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

Remi Yoshikata; Khin Zay Yar Myint; Junichi Taguchi

doi:10.1371/journal.pone.0288946

. 2024 Mar 27;19(3):e0288946. doi: 10.1371/journal.pone.0288946

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

Remi Yoshikata ^1,², Khin Zay Yar Myint ^1,^*, Junichi Taguchi ¹

Editor: Elingarami Sauli³

PMCID: PMC10971664 PMID: 38536793

Abstract

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.

Introduction

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 [1]. Equol then enters the blood stream after being absorbed from the intestinal wall and is distributed to the target organs [2, 3]. Similar to other dietary end-products, it is chemically modified in the liver and mainly excreted in the urine, with only a small amount excreted into the stool [4]. It can be detected in the blood 8 hours after ingestion of isoflavones and reaches a peak concentration after 12 to 24 hours. After 72 hours, its blood level becomes negligible, as it is rarely reserved in the body [4].

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 [5, 6]. It has the highest potential for inducing health benefits among soy isoflavones, as it possesses estrogenic and antiestrogenic actions as well as antiandrogenic and antioxidant actions [7–9]. It is associated with the relief of menopausal symptoms and a reduced risk of related conditions, including osteoporosis [10–14], in women. For men, it was found to be associated with a reduced risk of prostate cancers [15, 16]. In addition, it was reported to have positive and antiaging effects on skin structures in both men and women [17, 18].

However, not everyone has the ability to produce equol [5]. The ability to produce equol depends on age, gender, genetics, dietary contents, and other factors [19–21]. Therefore, health benefits are not observed in some people even if they consume soy isoflavones. Even in those with equol-producing ability, individual and diurnal variations and the use of antibiotics greatly influence the desirable level of equol in the body [22–24]. For those reasons, there have been attempts to induce equol actions through supplements containing equol or with probiotics such as Lactobacilli and Bifidobacteria [25].

There are inconsistencies in the definitions of equol producers. For instance, some researchers used the minimum detectable levels of either serum or urinary equol [22, 26–28], which could vary with type of measurement. Some researchers defined equol producers with a cut-off value for either serum or urinary equol [29–32]. Some others recommended using the precursor and product ratio for accuracy. For example, Setchell (2006) et al. defined equol producers as those having a log10-transformed urinary equol to daidzein ratio of -1.75 and above after a soy challenge [33].

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 [34]. There, they found out that those having a log10-transformed urinary equol to daidzein ratio of -1.42 and above can be classified as equol producers. 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. In this study, we aimed to examine cut-off value of equol producers, the relationship of blood and urinary equol, as well as their relationships with other parameters, such as sex, age, endocrine function, glucose metabolism, lipid metabolism, and renal function, in both sexes among the different age groups.

Materials and methods

Participants

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.

Ethical considerations

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.

Isoflavone measurements

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 [35, 36]. Therefore, it has been used extensively in quantitative measurements of isoflavones in several studies [37–41]. Based on a previous epidemiological study in 4,412 Japanese women [34], equol producers were defined as those whose log ratio of urinary equol and daidzein concentration or log (equol/daidzein) was -1.42 or higher.

Metabolic parameters

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).

Statistical analysis

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.

Results

Evaluation of equol producer status

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 [34]. The log10-transformed ratios of urinary equol to daidzein concentrations were plotted as shown in Fig 1 across all participants (n = 466), as well as in men (n = 292) and women (n = 174) separately. In all distributions, we observed that the cut-off values were approximately -1.4, without significant distinction between men and women. Based on this definition, among 466 participants, 195 were equol producers (42%). The proportion of equol producers in women was 47%, whereas that in men was 39%.

Fig 1 — 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.

Comparisons of parameters between men and women participants

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.

Table 1. Comparisons of parameters between men and women participants.

	Men (n = 292)		Women (n = 174)		p value
Equol producers	114	(39%)	82	(47%)	0.136^a
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

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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 ^a: chi-squared test; statistically significant p values are bold. <LOD = lower than the limit of detection, HOMA-IR = Homeostatic Model Assessment for Insulin Resistance

Relationship between serum and urine equol levels

Fig 2 showed strong positive relationships between the serum and urine equol levels in both men (r = 0.75, R² = 0.56, p<0.001) and women equol producers (r = 0.63, R² = 0.39, p<0.001). However, such a relationship was not observed in equol nonproducers in either sex (r = 0.24, R² = 0.0576, p<0.01, and r = 0.03, R² = 0.0008, p = 0.8, respectively). Equol nonproducers tended to have greater variances of urinary equol with reference to serum equol concentration, or they had almost no relationship between these two parameters. The relationship between serum and urinary equol concentrations was weaker in women equol producers, as their regression slope was lenient than that of men equol producers, although there were some individual differences.

Comparison of other parameters between equol producers and nonproducers in men

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.

Table 2. Comparison between men equol producers and nonproducers.

	Equol 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

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Continuous variables are expressed as medians (interquartile ranges) and were compared by the Mann‒Whitney U test except for ^a: chi-squared test; statistically significant p values are bold. HOMA-IR = Homeostatic Model Assessment for Insulin Resistance

Fig 3 — 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.

Comparison between equol producers and nonproducers in women

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).

Table 3. Comparison between equol producers and nonproducers in women.

	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

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Fig 4 — The proportions of abnormal values were compared using the chi-squared test.

Discussion

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 [34]. Therefore, this is the first study that could reproduce the same results in both men and women. Additionally, we found that urinary and serum equol concentrations were significantly correlated in equol producers but not in nonproducers. This also highlighted the important concept that it could be difficult to differentiate the equol producer phenotypes relying on either absolute serum or urinary equol concentrations. Therefore, Setchell et al proposed a robust method of defining an equol producer based upon the precursor-product relation using the log10-transformed ratio of urinary equol to its precursor daidzein [33].

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 [34]. By using both the precursor and product relation to determine the equol producer phenotype, we also minimized errors due to large variance in equol concentrations due to differences in dietary isoflavone intake, pharmacokinetics, and methodologies for measuring the intrinsic equol. This definition identified 195 participants among 466 as equol producers (42%), which was similar to the Japan Nurses’ Health Study on 4,412 participants that has used the same cut-off value (41.5%). The proportion of equol producers in women was larger than that in men (47% versus 39%). The lenient nature of the relationship between serum and urinary equol levels in women might have reflected the stable serum equol concentration in women.

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 [42]. These positive effects were due to the estrogenic and antioxidant action of equol.

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 [15, 16, 43, 44]. Therefore, this study has also added knowledge to equol benefits on men’s prostate health. Additionally, it was a significant finding that abnormal LDL cholesterol levels were associated with men equol nonproducers. Furthermore, men equol nonproducers tended to have abnormal HDL cholesterol and uric acid levels, which needs to be explored in larger studies.

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 [45].

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.

Conclusion

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.

Supporting information

S1 File. Consent form.

(PDF)

pone.0288946.s001.pdf^{(27.2KB, pdf)}

S2 File. Strobe checklist.

(DOCX)

pone.0288946.s002.docx^{(29.9KB, docx)}

S3 File. Cut-off values to determine abnormal results for each parameter.

(TIF)

pone.0288946.s003.tif^{(120.1KB, tif)}

S4 File. Minima data set.

(XLSX)

pone.0288946.s004.xlsx^{(166.6KB, xlsx)}

Acknowledgments

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.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Advanced Medical Care, Inc. and Himedic Medical Club gave the administrative support for this study but the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0288946.r001

Decision Letter 0

Elingarami Sauli

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

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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

3. Please include the abbreviation for PSA in the abstract.

4. Male/female and men/women are mixed in the paper. Please unify them.

5. Figure1: "Density" on the vertical axis should be changed to an appropriate term.

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.

7. Table1：Please also indicate the median age.

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.

9. Table3: Please indicate the median age of each of the producers and non-producers.

Reviewer #2: Thank you very much for allowing me to review this important research undertaking. I have some comments:

Introduction:

1. The first paragraph only accounts for a single reference. I wonder if all those information were all lifted from the same source.

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.

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. Absence of the information is critical to investigate potential relationship between equol phenotype and those outcomes.

Other points:

Why blood isoflavone levels should be corrected by creatinine?

Significant digits should be unified through the texts and tables.

The authors stated “However, it would not affect the results of this epidemiological study significantly”, but this does not make sense.

Figure 3A should be with error bars.

In Figure 3B and Figure 4, numbers in each category and p-values should be given.

**********

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

**********

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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.

PLoS One. 2024 Mar 27;19(3):e0288946. doi: 10.1371/journal.pone.0288946.r002

Author response to Decision Letter 0

29 Jan 2024

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Thank you for your advice. We will deposit the raw data there when the manuscript was accepted.

Thank you for your advice. We have added those points in the ethical consideration section.

Thank you for your advice. We have added those points in the submission form.

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Thank you for your advice. We have added those points in the cover letter.

Thank you for your advice. We will deposit the raw data there when the manuscript was accepted.

Thank you for your advice. We have added the data set as Supporting information.

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

Major points

・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.

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:

1. The first paragraph only accounts for a single reference. I wonder if all those information were all lifted from the same source.

Thank you for your insightful advice. References were added in the first paragraph of the introduction.

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:

Thank you for your valuable advice. We have addressed the points in the discussion section.

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.

Background of participants is highly unclear.

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.

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

________________________________________

Attachment

Submitted filename: Response to reviewers_PONE-D-23-20874.docx

pone.0288946.s005.docx^{(30.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0288946.r003

Decision Letter 1

Elingarami Sauli

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,

==============================

Be sure to include the following when submitting your responses to reviewer comments; proper labeling/naming of tables, figures, and concentrations. Also remember to include information on dietary habits, without forgetting proper discussion and conclusion of your findings/results.

==============================

Please submit your revised manuscript by Mar 29 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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?

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?

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?

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

**********

6. Review Comments to the Author

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 (8) means prior to the author names.

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 (2, 0). As far as I understood it the first number belongs to equol non-producers and the second number to equal producers. Furthermore, in case of “80s”, how can you provide a standard deviation when only one person belonged to the group. Please also specify which type of standard deviation is provided.

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 (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 #3: No

Reviewer #4: No

Reviewer #5: Yes: Yong Min Cho

Reviewer #6: No

**********

PLoS One. 2024 Mar 27;19(3):e0288946. doi: 10.1371/journal.pone.0288946.r004

Author response to Decision Letter 1

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.

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 [34]. Therefore, this is the first study that could reproduce the same results in both men and women. Additionally, we found that urinary and serum equol concentrations were significantly correlated in equol producers but not in nonproducers. This also highlighted the important concept that it could be difficult to differentiate the equol producer phenotypes relying on either absolute serum or urinary equol concentrations."

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 [40]. These positive effects were due to the estrogenic and antioxidant action of equol."

Background of participants is highly unclear.

"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.

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.

________________________________________

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.

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.

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. [35, 36]. Therefore, it has been used extensively in quantitative measurements of isoflavones in several studies　[37-41]."

Page 24. 3rd line. Association → relationship

Thank you for your comment. We corrected the term as you suggested.

________________________________________

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."

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.

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.

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.

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 (8) means prior to the author names.

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.

Thank you for your comment. We have corrected the points as you suggested.

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.

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

pone.0288946.s006.docx^{(34.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0288946.r005

Decision Letter 2

Elingarami Sauli

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Elingarami Sauli, PhD

Academic Editor

PLOS ONE

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PLoS One. doi: 10.1371/journal.pone.0288946.r006

Acceptance letter

Elingarami Sauli

13 Mar 2024

PONE-D-23-20874R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Consent form.

(PDF)

pone.0288946.s001.pdf^{(27.2KB, pdf)}

S2 File. Strobe checklist.

(DOCX)

pone.0288946.s002.docx^{(29.9KB, docx)}

S3 File. Cut-off values to determine abnormal results for each parameter.

(TIF)

pone.0288946.s003.tif^{(120.1KB, tif)}

S4 File. Minima data set.

(XLSX)

pone.0288946.s004.xlsx^{(166.6KB, xlsx)}

Attachment

Submitted filename: Response to reviewers_PONE-D-23-20874.docx

pone.0288946.s005.docx^{(30.8KB, docx)}

Attachment

Submitted filename: Response to reviewers_PONE-D-23-20874_V3.docx

pone.0288946.s006.docx^{(34.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

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

Remi Yoshikata

Khin Zay Yar Myint

Junichi Taguchi

Roles

Abstract

Introduction

Materials and methods

Participants

Ethical considerations

Isoflavone measurements

Metabolic parameters

Statistical analysis

Results

Evaluation of equol producer status

Fig 1. Assessing the cut-off values for Log10-transformed urinary equol: Daidzein ratios.

Comparisons of parameters between men and women participants

Table 1. Comparisons of parameters between men and women participants.

Relationship between serum and urine equol levels

Fig 2. Relationship between serum and urine equol levels in men and women participants.

Comparison of other parameters between equol producers and nonproducers in men

Table 2. Comparison between men equol producers and nonproducers.

Fig 3. Comparison of blood parameters between equol producers and nonproducers in men.

Comparison between equol producers and nonproducers in women

Table 3. Comparison between equol producers and nonproducers in women.

Fig 4. Comparison of blood parameters between equol producers and nonproducers in women.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Elingarami Sauli

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Author response to Decision Letter 0

Decision Letter 1

Elingarami Sauli

Roles

Author response to Decision Letter 1

Decision Letter 2

Elingarami Sauli

Roles

Acceptance letter

Elingarami Sauli

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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