine or ADMA. For homocysteine, changes from baseline were 0.32 micromol/L (range: -0.31-0.92; 95% CI 0.13-0.72), and 0.29 micromol/L (range: -0.45-1.09; 95% CI 0.01-0.63, P = 0.286) for isoflavone treatment and placebo, respectively. For ADMA concentrations, changes from baseline were -0.02 micromol/L (range: -0.08-0.03; 95% CI -0.04-0.01, and 0.00 micromol/L (range: -0.05-0.03; 95% CI -0.03-0.01, P = 0.397) for isoflavone treatment and placebo, respectively. There was no association between plasma total homocysteine and ADMA. Changes from baseline in plasma ADMA and folate were negatively correlated (r = -0.18, P = 0.017). These results challenge the overall health effect of isoflavone supplementation in healthy postmenopausal women.

The aim of this work was to study the effect of 1 year of storage at different temperatures and 1 month of storage at different water activities on the content and profile of isoflavones, antioxidant activity, and in vitro protein digestibility of defatted soy flours (DSF) and soy protein isolates (SPI). The storage for up to 1 year, at temperatures from -18 to 42 degrees C, had no effect on the total content of isoflavones, but the profile changed drastically at 42 degrees C, with a significant decrease of the percentage of malonylglucosides with a proportional increase of beta-glucosides. A similar effect was observed for SPI stored at a(w) = 0.87 for 1 month. For DSF, however, there was observed a great increase in aglycons (from 10 to 79%), probably due to the action of endogenous beta-glucosidases. The antioxidant activity decreased after storage at 42 degrees C, but the in vitro protein digestibility did not change.

Three biological activities of 12 soy isoflavones were investigated in terms of the structure-activity relationship. Seven molecular descriptors and one absorption predictor were significantly related to the inhibition of cytochrome P4501 activity in HepG2 cells by soy isoflavones, yielding water solubility [correlation coefficient (gamma)=-0.779] and hydrophobicity (gamma=0.718) as more relevant molecular properties. By contrast, the inducing activities of 12 soy isoflavones both on quinone reductase in HepG2 cells and proliferation of the osteoblastic MG-63 cells demonstrated significant correlation to the hydroxyl and methoxyl substituents on the isoflavone skeleton (p<0.01).

Consumption of soy protein has been associated with altered risk of developing endocrine-regulated cancers. This study was designed to assess the independent effect of soy relative to animal protein and soy-derived isoflavones on circulating estrogen and androgen concentrations in postmenopausal women and older men. Forty-two subjects (> 50 yr) with low-density lipoprotein cholesterol levels of > or = 3.36 mmol/l were fed each of 4 diets in randomized order for 6 wk/phase. All food and drink were provided. Diets contained 25 g soy or common sources of animal protein/4.2 MJ containing trace or 50 mg isoflavones/4.2 MJ. At the end of each diet phase, concentrations of estrone sulfate, estrone, estradiol, testosterone, androstendione, dihydrotestosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate were measured. In postmenopausal women, concentrations of estrone were higher and its precursor, dehydroepiandrosterone, lower after consuming the soy compared with animal protein diets (P = 0.0396 and 0.0374, respectively). There was no significant effect of isoflavones on any of the hormones measured. In older men, dehydroepiandrosterone sulfate concentrations were lower after consuming the isoflavone (P = 0.0106) and higher after soy, compared with the animal protein diets (P = 0.0118). These data suggest that relatively large amounts of soy protein or soy-derived isoflavones had modest and limited sex-specific effects on circulating hormone levels.

Circulating hormones are associated with mammographic density, an intermediate marker of breast cancer risk. Differences in circulating hormones, including estrone and testosterone, have been observed in premenopausal women based on their capacity to metabolize daidzein, an isoflavone found predominantly in soybeans. Equol and O-desmethylangolensin (O-DMA) are products of intestinal bacterial metabolism of daidzein. There is interindividual variability in the capacity to produce daidzein metabolites; individuals can be equol producers or non-producers and O-DMA producers or non-producers. We tested the hypothesis that daidzein-metabolizing phenotypes are associated with mammographic density. Participants were recruited from among 92 sedentary, postmenopausal women, ages 50 to 75 years, who participated in a 1-year physical activity intervention. Pre-intervention mammographic density was determined using a computer-assisted, gray-scale thresholding technique. Fifty-five of these women consumed supplemental soy protein (>10 mg daidzein/d) for 3 days and collected a first-void urine sample on the fourth day to determine daidzein-metabolizing phenotypes. Equol and O-DMA concentrations were measured using gas chromatography-mass spectrometry. Associations between daidzein-metabolizing phenotypes and percent mammographic density were adjusted for age, maximum adult weight, gravidity, family history of breast cancer, and serum follicle-stimulating hormone and free testosterone concentrations. Mammographic density was 39% lower in equol producers compared with non-producers (P = 0.04). O-DMA producers had mammographic density 69% greater than non-producers (P = 0.05). These results suggest that particular intestinal bacterial profiles are associated with postmenopausal mammographic density, and these associations are not entirely explained by differences in reproductive or anthropometric characteristics or circulating hormones.

Three experiments were conducted to characterize the metabolism of isoflavones from soy milk in women: two meals in 2 wk separated by a 1-wk washout period (Experiment 1), one meal feeding (Experiment 2) and six consecutive days of feeding (Experiment 3). Urine and plasma samples were extracted directly or predigested before extraction with H-2 beta-glucuronidase/sulfatase or B-3 beta-glucuronidase so that isoflavone glucuronide and sulfate conjugates could be determined by difference. Among the three experiments, no significant differences were found in the proportion of glucuronide, sulfate and aglycone isoflavones recovered from plasma samples taken 3 h after isoflavone dosing or in 24-h urine samples taken after isoflavone dosing. In the 6-d feeding study, samples taken on d 5 and 6 did not differ significantly in isoflavone content or proportion of the metabolites studied. The percentages of daidzein and genistein glucuronides were 73 +/- 4 and 71 +/- 5% of total daidzein and genistein excreted in urine, and 62 +/- 4 and 53 +/- 6% of total daidzein and genistein present in plasma, respectively. Percentages of aglycone daidzein and genistein were 4 +/- 1 and 5 +/- 1% of total daidzein and genistein in urine, and 18 +/- 2 and 26 +/- 7% of total daidzein and genistein present in plasma, respectively. These studies showed that about one fifth of circulating isoflavones are aglycones. Concentrations of isoflavones chosen for in vitro studies should take this into account. Because the glucuronide isoflavones predominate in vivo, these metabolites should not be overlooked as possible contributors to observed effects of isoflavones.

Background: Despite significant interest in the risks and benefits of phytoestrogens to human health, few data exist on their pharmacokinetics in humans., Objective: We investigated the pharmacokinetics of the (13)C isotopic forms of daidzein and genistein in healthy humans, specifically addressing intraindividual variability, effect of increasing intake, and influence of prolonged exposure to a soy food diet., Design: Premenopausal women (n = 16) were administered 0.4 mg [(13)C]daidzein or [(13)C]genistein/kg body wt orally on 3 occasions, including once after eating soy foods for 7 d. On a further occasion the dose was doubled. Plasma and urinary [(13)C]isoflavone concentrations were measured by mass spectrometry., Results: Serum concentrations of [(13)C]genistein and [(13)C]daidzein peaked after 5.5 and 7.4 h, respectively. The systemic bioavailability and maximum serum concentration of [(13)C]genistein were significantly greater than those of [(13)C]daidzein. The bioavailability of both isoflavones did not increase linearly when the dietary intake was doubled. The mean volume of distribution normalized to bioavailability (V(d)/F), clearance rate, and half-life of [(13)C]daidzein were 336.25 L, 30.09 L/h, and 7.75 h, respectively; the corresponding values for [(13)C]genistein were 258.76 L, 21.85 L/h, and 7.77 h. The average recovery of [(13)C]daidzein and [(13)C]genistein in urine was 30.1% and 9.0% of the dose ingested, respectively., Conclusions: The serum pharmacokinetics of [(13)C]daidzein and [(13)C]genistein were reproducible among healthy women, and genistein was more bioavailable than was daidzein. Pharmacokinetics were unaffected by chronic exposure to soy foods. Urinary isoflavone concentrations correlated poorly with maximal serum concentrations, indicating the limitations of urine measurements as a predictor of systemic bioavailability. The bioavailability of both isoflavones was nonlinear at higher intakes, suggesting that uptake is rate-limiting and saturable.

The way of nutrition and particular components of the diet have substantial influence on the development of many diseases. It has been proven by large epidemiological studies dealing with the incidence of cardiovascular diseases and tumours. What is more, the diet may have also an important role in the secondary prevention of myocardial infarction. The role of a soy as a component of the diet with potentially favorable action on human health was discussed in this paper. Special attention was paid to mechanisms of action of soy phytoestrogens and their influence on development of ischaemic heart disease, tumours, osteoporosis and other symptoms related to menopause.

Objective: Although the mechanisms by which dietary soy inhibits atherosclerosis are unclear, one line of evidence implicates an important role for its phytoestrogenic isoflavones. We sought to determine whether soy isoflavones exert atheroprotective effects through estrogen receptor-dependent processes and, if so, which estrogen receptor subtype (ie, alpha or beta) is involved., Methods and Results: We compared the effects of diets rich in soy protein that were either isoflavone depleted (0.04 mg/g protein isolate) or isoflavone-replete, or Soy(+IF) (1.72 mg/g protein isolate) in apolipoprotein E-deficient (ee) mice that had been crossed with estrogen receptor-alpha- and -beta-deficient mice to produce double-knockout alphaalphaee and betabetaee mice and (estrogen receptor) wild-type controls (AAee and BBee). Both male and ovariectomized female mice were studied (n=10 to 17 per treatment group; total n=201). After 16 weeks, atherosclerosis was assessed by quantifying the aortic content of esterified cholesterol. Atherosclerosis was reduced 20% to 27% (P<0.05) by Soy(+IF) in betabetaee, BBee, and AAee mice but was unaffected in alphaalphaee mice. The inhibitory effect of Soy(+IF) was unrelated to sex, total plasma cholesterol, VLDL, LDL, and HDL cholesterol., Conclusions: The results indicate a necessary role for estrogen receptor-alpha-dependent processes in mediating the atheroprotective effects of dietary soy isoflavones.

Objective: The objective of this study was to assess the independent effect of soy relative to common sources of animal protein and soy-derived isoflavones on blood lipids., Methods and Results: Forty-two subjects with LDL cholesterol levels > or =3.36 mmol/L were fed each of four diets in randomized order for 6 weeks per phase. Diets contained a minimum of 25 g animal protein or isolated soy protein/4.2 MJ, with each containing trace amounts or 50 mg of isoflavones/4.2 MJ. Soy protein had a modest effect on total, LDL and HDL cholesterol, and triglyceride concentrations (-2%, P=0.017; -2%, P=0.042; +3%; P=0.034, -11%, P<0.001, respectively). Soy protein had no significant effect on plasma lipids in individuals with LDL cholesterol <4.14 mmol/L and significantly reduced total and LDL cholesterol and triglyceride concentrations in individuals with LDL cholesterol > or =4.14 mmol/L (-4%, P=0.001; -5%, P=0.003; -15%, P<0.001, respectively). No significant effect of isoflavones on plasma lipid levels was observed either constituent to the soy protein or supplemental to the animal protein., Conclusions: Although potentially helpful when used to displace products containing animal fat from the diet, the regular intake of relatively high levels of soy protein (>50 g/day) had only a modest effect on blood cholesterol levels and only in subjects with elevated LDL cholesterol levels (> or =4.14 mmol/L). Soy-derived isoflavones had no significant effect.

Isoflavones, rich in soybean, are currently receiving much attention because of their potential role in preventing and treating cancer and other human chronic diseases. The present review provides an overview of the recent results in this research field. Data from epidemiological reports and laboratories have shown that isoflavones have multi-biological and pharmacological effects in animals and humans. These include estrogenic and antiestrogenic effects, cell signalling conduction, as well as cell growth and death. Based on these properties, soy protein and isoflavones have been associated with reduced incidences of breast and prostate cancers, cardiovascular diseases or osteoporosis, and exhibit some other favorable effects. The mechanism through which isoflavones may exert the above-mentioned functions are not only based on the estrogenic properties of isoflavones, but also on their role as protein tyrosine kinase inhibitors, as regulators of gene transcription, modulators of transcription factors, antioxidants, as well as by altering some enzyme activities. However, to draw a clear conclusion regarding the clinical use of isoflavones further investigation would be required, although only a few effects of short- or long-term use of soy proteins are known in humans.

Phytoestrogens, plant chemicals classified as isoflavones, coumestans and lignans, display estrogen-like activity because of their structural similarity to human estrogens and exhibit high affinity binding for the estrogen receptor beta. They are common components of food items such as grains, beans, fruits and nuts. Isoflavones are primarily found in soybeans and foods made from soy. In particular, significant therapeutic properties have been generally attributed to soy isoflavones, but most of the claims have been poorly, or not at all, confirmed by well designed clinical trials. Such is the case of the purported role of soy isoflavones in reducing plasma cholesterol levels. This link is now not supported by many authors or by appropriately designed clinical studies. The role of isoflavones in cancer prevention, particularly of tumours under endocrine control (breast, prostate and others) is again only supported by weak to nonexisting clinical evidence. A similarcase is that of the prevention/treatment of postmenopausal symptoms and osteoporosis. Disturbing data have been reported on potential negative effects of soy isoflavones on cognitive function in the aged, particularly relating to tofu intake. Recent studies have finally indicated a potential role for soy isoflavones in inducing chromosomal changes in cells exposed in vitro and potentiating chemical carcinogens. These findings may not, however, be extrapolated to clinical conditions. Available data do not appear to unequivocally support beneficial effects of soy isoflavones, and warn against their wide use, in the absence of satisfactory clinical findings.

The soy isoflavones, daidzein and genistein, and the lignans, matairesinol and secoisolariciresinol, are phytoestrogens metabolized extensively by the intestinal microflora. Considerable important evidence is already available that shows extensive interindividual variation in isoflavone metabolism, and we have investigated the extent of this variation in a crossover study of a soy-containing food low or high in isoflavones (each treatment period lasted for 17 days, and the 2 treatment periods were separated by a 25-day washout period) in 24 healthy subjects [19 women and 5 men, mean age 30 yr, range 19-40, mean body mass index 22.5 +/- 3.5 (SD) kg/m2]. There was a 16-fold variation in total isoflavonoid excretion in urine after the high-isoflavone treatment period. The variation in urinary equol excretion was greatest (664-fold), and subjects fell into two groups: poor equol excretors and good equol excretors (36%). A significant negative correlation was found between the proportion of energy from fat in the habitual diet and urinary equol excretion (r = -0.55; p = 0.012). Good equol excretors consumed less fat as percentage of energy than poor excretors (26 +/- 2.3% compared with 35 +/- 1.6%, p < 0.01) and more carbohydrate as percentage of energy than poor excretors (55 +/- 2.9% compared with 47 +/- 1.7%, p < 0.05). Interindividual variation in the urinary excretion of O-desmethyl-angolensin (O-DMA) was also apparent (76-fold after the high-isoflavone treatment period), but there was no relationship between equol excretion and O-DMA excretion. Enterolactone was the major lignan metabolite in urine and plasma but showed less interindividual variation than equol and O-DMA. It is suggested that the dietary fat intake decreases the capacity of gut microbial flora to synthesize equol.

This study compared the bioavailability of conjugates of the soy isoflavones genistein and daidzein in rats. Rats were given a single oral dose of a soy extract that provided 74 micromol genistein and 77 micromol daidzein/kg body wt (as conjugates). Plasma samples were obtained from treated and untreated rats; urine and fecal samples were obtained before and after treatment. Isoflavones, equol (the main end product of bacterial degradation of daidzein), and 4-ethyl phenol (the main end product from genistein) were measured by HPLC. The plasma daidzein concentration was maximal at 2 h (9.5 +/- 0.71 micromol/L) and was almost double that of genistein (P = 0.009). Between 2 and 15 h, the plasma daidzein concentration declined by 32%, but the concentration of genistein changed little. At 15 h, the concentrations of daidzein and genistein were not significantly different. Urinary excretion of daidzein over the 48-h postdose period was 17.4 +/- 1.2% of the dose, but only 11.9 +/- 1.1% of the genistein dose was excreted in urine. Equol excretion was 5.0 +/- 1.5% of the daidzein dose, but 41.9 +/- 5.0% of the genistein dose was excreted as 4-ethyl phenol. Fecal daidzein accounted for 2.3 +/- 0.5% and fecal genistein for 3.4 +/- 0.4% of the respective doses. It is concluded that conjugates of daidzein are more bioavailable than those of genistein, probably because of the greater resistance of the former to degradation by gut bacteria.

Soy-based infant formulas have been in use for >30 y. These formulas are manufactured from soy protein isolates and contain significant amounts of phytoestrogens of the isoflavone class. As determined by HPLC, the isoflavone compositions of commercially available formulas are similar qualitatively and quantitatively and are consistent with the isoflavone composition of soy protein isolates. Genistein, found predominantly in the form of glycosidic conjugates, accounts for >65% of the isoflavones in soy-based formulas. Total isoflavone concentrations of soy-based formulas prepared for infant feeding range from 32 to 47 mg/L, whereas isoflavone concentrations in human breast milk are only 5.6 +/- 4.4 microg/L (mean +/- SD, n = 9). Infants fed soy-based formulas are therefore exposed to 22-45 mg isoflavones/d (6-11 mg x kg body wt(-1) x d(-1)), whereas the intake of these phytoestrogens from human milk is negligible (<0.01 mg/d). The metabolic fate of isoflavones from soy-based infant formula is described. Plasma isoflavone concentrations reported previously for 4-mo-old infants fed soy-based formula were 654-1775 microg/L (mean: 979.7 microg/L: Lancet 1997:350;23-7), significantly higher than plasma concentrations of infants fed either cow-milk formula (mean +/- SD: 9.4 +/- 1.2 microg/L) or human breast milk (4.7 +/- 1.3 microg/L). The high steady state plasma concentration of isoflavones in infants fed soy-based formula is explained by reduced intestinal biotransformation, as evidenced by low or undetectable concentrations of equol and other metabolites, and is maintained by constant daily exposure from frequent feeding. Isoflavones circulate at concentrations that are 13,000-22,000-fold higher than plasma estradiol concentrations in early life. Exposure to these phytoestrogens early in life may have long-term health benefits for hormone-dependent diseases.

The importance of estrogens in homeostatic regulation of many cellular and biochemical events is well illustrated by the pathophysiologic changes that occur with estrogen deficiency. Many of the major diseases of Western populations are hormone dependent and epidemiologic data have shown a strong association between their incidence and diet. In particular, the importance of a plant-based diet is evident from the current dietary recommendations that emphasize an increase in the proportion and amount of fruit and vegetables that should be consumed. Although interpretation of the role of individual components of the diet is difficult from epidemiologic and dietary studies, it is recognized that there are many plant-derived bioactive nonnutrients that can confer significant health benefits. Among these phytochemicals is the broad class of nonsteroidal estrogens called phytoestrogens, and in the past decade there has been considerable interest in the role of isoflavones because of their relatively high concentrations in soy protein. The isoflavones in modest amounts of ingested soy protein are biotransformed by intestinal microflora, are absorbed, undergo enterohepatic recycling, and reach circulating concentrations that exceed by several orders of magnitude the amounts of endogenous estrogens. These phytoestrogens and their metabolites have many potent hormonal and nonhormonal activities that may explain some of the biological effects of diets rich in phytoestrogens.

Soybeans contain isoflavones, which have been associated with many health benefits, including decreased cancer risk. The purpose of our study was to measure urinary isoflavonoid excretion in response to daily consumption of soy that contained 0-36 mg isoflavones--a lower range than used in previous studies--and to compare urinary isoflavonoid excretion between equol excreters and nonexcreters. Fourteen men and women aged 20-40 y participated in the study. Half of the subjects were identified previously as equol excreters and the other half as equol nonexcreters. This randomized, double-blind, crossover study consisted of four 9-d diet treatment periods. During each treatment period participants consumed a low-photoestrogen controlled diet and a beverage containing 0, 5, 10, or 20 g soy protein. Urine collected on the last 3 d of each treatment period was analyzed for isoflavonoid (equol, O-desmethylangolensin, genistein, and daidzein) and lignan (enterodiol and enterolactone) contents by using isotope-dilution gas chromatography-mass spectrometry. There was a highly linear dose response of urinary isoflavonoid excretion to soy consumption, which did not differ significantly between equol excreters and nonexcreters. There were no significant differences in lignan excretion between the two diet treatments. Our results indicate that urinary isoflavonoid excretion is dose dependent in humans at low to moderate levels of soy consumption.