Equol is produced from daidzein by the action of gut bacteria on soy isoflavones. However, not all people can produce equol, and metabolism differs even among the producers. We aimed to examine the equol producer status in both men and women, and investigate the relationships among the serum and urinary isoflavones as well as to other biomedical parameters. In this study, we measured the equol and daidzein concentrations from the blood and urine of 292 men and 174 women aged between 22 and 88 years by liquid chromatography-tandem mass spectrometry (LC‒MS/MS). We then analysed the cut-off value for equol producers in both sexes, the relationship of serum and urinary equol concentrations, and other parameters, such as sex, age, endocrine function, glucose metabolism, lipid metabolism, and renal function with regards to equol-producing ability, among the different age groups. Equol producers were defined as those whose log ratio of urinary equol and daidzein concentration or log (equol/daidzein) was -1.42 or higher. Among 466 participants, 195 were equol producers (42%). The proportion of equol producers was larger in women. The cut-off value for equol producers was consistent in both sexes. Positive relationships were noted between serum and urinary equol levels in equol producers of both sexes; however, such a relationship was not detected in nonproducers. Lipid and uric acid abnormalities were more common with non equol producers in both men and women. Prostate specific antigen (PSA) levels in men were significantly lower in equol producers, especially in those in their 40 s. This study suggests a relationship between equol-producing ability and reduced risk of prostate disease as well as positive effects of equol on blood lipids and uric acid levels. However, lack of dietary information and disperse age groups were major drawbacks in generalizing the results of this study.
Dietary isoflavones are metabolized in the lower part of the small and large intestine into three main ingredients of soy isoflavones: daidzein, genistein and glycitein. There, the end-product or active metabolite of daidzein called 'equol' could be produced only in certain people who have functional equol-producing bacteria [[
Equol exists as enantiomers, R-equol and S-equol. However, S-equol is the only product that can be identified in the blood and urine of humans and animals [[
However, not everyone has the ability to produce equol [[
There are inconsistencies in the definitions of equol producers. For instance, some researchers used the minimum detectable levels of either serum or urinary equol [[
However, the above research findings were among the small group of people where soy challenge was relatively easier to implement. We need a robust definition to determine equol producers in larger populations where soy challenge is not feasible, such as those undergoing annual health screening. In 2018, Ideno et al. conducted an epidemiological study without soy challenge by measuring the urinary equol and daidzein levels of 4,412 Japanese women in the Japan Nurses' Health Study by the liquid chromatography-tandem mass spectrometry (LC-MS/CS) method [[
This cross-sectional study was conducted among 466 healthy men and women (292 men and 174 women) aged between 22 and 88 (mean age 55) years who had undergone annual health screening from June 2016 to December 2017 at the Himedic Kyoto University Hospital, a membership medical support facility where management and research are collaboratively carried out by Resorttrust, Inc and Kyoto University. All the participants hold the memberships of that facility. We were unable to obtain the information on the soy consumption habits or the last meals of the participants as they were not assessed in the ordinary health screening program.
We included the data of the participants from all the men and women within the study period who were informed and provided a written general consent (S1 File) for the use of their health screening data for secondary purposes. The data were fully anonymized before they were assessed for research purpose on November 24, 2021. Although the study was conducted at the Kyoto University Hospital, we need to submit the proposal to a third-party ethical review board according to the regulations of Kyoto University Hospital for the use of secondary data by researchers from different affiliations. Therefore, the study was approved by the Institutional Review Board of The University of Tokyo.
Equol and daidzein concentrations in the serum and urine were determined by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, measured by LSI Medience Corporation (Tokyo, Japan). In brief, 100 μL of serum or 10-fold diluted urine was mixed with internal standards, followed by the addition of 150 μL of an β-glucuronidase enzyme solution for deconjugation (Roche Biochemical, Mannheim, Germany). Following a one-hour deconjugation reaction at 37°C, free equol, daidzein, and genistein were purified using solid-phase extraction (Oasis PRiME HLB, Waters, Milford, MA). Subsequently, liquid chromatography (LC) -tandem mass spectrometry (LCMS-8050, Shimadzu, Japan) was employed with a reverse-phase LC column (ACQUITY UPLC HSS T3, 1.8 μm, 2.1 mm × 100 mm, Waters, Milford, MA) for analysis. Data processing was conducted using Mass Hunter software (Agilent, Santa Clara, CA). The peak areas were normalized using internal standards, and the concentration of each analyte was determined through a standard curve. The limits of detection (LODs) were 1 ng/mL for serum equol and daidzein, 10 ng/mL for urine equol, and 20.0 ng/mL for urine daidzein. Liquid chromatography‒mass spectrometry (LC‒MS) is an indispensable tool for quantitative and qualitative analysis in a wide range of fields, from pharmaceuticals and food science to environmental analysis. The advantage of LC-MS/MS over other methods such as glass chromatography-based methods for detection of isoflavones is that all the conjugated and unconjugated isoflavones and their metabolites can be separated and analysed faster and more efficiently [[
Blood samples were obtained after an overnight fast to determine the fasting blood glucose level (FBG), hemoglobin A1c (HbA1c), glycated albumin, 1.5-Anhydro-D-glucitol, insulin, C-peptide, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), total cholesterol, low density lipoprotein cholesterol (LDL-C), triglycerides(TG), high density lipoprotein cholesterol (HDL-C), uric acid(UA), urinary creatinine(UCRE), high sensitivity C-reactive protein (hs-CRP), estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), free-triiodothyronine (f-T3), free-thyroxine (f-T4).
We first calculated the effected size for equol producer proportions by gender using Setchell's study on 23 men and 18 women, with equol producer proportions of 65% for men and 28% for women. Using that effect size (w = 0.162069), with the assumption of a two-sided 5% significance level and power of 80%, and calculated that a minimum of 415 subjects in total was necessary for the comparison of equol producers in gender by Chi-squared test. Considering 10% opt-out rate, we included data from 466 subjects. Serum and urinary S-equol and daidzein concentrations were expressed as micrograms/liter and when analysed were expressed as medians and interquartile ranges. The ratio of urinary equol to daidzein was calculated, transformed, and expressed as log10. We assessed the distributions of log10-transformed urinary equol: daidzein concentrations in histograms and used mixed models to examine any difference between the sexes to determine the equol producer status. According to the results, subjects with a ratio above −1.42 on this scale were classified as equol producers. We calculated the creatinine-corrected values of isoflavones to examine the associations between serum and urinary equol concentrations by linear regression analysis. The distribution of normality of the parameters was assessed with the Kolmogorov–Smirnov test, box plots, and histograms. We then compared the differences in parameters with regard to equol-producing ability using the Mann‒Whitney U test. The proportions of abnormal values were compared using the chi-squared test. The cut-off values for each parameter were stated in the S3 File. All continuous variables are expressed as medians and interquartile ranges, and categorical variables are expressed as numbers and percentages. The calculations and Figure generation, except for the generation of the mixed model histograms, were performed using Microsoft Excel (Microsoft Corporation, 2019). The sample size calculations, and mixed model histograms were generated using R software (R 4.3.0, R Core Team, 2023). All tests were two-sided, and statistical significance was set to p < 0.05.
We applied the same finite fixed model as the previous study to determine the optimal cut-off value to distinguish between equol producers and nonproducers [[
Graph: Determination of cut-off values in (A) all participants (N = 466), (B) men (n = 292), and (C) women (n = 174) using mixed model analysis. In all distributions, the cut-off values were approximately -1.4, without significant distinction between the men and women, i.e., at the value of -1.42.
Table 1 shows that women participants had higher equol concentrations in both serum and urine, glycated albumin, HDL-C, E2, LH, and FSH, which is consistent with other studies. Men had higher fasting blood glucose, 1.5-Anhydro-D-glucitol, C-peptide, HOMA-IR, TG, UA, UCRE, f-T3 and f-T4.
Graph
Table 1 Comparisons of parameters between men and women participants.
Men (n = 292) Women (n = 174) p value Equol producers 114 (39%) 82 (47%) 0.136a Age (years) 54 (22–86) 54 (28–88) 0.377 a Serum equol (ng/dL) 0.6 (0.34, 3.37) 1.03 (0.61, 7.23) <0.001 Serum daidzein (ng/dL) 11.74 (3.96, 35.35) 17.85 (7.16, 44.36) 0.004 Urinary equol (μg/gCr) 7.52 (3.7, 209) 14.11 (6.8, 600.8) <0.001 Urinary daidzein (μg/gCr) 1490.57 (472.11, 4275.9) 1876.56 (761, 5997) 0.07 log (Equol/Daidzein) -1.99 (-2.74, 4275.9) -1.59 (-2.49, -0.3) 0.121 Estradiol (pg/mL) <LOD (0, 0) 0.25 (0.25, 66.55) <0.001 Luteinizing hormone (mIU/mL) <LOD (0, 5.13) 22.55 (7.1, 32.68) <0.001 Follicular stimulating hormone (mIU/mL) <LOD (0, 5.78) 47.65 (7.48, 70.23) <0.001 C-reactive protein (mg/dL) 0.05 (0.05, 0.1) 0.05 (0.05, 0.1) 0.167 Thyroid stimulating hormone (μIU/mL) 1.65 (1.1, 2.51) 1.83 (1.21, 2.63) 0.106 Free thyroxine (ng/dL) 1.28 (1.18, 1.4) 1.21 (1.13, 1.32) <0.001 Free triiodothyronine (pg/mL) 3.14 (2.88, 3.41) 2.8 (2.63, 3.06) <0.001 Fasting blood glucose (mg/dL) 94 (88, 103) 90 (85.3, 95.75) <0.001 Hemoglobin A1c (%) 5.60 (5.00, 6.00) 5.70 (5.00, 6.00) 0.814 Glycated albumin (%) 1.30 (1.30, 1.40) 1.40 (1.30, 1.50) <0.001 1.5-Anhydro-D-glucitol (μg/mL) 18.7 (13.48, 22.53) 15.95 (12.8, 19.48) <0.001 Insulin (μU/mL) 5 (3.38, 7.60) 4 (2.70, 5.30) 0.423 C-peptide (ng/mL) 1.62 (1.16, 2.08) 1.16 (0.91, 1.59) <0.001 HOMA-IR (Ratio) 1.17 (0.76, 1.85) 0.91 (0.60, 1.32) <0.001 Insulin resistance, n (%) 40 (14%) 14 (8%) 0.093 a Total cholesterol (mg/dL) 197.5 (175.8, 226) 202 (181, 223) 0.289 Low density lipoprotein cholesterol (mg/dL) 115 (99, 138) 116.5 (97, 137 0.878 Triglycerides (mg/dL) 114.5 (74, 177.25) 69.5 (53.3, 106.5) <0.001 High density lipoprotein cholesterol (mg/dL) 55 (47, 65) 69 (55.3, 78) <0.001 Uric acid (mg/dL) 6.15 (5.4, 6.9) 4.65 (3.9, 5.38) <0.001 Urinay creatinine (mg/dL) 122 (80.75, 173) 72 (50.3, 111.5) <0.001
1 Categorical variables were expressed as numbers (percentages). Continuous variables are expressed as medians (interquartile ranges) and were compared by the Mann‒Whitney U test except for Fig 2 showed strong positive relationships between the serum and urine equol levels in both men (r = 0.75, R Graph: Linear regression analysis in (A) men equol producers (n = 114) and nonproducers (n = 178), (B) women equol producers (n = 81) and nonproducers (n = 93). EQP = equol producers, EQNP = equol nonproducers.
Table 2 shows that PSA levels in men were significantly lower in equol producers (0.8 v.s. 1.0 ng/ml, p = 0.004), especially in men equol producers in their 40s (0.82 vs. 1.13 ng/ml, p<0.001) and 60 s (0.64 vs. 1.02 ng/ml, p<0.001), as shown in Fig 3A. In addition, a significant proportion of men with high LDL cholesterol levels were equol nonproducers (48.9% vs. 36.8%, p = 0.043), as shown in Fig 3B. Graph: A: Comparisons of prostate-specific antigen levels between equol producers and nonproducers in each age group of men. PSA levels were expressed as medians and compared using the Mann‒Whitney U test. B: Comparisons of abnormal metabolic values between equol producers and nonproducers in men. The proportions of abnormal values were compared using the chi-squared test. Graph Table 2 Comparison between men equol producers and nonproducers.
2 Continuous variables are expressed as medians (interquartile ranges) and were compared by the Mann‒Whitney U test except for Among women, no significant quantitative differences were observed between equol nonproducers except for isoflavone parameters (Table 3). However, equol nonproducers had more abnormal LDL cholesterol, triglyceride, and uric acid levels (Fig 4). Graph: The proportions of abnormal values were compared using the chi-squared test. Graph Table 3 Comparison between equol producers and nonproducers in women.
3 Continuous variables are expressed as medians (interquartile ranges) and were compared by the Mann‒Whitney U test except for We found that the cut-off value of the log-transformed urinary equol to daidzein ratio was almost consistent in both sexes, i.e., at the value of -1.42. Therefore, our definition of equol producers as those having a log 10-transformed ratio of urinary equol to daidzein of -1.42 or higher was relevant in our study population including both men and women. Previously, this cut-off value was reported in women participants only [[ The cut-off value of equol producers in Setchell's study was -1.75 after a standard 3-day challenge of soy foods containing isoflavones. Here, in our study, we used -1.42 as the cut-off value, as we did not perform the soy challenge, to reflect the real-time measurement as in the Japan Nurses' Health Study [[ Studies on the benefits of soy isoflavones have yielded inconsistent results. This could be most likely due to the variations in equol producer phenotypes. Even in the studies that assessed the equol producer phenotypes, some results failed to reach statistical significance due to small sample sizes. For example, in this study, women equol nonproducers tended to have higher LDL cholesterol, triglyceride, high sensitivity C-reactive protein and uric acid levels, but the results were not statistically significant. However, in our previous study on 743 healthy women, equol producers in their 50s and 60 s, the age groups with declining estrogen levels, had favorable blood levels of lipids, uric acid, bone resorption markers, high sensitivity C-reactive protein, and homocysteine [[ In this study, men equol producers had significantly lower PSA levels than nonproducers, especially in men in their 40s and 60 s. Additionally, abnormal PSA values were rarely associated with men equol producers. Several studies have reported that equol can decrease serum PSA levels by its antiandrogenic action on 5-alpha-dehydro testosterone, decrease prostate size, and thus reduce the risk of prostate cancer [[ This is the first study to confirm the use of the precursor-product relation as a robust cut-off value to identify the equol producer phenotype in men and women using the LC‒MS/MS method. It is also the first study that examined the differences between urine and serum equol concentrations in both women and men in equol producers and nonproducers. As equol producers were associated with better health benefits, findings in this study can be applied in integrating equol producer tests in the assessment of well-being for both sexes, especially in the middle age men and women with declining sex hormones. Determination of equol producers can also promote the healthy eating habits, and lifestyle promotion efforts [[ However, this study has the following limitations. First, we did not have the detailed characteristics of the study participants, including medical history, anthropometric measures, and dietary habits. Therefore, that might affect the outcomes of the study. Especially the dietary habits might affect the proportion of equol producers since the isoflavone concentration in the body fluctuates with soy intake and is influenced by many other dietary factors. Second, the sample size varied among the different age groups. Age is one of the most important determinants affecting the parameters that we assessed. Therefore, we need to be cautious about generalizing the results in all age groups and need to consider further studies based on the sample size calculations using the effect sizes in this study. Furthermore, the effect size we used in the sample size calculation was only based on the phenotype differences between genders and not applied for other outcomes. Especially, 37% difference between genders is not plausible. Lastly, as it is a cross-sectional study, causality cannot be determined. The proportion of equol producers was larger in women. The cut-off value of -1.42 for equol producers was consistent in both sexes. Positive relationships were noted between serum and urinary equol levels in equol producers of both sexes; however, such a relationship was not detected in nonproducers. The equol-producing ability tended to be higher in women, suggesting a relationship between estrogen and equol-producing ability. In men, equol-producing subjects had significantly lower PSA levels, suggesting a relationship between equol-producing ability and reduced risk of prostate disease. In addition, both women and men equol producers have positive effects on blood lipids and uric acid levels. However, we need more robust clinical trials in the representative samples of different age groups including dietary assessments to determine the health benefits of equol in both men and women. S1 File Consent form. (PDF) S2 File Strobe checklist. (DOCX) S3 File Cut-off values to determine abnormal results for each parameter. (TIF) S4 File Minima data set. (XLSX) Sauli Elingarami Academic Editor 15 Dec 2023 PONE-D-23-20874Comparison of blood and urine concentrations of equol by LC‒MS/MS method and factors associated with equol production in 466 Japanese men and womenPLOS ONE Dear Dr. Myint, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== When responding to reviewer comments, please make sure to clearly detail your problem statement, introduction (with proper current citations), including thorough discussion of your results, which should also be properly written/presented. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This cross-sectional study calculated a cutoff value for the benefit of equol-producers in Japanese men and women. While it is worth reporting that the cutoff was the same for both sexes (-1.42), the authors need to consider the following points. Major points 1. The authors emphasize that there was a favorable trend in the equol producers, but this is not evident from the results or the figures. Therefore, the following corrections are needed, especially in Figure 3B and Figure 4: ・The cutoff values for each parameter should be stated (e.g., no cutoff value listed for High LDL cholesterol). ・List all P values as in the Table. ・Unify the description of the parameters in Table3B and Table4. 2. Conclusion: Please describe what you found out from this study (e.g., cutoff value of -1.42 for both men and women, association between blood and urine equol levels only in the producers, etc.), rather than describing the strengths of the study. Minor points Reviewer #2: Thank you very much for allowing me to review this important research undertaking. I have some comments: Introduction: Methods 1. Were the participants asked if they were soy drinkers and the frequency of drinking soy? This is an important variable as this might have an effect on the outcome measured. Discussion: Overall, the discussion is a weak. It should discuss public health implication of the findings and how can the government address the health issue on hand. Also, strengths of the study should also be included before the limitation part of the discussion.The limitation part also lacks in details in terms of the study design limitations. Kindly improve including the objectives. Reviewer #3: In the study, urinary and blood isoflavones were determined in participants of annual health checkups. The associations between equol production and other various outcomes were investigated. The findings on the relationship between blood and urinary levels are not surprising even though they may examine them in each sex and equol metabolic phenotype. The cut-off value for equol phenotype was previously proposed by Ideno et al. and the authors in this study employed them and no additional investigation was conducted. Thus they found the threshold around -1.4 in log-scale and it does not give new insights. The authors appeal that they found beneficial effects of equol on PSA in male population. In this study, the authors conducted comparisons of various outcomes other than PSA, and thus it is likely a statistical chance. Indeed, other outcomes showed statistically significant differences between equol phenotypes, but the authors focused only on PSA, this is cherry-picking and p-value hacking. Background of participants is highly unclear. During Jun 2016¬ to Dec 2017, only 466 persons visited the health screening? Total number of examined persons should be given. In addition, the participants were recruited at Kyoto University but the IRB approval was provided by University of Tokyo. Why? Further, ranges of ages are too wide to evaluate the possible subclinical effects. Sample size estimation was not appropriate. The effect size was only based on the phenotype differences between genders and not applied for other outcomes. Especially, 37% difference between genders is not plausible. In addition, various outcomes examined in this study are affected by other background characteristics of participants. 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We have added the data set as Supporting information. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: We will update your Data Availability statement to reflect the information you provide in your cover letter. Reviewer's Responses to Questions Comments to the Author Reviewer #1: This cross-sectional study calculated a cutoff value for the benefit of equol-producers in Japanese men and women. While it is worth reporting that the cutoff was the same for both sexes (-1.42), the authors need to consider the following points. Major points 1. The authors emphasize that there was a favorable trend in the equol producers, but this is not evident from the results or the figures. Therefore, the following corrections are needed, especially in Figure 3B and Figure 4: ・The cutoff values for each parameter should be stated (e.g., no cutoff value listed for High LDL cholesterol). ・List all P values as in the Table. ・Unify the description of the parameters in Table3B and Table4. Thank you for your valuable advice. The above points have been addressed in the figures and tables. 2. Conclusion: Please describe what you found out from this study (e.g., cutoff value of -1.42 for both men and women, association between blood and urine equol levels only in the producers, etc.), rather than describing the strengths of the study. Thank you for your insightful advice. The above points have been addressed in the conclusion. Minor points 3. Please include the abbreviation for PSA in the abstract. Thank you for your insightful advice. The above points have been addressed in the abstract. 4. Male/female and men/women are mixed in the paper. Please unify them. Thank you for your insightful advice. The above points have been addressed. 5. Figure1: "Density" on the vertical axis should be changed to an appropriate term. Thank you for your advice. The above points have been addressed in the figure 1. 6. Table 1: Please include the abbreviation of the parameter in the footnote as well. Or, please provide the full name of the parameter in the table. Thank you for your insightful advice. The above points have been addressed in table 1. 7. Table1：Please also indicate the median age. Thank you for your insightful advice. The above points have been addressed in table 1. 8. Table2: Please indicate the median age of each of the producers and non-producers. Please adjust the decimal point of the parameter to Table3. Thank you for your insightful advice. The above points have been addressed in table 3. 9. Table3: Please indicate the median age of each of the producers and non-producers. Thank you for your insightful advice. The above points have been addressed in table 3. Reviewer #2: Thank you very much for allowing me to review this important research undertaking. I have some comments: Introduction: Thank you for your insightful advice. References were added in the first paragraph of the introduction. 2. The introduction part is a bit weak. There should be a strong statement of the problem on the reasons why equol should be given priority and then transitioning to the research gap. Recent researches on epidemiological research on equol should also be stated to describe current landscape on such research topic. Thank you for your insightful advice. Epidemiological research on equol, statement of the problem and rationale for research objective have been added in the introduction. Methods 1. Were the participants asked if they were soy drinkers and the frequency of drinking soy? This is an important variable as this might have an effect on the outcome measured. Thank you for your insightful advice. We were not able to collect dietary assessments in this research and revised the methods and limitation sections for this. Discussion: Overall, the discussion is a weak. It should discuss public health implication of the findings and how can the government address the health issue on hand. Also, strengths of the study should also be included before the limitation part of the discussion.The limitation part also lacks in details in terms of the study design limitations. Kindly improve including the objectives. Thank you for your valuable advice. We have addressed the points in the discussion section. Reviewer #3: In the study, urinary and blood isoflavones were determined in participants of annual health checkups. The associations between equol production and other various outcomes were investigated. The findings on the relationship between blood and urinary levels are not surprising even though they may examine them in each sex and equol metabolic phenotype. The cut-off value for equol phenotype was previously proposed by Ideno et al. and the authors in this study employed them and no additional investigation was conducted. Thus they found the threshold around -1.4 in log-scale and it does not give new insights. The authors appeal that they found beneficial effects of equol on PSA in male population. In this study, the authors conducted comparisons of various outcomes other than PSA, and thus it is likely a statistical chance. Indeed, other outcomes showed statistically significant differences between equol phenotypes, but the authors focused only on PSA, this is cherry-picking and p-value hacking. Background of participants is highly unclear. During Jun 2016¬ to Dec 2017, only 466 persons visited the health screening? Total number of examined persons should be given. In addition, the participants were recruited at Kyoto University but the IRB approval was provided by University of Tokyo. Why? Further, ranges of ages are too wide to evaluate the possible subclinical effects. Thank you for your advice. These participants were the members of the facility that we carried out research, not the entire hospital, therefore, the number is low. Although the study was conducted at the Kyoto University Hospital, we need to submit the proposal to a third-party ethical review board according to the regulations of Kyoto University Hospital for the use of secondary data by researchers from different affiliations. We have added those points in the method section. We have deleted the age ranges from the table as the numbers of subjects in each age range were too small if we change to narrower ranges of ages. Sample size estimation was not appropriate. The effect size was only based on the phenotype differences between genders and not applied for other outcomes. Especially, 37% difference between genders is not plausible. In addition, various outcomes examined in this study are affected by other background characteristics of participants. Absence of the information is critical to investigate potential relationship between equol phenotype and those outcomes. Thank you for your valuable advice. We have added those points as limitations of our study. Other points: Why blood isoflavone levels should be corrected by creatinine? Thank you for your valuable advice. We have corrected these levels. Significant digits should be unified through the texts and tables. Thank you for your valuable advice. We have unified them. The authors stated "However, it would not affect the results of this epidemiological study significantly", but this does not make sense. Thank you for your valuable advice. We have added those points as limitations of our study. Figure 3A should be with error bars. Thank you for your valuable advice. We have added error bars in figure 3A. In Figure 3B and Figure 4, numbers in each category and p-values should be given. Thank you for your valuable advice. We have added those points in figure 3B and figure 4. ________________________________________ 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No ________________________________________ [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachment Submitted filename: Response to reviewers_PONE-D-23-20874.docx Sauli Elingarami Academic Editor 13 Feb 2024 PONE-D-23-20874R1Comparison of blood and urine concentrations of equol by LC‒MS/MS method and factors associated with equol production in 466 Japanese men and womenPLOS ONE Dear Dr. Myint, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Be sure to include the following when submitting your responses to reviewer comments; proper labeling/naming of tables, figures, and concentrations. 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For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Elingarami Sauli, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the "Comments to the Author" section, enter your conflict of interest statement in the "Confidential to Editor" section, and submit your "Accept" recommendation. Reviewer #3: (No Response) Reviewer #4: All comments have been addressed Reviewer #5: (No Response) Reviewer #6: (No Response) *** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: No Reviewer #4: Yes Reviewer #5: Yes Reviewer #6: Partly *** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: No Reviewer #4: Yes Reviewer #5: Yes Reviewer #6: Yes *** 4. Have the authors made all data underlying the findings in their manuscript fully available? The Reviewer #3: Yes Reviewer #4: Yes Reviewer #5: Yes Reviewer #6: Yes *** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #3: Yes Reviewer #4: Yes Reviewer #5: Yes Reviewer #6: No *** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #3: Authors' responses are not by point by point. I cannot understand what revisions were made to my comments. Reviewer #4: Authors have appropriately addressed the comments and suggestions from the three reviewers in the first round of review. I have no more comment. Reviewer #5: This study provides important information about isoflavone intake and health effects via the epidemiologic cross-sectional designed investigation. To report its achievements through Plos One, please consider and revise about comments below. The conclusion should be included in one sentence in the abstract. You need a sentence like this. "This study suggests (or found) that ~." Page 6. Please describe the abbreviation "LC/MS/MS" here (not page 9), because it was firstly used in this manuscript. Several expressions on the page 12 are not good on the Results section. Expressions such as "consistent with our definition", "relevant in our population" must be moved to the Discussion section. Page 15. 1st sentence. observation → relationship Figure 3A. Numbers of each age range are too small. I recommend unifying 20s and 30s as well as 70s and 80s. Page 23. The sentences "In addition, ~ examined in this study." are a duplication with the first limitation. Authors emphasize LC/MS/MS method such an extent to include the title and key words. Then please explain the meaning of this method in your study. It is not a newly developed or validated method in this study, and readers may be curious about this method whether any special strength or not than other methods (i.e. GC-MS or anything else). Page 24. 3rd line. Association → relationship Reviewer #6: The article describes the investigation of the equol level in blood and urine samples from 466 Japanese men and women. Unfortunately, no information about the dietary habits are available which limits the suitability of this study. In my opinion it is important to state this limitation already in the abstract. Cut-off value: I am sorry, but I do not understand how the authors "analysed" the cut-off value. As far as I understood it, they just took it from a previous publication. Please clarify it. Furthermore, I have a problem with the statements of the authors drawing conclusions with the health data. First of all, there are no information about the dietary habits of the participants, so whether or not equol was detected in blood or urine greatly depends on the last consumption of isoflavone containing food. Secondly, the number of participants in the different age groups were rather limited. In my opinion, it is important to point out these limitations already in the abstract. The authors provided the minimal data set, but without an explanation, it is not clear to me what the individual columns are standing for. #Please check the English language in the whole manuscript, e.g., sometimes you use articles where there should be no article (page 12, line 8 – there should not be an article before "between" "men and women"). Keywords: As far as I have learnt it the key words should be different than the words used in the title. Page 6 at the end of the page: What do you mean with "LC/MS/CS"? Do you mean "LC-MS/MS"? Page 7 – line 2: I have learnt that "but" should not be used at the beginning of the sentence. Please consider using "However" instead. Page 9: I am missing some details on the used method. Ideally you should provide them in the manuscript itself, but at least you should provide a reference. Page 9 – metabolic parameters: Please check, but there should always be a space before the brackets (e.g., "glucose level (FBG)"). Furthermore, in English the compound names should be written with small starting letters (e.g., estradiol). Please check this in the whole manuscript. Page 12 – evaluation of equol producer status: You state "we applied the same finite fixed model as the previous study", but you do not provide a reference. Please add it here. Page 12 – line 6: Please check, but you sometimes used "n" and sometimes "N", please be consistent. Table 1-3: Why did you change the order in these tables? I would use always the same order since these tables state the same information, but in different groups (men & women, men and women). In Table 1: Why did you report serum concentrations as µg/g Cr? This unit belongs to the urinary concentrations, but not to serum. In the other two tables (2 and 3) you used the unit "ng/dL". Furthermore, please never report analytical results of "zero" as in case of estradiol. Always state "lower than the limit of detection < LOD). Please explain all used abbreviations – also "BMI" (page 23). Supporting information file 3: Please check the HDL-cholesterol level – is there really a 10 fold difference between men and women? All other values are exactly the same between men and women References: Please check the references carefully. "in vitro", "in vivo" should be written in general written in italics. Reference 5: It should be a "beta (β)" symbol not a "ß" which is a kind of "s" in German. Several times not only the year, but also the month of publication is provided. I think that it is not necessary to state the month as well. Reference 11: Please check – I am not sure what the number eight ( Reference 43: I think that here something is missing. Please check. Figures and Tables should be understandable without reading the manuscript itself. Therefore, please explain the used abbreviations (e.g., "EQP" and "EQNP" in Figure 2). Figure 1: You changed the nomenclature in the whole manuscript to "men" and "women"; but in this figure you still you "male" and "female". Please correct it here as well. Figure 2: Please state the units reported in theses graphs. It is not clear to me what you did. Furthermore, in each subgraph for male and female two Rsquare values are provided, but only in case of male it is specified to which group it belongs. Moreover, you changed the nomenclature in the whole manuscript to "men" and "women"; but in this figure you still you "male" and "female". Please correct it here as well. Figure 3A Please check the numbers provided below the figure. In case of "20s" should it not be (0, 2) instead of ( Please unify, because in Figure 3B and 4 first equol-non-producers are provided, whereas in Figure 3A you changed the order and provide first equol producers and then equol-non-producers. In my opinion it would be less confusing if the order is always the same. *** 7. PLOS authors have the option to publish the peer review history of their article (https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public.
Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our https:// Reviewer #3: No Reviewer #4: No Reviewer #5: Yes: Yong Min Cho Reviewer #6: No *** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 28 Feb 2024 Reviewer #3: Authors' responses are not by point by point. I cannot understand what revisions were made to my comments. We apologize for not addressing point by point. Let us respond your previous comments as follows. Responses for the previous comments by Reviewer #3. Reviewer #3: In the study, urinary and blood isoflavones were determined in participants of annual health checkups. The associations between equol production and other various outcomes were investigated. The findings on the relationship between blood and urinary levels are not surprising even though they may examine them in each sex and equol metabolic phenotype.The cut-off value for equol phenotype was previously proposed by Ideno et al. and the authors in this study employed them and no additional investigation was conducted. Thus, they found the threshold around -1.4 in log-scale and it does not give new insights. Thank you for your insightful comments. We have mentioned the distinction in the discussion as follows. " Previously, this cut-off value was reported in women participants only [ The authors appeal that they found beneficial effects of equol on PSA in male population. In this study, the authors conducted comparisons of various outcomes other than PSA, and thus it is likely a statistical chance. Indeed, other outcomes showed statistically significant differences between equol phenotypes, but the authors focused only on PSA, this is cherry-picking and p-value hacking. Thank you for your comment. We have discussed other outcomes as follows. " Studies on the benefits of soy isoflavones have yielded inconsistent results. This could be most likely due to the variations in equol producer phenotypes. Even in the studies that assessed the equol producer phenotypes, some results failed to reach statistical significance due to small sample sizes. For example, in this study, women equol nonproducers tended to have higher LDL cholesterol, triglyceride, high sensitivity C-reactive protein and uric acid levels, but the results were not statistically significant. However, in our previous study on 743 healthy women, equol producers in their 50s and 60 s, the age groups with declining estrogen levels, had favorable blood levels of lipids, uric acid, bone resorption markers, high sensitivity C-reactive protein, and homocysteine [ Background of participants is highly unclear. During Jun 2016¬ to Dec 2017, only 466 persons visited the health screening? Total number of examined persons should be given. In addition, the participants were recruited at Kyoto University but the IRB approval was provided by University of Tokyo. Why? Thank you for your advice. These participants were the members of the facility that we carried out research, not the entire hospital, therefore, the number is low. Although the study was conducted at the Kyoto University Hospital, we need to submit the proposal to a third-party ethical review board according to the regulations of Kyoto University Hospital for the use of secondary data by researchers from different affiliations. We have added those points in the method section as follows. "Although the study was conducted at the Kyoto University Hospital, we need to submit the proposal to a third-party ethical review board according to the regulations of Kyoto University Hospital for the use of secondary data by researchers from different affiliations. Therefore, the study was approved by the Institutional Review Board of The University of Tokyo (Supplementary data file 2)." Further, ranges of ages are too wide to evaluate the possible subclinical effects. Thank you for your comment. We have deleted those age ranges in the tables 1-3. Sample size estimation was not appropriate. The effect size was only based on the phenotype differences between genders and not applied for other outcomes. Especially, 37% difference between genders is not plausible. In addition, various outcomes examined in this study are affected by other background characteristics of participants. Absence of the information is critical to investigate potential relationship between equol phenotype and those outcomes. Thank you for your valuable advice. We have added those points as limitations of our study as follows. " Furthermore, the effect size we used in the sample size calculation was only based on the phenotype differences between genders and not applied for other outcomes. Especially, 37% difference between genders is not plausible." Other points: Why blood isoflavone levels should be corrected by creatinine? Thank you for your valuable advice. We have corrected these levels in the tables 1-3. Significant digits should be unified through the texts and tables. Thank you for your valuable advice. We have unified them. The authors stated "However, it would not affect the results of this epidemiological study significantly", but this does not make sense. Thank you for your valuable advice. We have added those points as limitations of our study as follows. "First, we did not have the detailed characteristics of the study participants, including medical history, anthropometric measures, and dietary habits. Therefore, that might affect the outcomes of the study. Especially the dietary habits might affect the proportion of equol producers since the isoflavone concentration in the body fluctuates with soy intake and is influenced by many other dietary factors." Figure 3A should be with error bars. Thank you for your valuable advice. We have added error bars in figure 3A. In Figure 3B and Figure 4, numbers in each category and p-values should be given. Thank you for your valuable advice. We have added those points in figure 3B and figure 4. ________________________________________ Reviewer #4: Authors have appropriately addressed the comments and suggestions from the three reviewers in the first round of review. I have no more comment. Thank you for your comment. We really appreciate your valuable insights and suggestions. ________________________________________ Reviewer #5: This study provides important information about isoflavone intake and health effects via the epidemiologic cross-sectional designed investigation. To report its achievements through Plos One, please consider and revise about comments below. The conclusion should be included in one sentence in the abstract. You need a sentence like this. "This study suggests (or found) that ~." Thank you for your comment. We added that sentence in the abstract as follows. "This study suggests a relationship between equol-producing ability and reduced risk of prostate disease as well as positive effects of equol on blood lipids and uric acid levels." Page 6. Please describe the abbreviation "LC/MS/MS" here (not page 9), because it was firstly used in this manuscript. Thank you for your comment. We described the abbreviation in page 6 as you suggested. Several expressions on the page 12 are not good on the Results section. Expressions such as "consistent with our definition", "relevant in our population" must be moved to the Discussion section. Thank you for your comment. We moved that section to the Discussion section as you suggested. Page 15. 1st sentence. observation → relationship Thank you for your comment. We corrected the term as you suggested. Figure 3A. Numbers of each age range are too small. I recommend unifying 20s and 30s as well as 70s and 80s. Thank you for your comment. We have combined the age ranges of 30s and 40s and 70s and 80s and revised figure 3A. Page 23. The sentences "In addition, ~ examined in this study." are a duplication with the first limitation. Thank you for your comment. We have deleted that sentence from limitation. Authors emphasize LC/MS/MS method such an extent to include the title and key words. Then please explain the meaning of this method in your study. It is not a newly developed or validated method in this study, and readers may be curious about this method whether any special strength or not than other methods (i.e. GC-MS or anything else). Thank you for your comment. We added those facts in the method section as follows. "In brief, 100 µL of serum or 10-fold diluted urine was mixed with internal standards, followed by the addition of 150 µL of an β-glucuronidase enzyme solution for deconjugation (Roche Biochemical, Mannheim, Germany). Following a one-hour deconjugation reaction at 37°C, free equol, daidzein, and genistein were purified using solid-phase extraction (Oasis PRiME HLB, Waters, Milford, MA). Subsequently, liquid chromatography (LC) -tandem mass spectrometry (LCMS-8050, Shimadzu, Japan) was employed with a reverse-phase LC column (ACQUITY UPLC HSS T3, 1.8 µm, 2.1 mm × 100 mm, Waters, Milford, MA) for analysis. Data processing was conducted using Mass Hunter software (Agilent, Santa Clara, CA). The peak areas were normalized using internal standards, and the concentration of each analyte was determined through a standard curve." "The advantage of LC-MS/MS over other methods such as glass chromatography-based methods for detection of isoflavones is that all the conjugated and unconjugated isoflavones and their metabolites can be separated and analysed faster and more efficiently. [ Page 24. 3rd line. Association → relationship Thank you for your comment. We corrected the term as you suggested. ________________________________________ Reviewer #6: The article describes the investigation of the equol level in blood and urine samples from 466 Japanese men and women. Unfortunately, no information about the dietary habits are available which limits the suitability of this study. In my opinion it is important to state this limitation already in the abstract. Thank you for your comment. We added that sentence in the abstract as follows. " However, lack of dietary information and disperse age groups were major drawbacks in generalizing the results of this study." Cut-off value: I am sorry, but I do not understand how the authors "analysed" the cut-off value. As far as I understood it, they just took it from a previous publication. Please clarify it. Thank you for your comment. We added the following points for what is unknown from the previous study and our hypothesis in the introduction before the objective of the study as follows. " However, their study was conducted only among women and there was no such study among men nor reproducibility of that definition has never been tested in both sexes. We hypothesized there were differences between men and women with regards to equol producing ability, i.e., they might have different cut-off values, as well as its relationship with other biomarkers inside the body." Furthermore, I have a problem with the statements of the authors drawing conclusions with the health data. First of all, there are no information about the dietary habits of the participants, so whether or not equol was detected in blood or urine greatly depends on the last consumption of isoflavone containing food. Secondly, the number of participants in the different age groups were rather limited. In my opinion, it is important to point out these limitations already in the abstract. Thank you for your comment. We have added that sentence in the abstract and conclusion section as follows. " However, lack of dietary information and disperse age groups were major drawbacks in generalizing the results of this study." "However, we need more robust clinical trials in the representative samples of different age groups including dietary assessments to determine the health benefits of equol in both men and women." The authors provided the minimal data set, but without an explanation, it is not clear to me what the individual columns are standing for. Thank you for your comment. We have added the explanation of the columns in the minimal data set in a new sheet. #Please check the English language in the whole manuscript, e.g., sometimes you use articles where there should be no article (page 12, line 8 – there should not be an article before "between" "men and women"). Thank you for your comment. We have corrected that point as you suggested. Keywords: As far as I have learnt it the key words should be different than the words used in the title. Thank you for your comment. We have changed the key words as you suggested. Page 6 at the end of the page: What do you mean with "LC/MS/CS"? Do you mean "LC-MS/MS"? Thank you for your comment. We have corrected the abbreviation as you suggested. Page 7 – line 2: I have learnt that "but" should not be used at the beginning of the sentence. Please consider using "However" instead. Thank you for your comment. We have corrected the vocabulary as you suggested. Page 9: I am missing some details on the used method. Ideally you should provide them in the manuscript itself, but at least you should provide a reference. Thank you for your comment. We added those facts and references in the method section as follows. "In brief, 100 µL of serum or 10-fold diluted urine was mixed with internal standards, followed by the addition of 150 µL of an β-glucuronidase enzyme solution for deconjugation (Roche Biochemical, Mannheim, Germany). Following a one-hour deconjugation reaction at 37°C, free equol, daidzein, and genistein were purified using solid-phase extraction (Oasis PRiME HLB, Waters, Milford, MA). Subsequently, liquid chromatography (LC) -tandem mass spectrometry (LCMS-8050, Shimadzu, Japan) was employed with a reverse-phase LC column (ACQUITY UPLC HSS T3, 1.8 µm, 2.1 mm × 100 mm, Waters, Milford, MA) for analysis. Data processing was conducted using Mass Hunter software (Agilent, Santa Clara, CA). The peak areas were normalized using internal standards, and the concentration of each analyte was determined through a standard curve." "The advantage of LC-MS/MS over other methods such as glass chromatography-based methods for detection of isoflavones is that all the conjugated and unconjugated isoflavones and their metabolites can be separated and analysed faster and more efficiently. [ Page 9 – metabolic parameters: Please check, but there should always be a space before the brackets (e.g., "glucose level (FBG)"). Furthermore, in English the compound names should be written with small starting letters (e.g., estradiol). Please check this in the whole manuscript. Thank you for your comment. We have corrected those vocabularies as you suggested. Page 12 – evaluation of equol producer status: You state "we applied the same finite fixed model as the previous study", but you do not provide a reference. Please add it here. Thank you for your comment. We have added the reference as you suggested. Page 12 – line 6: Please check, but you sometimes used "n" and sometimes "N", please be consistent. Thank you for your comment. We have made "n" consistent as you suggested. Table 1-3: Why did you change the order in these tables? I would use always the same order since these tables state the same information, but in different groups (men & women, men and women). Thank you for your comment. We have unified the order of the tables as you suggested. In Table 1: Why did you report serum concentrations as µg/g Cr? This unit belongs to the urinary concentrations, but not to serum. In the other two tables (2 and 3) you used the unit "ng/dL". Furthermore, please never report analytical results of "zero" as in case of estradiol. Always state "lower than the limit of detection < LOD). Thank you for your comment. We have corrected the units and used LOD in place of zero as you suggested. Please explain all used abbreviations – also "BMI" (page 23). Thank you for your comment. We have explained the abbreviations as you suggested. Supporting information file 3: Please check the HDL-cholesterol level – is there really a 10 fold difference between men and women? All other values are exactly the same between men and women Thank you for your comment. We have corrected the value as you pointed out. References: Please check the references carefully. "in vitro", "in vivo" should be written in general written in italics. Thank you for your comment. We have written them in italics as you suggested. Reference 5: It should be a "beta (β)" symbol not a "ß" which is a kind of "s" in German. Thank you for your comment. We have corrected the symbol as you suggested. Several times not only the year, but also the month of publication is provided. I think that it is not necessary to state the month as well. Thank you for your comment. We have omitted the months as you suggested. Reference 11: Please check – I am not sure what the number eight ( Thank you for your comment. We have corrected that point as you suggested. Reference 43: I think that here something is missing. Please check. Thank you for your comment. We have added some more information as you suggested. Figures and Tables should be understandable without reading the manuscript itself. Therefore, please explain the used abbreviations (e.g., "EQP" and "EQNP" in Figure 2). Figure 1: You changed the nomenclature in the whole manuscript to "men" and "women"; but in this figure you still you "male" and "female". Please correct it here as well. Thank you for your comment. We have corrected that point as you suggested. Figure 2: Please state the units reported in theses graphs. It is not clear to me what you did. Furthermore, in each subgraph for male and female two Rsquare values are provided, but only in case of male it is specified to which group it belongs. Moreover, you changed the nomenclature in the whole manuscript to "men" and "women"; but in this figure you still you "male" and "female". Please correct it here as well. Thank you for your comment. We have corrected the points as you suggested. Figure 3A Please check the numbers provided below the figure. In case of "20s" should it not be (0, 2) instead of ( Thank you for your comment. We have corrected the points as you suggested and combined 20s and 30s, as well as 70s and 80s as the numbers are quite low. Please unify, because in Figure 3B and 4 first equol-non-producers are provided, whereas in Figure 3A you changed the order and provide first equol producers and then equol-non-producers. In my opinion it would be less confusing if the order is always the same. Thank you for your comment. We have corrected the points as you suggested. Attachment Submitted filename: Response to reviewers_PONE-D-23-20874_V3.docx Sauli Elingarami Academic Editor 1 Mar 2024 Comparison of blood and urine concentrations of equol by LC‒MS/MS method and factors associated with equol production in 466 Japanese men and women PONE-D-23-20874R2 Dear Dr. Myint, We're pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. 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Kind regards, Elingarami Sauli, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Sauli Elingarami Academic Editor 13 Mar 2024 PONE-D-23-20874R2 PLOS ONE Dear Dr. Myint, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. 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Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Elingarami Sauli Academic Editor PLOS ONE We would like to acknowledge all the participants who have consented to use their health screening data for the purpose of medical research, Himedic Kyoto University Hospital, and all the persons who have contributed to making this research possible. By Remi Yoshikata; Khin Zay Yar Myint and Junichi Taguchi Reported by Author; Author; AuthorEquol producers (n = 114) Equol nonproducers (n = 178) p value Age (years) 54 (45, 64) 55 (46, 64) 0.6573 Serum equol (ng/mL) 7.64 (0.64, 21.54) 0.43 (0.3, 0.72) <0.001 Serum daidzein (ng/mL) 8.25 (2.97, 28.00) 13.01 (4.85, 36.84) 0.025 Urinary equol (μg/gCr) 539.91 (70.90, 2731.6) 4.73 (3.23, 8.23) <0.001 Urinary daidzein (μg/gCr) 875.25 (151.73, 2808.75) 2152.50 (956.15, 5324.83) <0.001 log (Equol/Daidzein) -0.30 (-0.64, 0.20) -2.57 (-2.98, -2.09) <0.001 C-reactive protein (mg/dL) 0.05 (0.05, 0.1) 0.05 (0.05, 0.1) 0.752 Thyroid stimulating hormone (μIU/mL) 1.51 (1.06, 2.45) 1.70 (1.13, 2.53) 0.366 Free thyroxine (ng/dL) 1.31 (1.20, 1.4) 1.27 (1.17, 1.4) 0.559 Free triiodothyronine (pg/mL) 3.16 (2.89, 3.4) 3.13 (2.88, 3.42) 0.907 Fasting blood glucose (mg/dL) 95.00 (89.00, 103) 94.00 (88, 101.75) 0.368 Haemoglobin A1c (%) 5.7 (5.50, 6.00) 5.6% (5.00, 6.00) 0.074 Glycated albumin (%) 1.3 (1.30, 1.40) 1.30 (1.30, 1.40) 0.929 1.5-Anhydro-D-glucitol (μg/mL) 18.55 (14.03, 22.90) 18.85 (13.13, 22.13) 0.455 Insulin (μU/mL) 5.20 (3.73, 7.78) 4.90 (3.13, 7.30) 0.472 C-peptide (ng/mL) 1.65 (1.23, 2.20) 1.57 (1.14, 2.00) 0.215 HOMA-IR (Ratio) 1.23 (0.85, 1.94) 1.14 (0.71, 1.81) 0.355 Insulin resistance, n (%) 18 (16%) 22 (12%) 0.511 a Total cholesterol (mg/dL) 192.50 (173.00, 225.75) 200.00 (179.25, 226.50) 0.291 Low density lipoprotein cholesterol (mg/dL) 109.00 (96, 134) 119.00 (100.25, 140.75) 0.114 Triglycerides (mg/dL) 129.50 (84.00, 187) 107.50 (72.25, 171.75) 0.088 High density lipoprotein cholesterol (mg/dL) 55.00 (46.00, 63.75) 56.00 (47.25, 65.75) 0.204 Uric acid (mg/dL) 6.20 (5.43, 6.90) 6.10 (5.40, 6.90) 0.899 Urinary creatinine (mg/dL) 117.50 (81.25, 174.50) 125.00 (80, 172.50) 0.840 Prostate specific antigen (ng/mL) 0.80 (0.34, 1.36) 1.00 (0.65, 1.53) 0.004 Equol producers (n = 82) Equol nonproducers (n = 92) p value Age 53.5 (54, 60.5) 55 (46.25, 63) 0. 8389 Serum equol (ng/mL) 6.77 (1.06, 30.93) 0.71 (0.52, 1.14) <0.001 Serum daidzein (ng/mL) 13.67 (5.73, 38.89) 20.41 (8.07, 51.81) 0.1 Urinary equol (μg/gCr) 691.98 (161.92, 2742.59) 7.94 (5.49, 12.20) <0.001 Urinary daidzein (μg/gCr) 1469.93 (292.07, 3864.86) 2907.89 (1048.95, 7666.67) <0.01 log (Equol/Daidzein) -0.3 (2.71, 3.54) -2.46 (2.79, 3.73) <0.001 Estradiol (pg/mL) 0.25 (0.25, 51.60) 0.25 (0.25, 70.50) 0.74 Luteinizing hormone (mIU/mL) 22.6 (7.10, 35.20) 22.55 (6.60, 31.30) 0.51 Follicular stimulating hormone (mIU/mL) 49.4 (9.80, 76.20) 47.65 (6.30, 68.20) 0.43 C-reactive protein (mg/dL) 0.05 (0.05, 0.10) 0.05 (0.05, 0.10) 0.57 Thyroid stimulating hormone (μIU/mL) 1.8 (1.07, 2.79) 1.83 (1.33, 2.55) 0.58 Free thyroxine (ng/dL) 1.22 (1.14, 1.32) 1.21 (1.11, 1.31) 0.3 Free triiodothyronine (pg/mL) 2.79 (2.67, 3.09) 2.8 (2.59, 3.05) 0.42 Fasting blood glucose (mg/dL) 90 (86, 94) 90 (85, 97) 0.64 Haemoglobin A1c (%) 5.6 (5.4, 5.8) 5.7 (5.5, 6) 0.05 Glycated albumin (%) 13.80 (13.10, 14.70) 14.00 (14.00, 15.00) 0.17 1.5-Anhydro-D-glucitol (μg/mL) 15.90 (13.50, 19.70) 15.95 (12.70, 19.40) 0.55 Insulin (μU/mL) 4.10 (3.00, 4.90) 4.00 (2.50, 5.80) 0.98 C-peptide (ng/mL) 1.16 (0.91, 1.58) 1.16 (0.92, 1.59) 0.83 HOMA-IR (Ratio) 0.91 (0.63, 1.16) 0.91 (0.55, 1.42) 0.92 Insulin resistance (%) 6 (7.40%) 8 (9%) 0.99 a Total cholesterol (mg/dL) 201 (183, 220) 202 (178, 224) 0.86 Low density lipoprotein cholesterol (mg/dL) 115 (98, 137) 116.5 (96, 137) 0.61 Triglycerides (mg/dL) 70 (55, 105) 69.5 (51, 110) 0.84 High density lipoprotein cholesterol (mg/dL) 71 (58, 80) 69 (55, 76) 0.3 Uric acid (mg/dL) 4.6 (4, 5.4) 4.65 (3.9, 5.3) 0.9 Urinary creatinine (mg/dL) 71 (51, 116) 72 (50, 100) 0.63
