The impact of equol-producing status in modifying the effect of soya isoflavones on risk factors for CHD: a systematic review of randomised controlled trials

Recent studies suggest that the ability to produce equol, a metabolite of the soya isoflavone daidzein, is beneficial to coronary health. Equol, generated by bacterial action on isoflavones in the human gut, is biologically more potent than dietary sources of isoflavones. Not all humans are equol producers. We investigated whether equol-producing status is favourably associated with risk factors for CHD following an intervention by dietary soya isoflavones. We systematically reviewed randomised controlled trials (RCT) that evaluated the effect of soya isoflavones on risk factors for CHD and that reported equol-producing status. We searched PubMed, EMBASE, Ovid Medline and the Cochrane Central Register for Controlled Trials published up to April 2015 and hand-searched bibliographies to identify the RCT. Characteristics of participants and outcomes measurements were extracted and qualitatively analysed. From a total of 1671 studies, we identified forty-two articles that satisfied our search criteria. The effects of equol on risk factors for CHD were mainly based on secondary analyses in these studies, thus with inadequate statistical power. Although fourteen out of the forty-two studies found that equol production after a soya isoflavone intervention significantly improved a range of risk factors including cholesterol and other lipids, inflammation and blood pressure variables, these results need further verification by sufficiently powered studies. The other twenty-eight studies primarily reported null results. RCT of equol, which has recently become available as a dietary supplement, on CHD and its risk factors are awaited.

Although there are clear cardiovascular benefits of isoflavones in vitro and in animal studies (9,11) , the evidence in humans is conflicting (12)(13)(14) . A growing hypothesis is that the ability of humans to metabolise daidzein to equol, referred to as 'equol producers', may contribute to the protective effects of soya (15,16) . Equol has a greater affinity for oestrogen receptors than its precursor daidzein (17) , a longer half-life and bioavailability in plasma than daidzein and genistein (3,18) , and more potent antioxidant activity than any other isoflavone (3) . Therefore, the potential beneficial effects of soya isoflavones for CHD and its risk factors may be greater among equol producers. While all tested animals, including rodents and monkeys, can produce equol, not all humans have the gut microflora required to convert daidzein to equol, a bioactive metabolite (15,19) .
Equol is a promising candidate for hindering the initiation and progression of atherosclerosis due to its ability to induce vasorelaxation and its anti-inflammatory and antioxidant activity (20) . Specifically, it induces vasorelaxation through enhancing the production of endothelium nitric oxide synthase-derived NO (21) . It can also inhibit NO derived by inducible nitric oxide synthase, expressed by immune cells during host defence, which is linked to atherosclerosis development (22) . Furthermore, equol prevents lipid and lipoprotein peroxidation, a crucial process in the pathogenesis of atherosclerosis (23,24) .
The purpose of the present review is to examine if there is a difference in the cardioprotective effect of soya isoflavones in humans based on the hosts' ability to produce equol. No previous reviews have thoroughly examined the impact of equolproducing status on risk factors for CHD. We conducted a comprehensive search of the scientific literature to identify randomised controlled trials (RCT) that evaluated the effects of soya isoflavones on risk factors for CHD and selected studies that included analyses based on equol producer status.

Literature search
The systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (25) . We initially searched PubMed (1950 to April 2015), EMBASE through Embase.com (1966( to April 2015, Ovid Medline (1946 to April 2015) and the Cochrane Library (Cochrane Central Register of Controlled Trials, 1999 to April 2015) for papers in any language using one or more textual or medical subject heading (MESH) terms for isoflavones (isoflavones, isoflavonoids, genistein, daidzein, equol), risk factors for CHD (cardiovascular disease, coronary heart disease, myocardial infarction, lipids, low-density lipoprotein-cholesterol, triglyceride, lipoproteins, hypercholesterolemia, lipid metabolism, blood pressure, glucose, vital signs, arterial stiffness, vascular stiffness, intima-media thickness, inflammation, endothelial function, endothelium, adipocytes) and RCT (randomised control study, clinical trial, placebo, intervention studies, pilot projects, sampling studies, twin studies, prospective studies, double blind study, single blind study, epidemiologic research design). We reviewed the reference lists of the collected articles to identify additional potentially relevant papers not identified by the original keyword search.

Study selection
Studies were selected for the systematic review if they met the following criteria: (1) RCT; (2) full-text was published in English; (3) analysed adult subjects who ingested soya with isoflavones or isolated isoflavones as an intervention; (4) analysed traditional risk factors for CHD (including lipids, inflammatory, blood pressure, glycaemic and body composition variables) as outcome measurements; (5) determined the equol producer status of the participants; and (6) stratified the outcome measurements by equol producer status. The exclusion criteria included reviews or commentaries.

Data synthesis and quality assessment
Searching, data extraction and the quality assessment were completed by two authors independently according to the inclusion criteria. Discrepancies were resolved by consensus. For each RCT, extracted data included sample size, baseline characteristics of the participants (sex, mean age, health status, demographics, equol producer status), study design, treatment regimen (dose, duration, isoflavone content, and type of soya intervention), and the assessment of the risk factor(s) for CHD.
The quality of the RCT methodology was graded using a fourteen-point evaluation tool for controlled clinical trials developed by the National Heart, Lung, and Blood Institute (26) . Questions were answered with a 'yes', 'no', 'not reported', 'cannot determine' or 'not applicable'. The evaluation was based on the primary outcome measurements of the RCT. The RCT were given an overall rating of 'good', 'fair' or 'poor' at the discretion of the reviewers based on the guidelines provided by this tool.

Synthesis of results
We categorised both the effects of soya isoflavones and equol producer status on the examined CHD risk factors as beneficial, negligible, or adverse (Tables 3-8). We analysed each risk factor independently; therefore the RCT were potentially categorised more than once. Twenty-two studies found statistically significant improvements in the risk factors for CHD after the soya isoflavone intervention compared with placebo. Of these, equol producer status further improved risk factors for CHD in six studies (including LDL-C, TAG, systolic blood pressure, diastolic blood pressure, flow-mediated dilation, soluble intercellular adhesion molecule-1, platelet-selectin and C-reactive protein). Equol producer status was comparable to the soya intervention in sixteen studies (including total cholesterol, LDL-C, HDL-cholesterol (HDL-C), TAG, apoB, systolic blood pressure, diastolic blood pressure, nitrate and nitrite, systemic arterial compliance, peak flow velocity, aortic augmentation index and IL-6).
Forty studies found no association between soya isoflavones and risk factors for CHD compared with placebo. Of these, equol producer status significantly improved risk factors for CHD in seven studies (including total cholesterol, LDL-C, TAG, apoA-I, apoB, lipoprotein (a), blood pressure, diastolic blood pressure, mean arterial pressure, carotid to femoral pulse wave velocity). As with the soya isoflavone intervention, equol producer status was insignificant in thirty-two studies and was adverse in one study.
Three studies found that soya isoflavones had a negative effect on the risk factors of CHD. Of these, equol producer status was negligible in two studies and magnified the adverse outcomes of the soya isoflavone intervention in one study (isoprostane excretion). Equol producer status was also associated with the adverse outcome of an increase in insulin-like growth factor binding protein-3.
Five studies were comprised of participants who were all equol producers ( Table 8); two of the studies found statistically significant beneficial effects of the isoflavone interventions on risk factors of CHD (including LDL-C, high-sensitivity C-reactive protein, TAG, inflammatory gene expression) while four studies observed negligible effects.
The RCT varied in quality, with the overall scores provided in Table 1 and the ratings summarised in Supplementary  Table S3. Failure to report sample size calculations, details on the randomisation and allocation concealment procedures, and lack of intention-to-treat analyses or other suitable statistical method of dealing with participant drop-out were the most frequent flaws. Six RCT were given a 'good' rating, twenty were given a 'fair' rating and sixteen were given a 'poor' rating.
The heterogeneity of the studies in terms of populations, treatment regimens, intended duration, and outcomes prevented us from quantitatively synthesising the evidence in the form of a meta-analysis. Besides, the total number of participants included in all forty-two of the studies together was 3796, which, along with varying interventions and populations, probably provides insufficient statistical power to quantitatively measure the effect of dietary interventions. Further, most of these forty-two studies were small and had fewer than fifty participants, and only eighteen out of the forty-two studies qualified to be 'fair' or 'good' quality. The six 'good'-quality papers (Hodis et al. (14) ; Liu et al. (39)(40)(41) ; van der Velpen et al. (53,54) ) come from three different trialswhile the Hodis study examined carotid artery intima-media thickness  * The quality of the RCT were evaluated based on the main outcomes reported. RCT were given a score of 'good', 'fair' or 'poor' after appraising the degree to which flaws in the study designs could affect the validity of the results. † Studies that are or potentially using shared study participants. ‡ Studies that are or potentially using shared study participants. § Studies that are or potentially using shared study participants. ‖ Studies that are or potentially using shared study participants. ¶ Studies that are or potentially using shared study participants. ** Studies that are or potentially using shared study participants. † † Studies that are or potentially using shared study participants.    (27) TAG Reduction in TAG in women with MetS (17·8 %, P = 0·04) after the soya intervention compared with placebo, unlike in women without MetS    (47) Isoprostane excretion There was a positive association between isoprostane excretion and isoflavones after the high soya diet intervention (P = 0·02) -There was a positive association between isoprostane excretion and the isoflavone intervention for EP (P = 0·03) but not NEP (P = 0·32) after the high-soya diet intervention   (27) DBP Reduction in DBP in women with and without MetS (5·4 %, P = 0·03 and 3·4 %, P = 0·0008, respectively) after the soya intervention + EP with and without MetS had reduced DBP (7·7 %, P = 0·02 and 3·3 %, P = 0·02, respectively) after the soya intervention compared with placebo. There were NS effects on NEP with or without MetS in DBP     (27) CRP, sICAM-1 Reduction in CRP in women with and without MetS (11·8 %, P = 0·04 and 30 %, P = 0·01, respectively) after the soya intervention compared with placebo. In women with MetS, reduction in sICAM-1 (5·2 %, P = 0·04) compared with placebo, unlike in women without MetS + Reduced CRP (21·4 %; P = 0·01) and sICAM-1 (7·3 %, P = 0·03) in EP with MetS compared with placebo after the soya intervention. Reduced CRP (30 %; P = 0·04) in EP without MetS compared with placebo. There were NS effects on NEP with or without MetS in any of these variables  (44) Neutrophil count, DNA damage markers (AP-site assay, comet assay), apoptosis markers (TUNEL assay, caspase-3 activation) The isoflavone intervention had a NS effect on the risk factors compared with placebo 0 EP status had a NS effect on the risk factors after the isoflavone intervention. Activated caspase-3 was higher in treated EP on day 1 but decreased through day 84, while it increased in NEP in this time period  (39)(40)(41) , while van der Velpen et al. (53,54) examined the effect of soya on the expression of inflammatory genes. Given these varying outcomes, we have chosen to not perform a meta-analysis in our present review.
The majority of the studies in the present review retrospectively categorised study participants by equol producer status and conducted a secondary analysis of the effect of equol on the risk factors for CHD. Therefore, these RCT were very unlikely to be sufficiently powered to detect a difference in CHD risk factors between equol producers and non-equol producers. We identified ten studies with study designs that included enrolment criteria based on equol producer status (28,(39)(40)(41)(49)(50)(51)(52)(53)(54) . Of these, three found equol producer status improved several CHD risk factors (LDL-C, LDL-C:HDL-C, TAG, platelet-selectin and inflammatory gene expression) after the soya intervention (39,50,54) while the remaining associations measured in the RCT were negligible.
There are numerous differences in the experimental design of the RCT that could explain the inconsistency in the outcomes. The isoflavone dose ranged in both quantity and consistency between RCT. In particular, the amount of daidzein in the intervention formulations, which indicates the magnitude of equol that could be metabolised from daidzein and bioavailable in equol producers, largely varied between studies. Additionally, the duration and frequency of exposure to the intervention were inconsistent. Curtis et al. (30) found that improvements in blood pressure, mean arterial pressure, and pulse wave velocity measures in equol producers were seen after 1 year but not at 6 months, suggesting that long-term exposure to isoflavones may be more beneficial.
The criteria used to define equol producers differed across the RCT included in our review, with variability in the biological samples used to measure equol, the concentration cutoffs selected to distinguish equol producers from non-equol producers, and the analytical methods used to measure equol. Setchell & Cole (68) proposed classifying equol producers Table 5. -, adverse effect of EP status on risk factors of CHD after soya intervention.
16  (29) IGF-BP3 The isoflavone intervention had a NS effect on IGF-BP3 compared with placebo 0 Equol excretion was positively associated with IGF-BP3 concentrations in postmenopausal women at the end of the placebo phase (r 0·895; P = 0·04) and isoflavone intervention (r 0·984; P = 0·002) -IGF, insulin-like growth factor; IGF-BP1, insulin-like growth factor binding protein-1; SHBG, sex hormone binding globulin; IGF-BP3, insulin-like growth factor binding protein-3. by a threshold log 10 -transformed ratio of S-(-)equol, a diastereoisomer of equol produced by the intestinal bacteria in humans, to its precursor daidzein of −1·75 in urine after a 3 d soya isoflavone challenge. This accounts for inconsistency in the technical measurements of equol and avoids classifying equol producers based on absolute measurements of equol, which exhibit greater variability (68) . Nine studies used this approach (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)56,62,64,66) , with four finding a beneficial effect of equol producer status on risk factors of CHD (39,54,56,66) and eight finding a negligible effect (39)(40)(41)53,56,62,64,66) . Further complicating the interpretation of the data are the potential sex differences in the metabolism of soya (69) , which could   affect the bioavailability of isoflavone metabolites between males and females. In a meta-analysis examining the effects of soya isoflavones on lipids, subjects with hypercholesterolaemia had greater reductions in men than in women (12) . While there were studies of mixed sex (n 11) or of only males (n 1), the present review consisted primarily of female-only RCT, which may have masked the effects of equol producer status on the outcome measurements. Nestel et al. (60) found that LDL-C was significantly reduced after supplementation with biochanin (a precursor of genistein) compared with placebo (P = 0·026); when results were stratified based on sex, males showed a significant reduction in median LDL-C levels of 9·5 % while females had no measurable difference. Equol producer status did not further reduce LDL-C, which the authors speculated was due to the small sample size of fifteen equol producers, with seven included in the biochanin intervention group (60) . The source of soya may also contribute to the variability in its effectiveness. The type of processing used for soya products during production can affect the isoflavone content (13) and modify other components of soya (70) . Additionally, soya protein isolate primarily contains isoflavone glucosides while fermented soya foods contain isoflavones mainly in the aglycone form (15,71) . Isoflavone aglycones are absorbed more efficiently than isoflavone glucosides in humans and may therefore be more effective in CHD prevention (72) . Daidzein in the aglycone form is also more readily converted to equol (15) . Clerici et al. (56) found that pasta enriched in isoflavone aglycones significantly reduced total cholesterol, LDL-C, high-sensitivity C-reactive protein, and arterial stiffness compared with placebo in study participants, with effects more pronounced in equol producers. Of the fourteen RCT that found a positive association between equol producer status and CHD risk factors, seven used interventions of foods and milk enriched with soya (27,30,35,55,56,59,66) .
Furthermore, baseline age and the health status of the participants may contribute to variability in the outcome measurements. Oestrogen receptor β has been found to be enhanced in extracted arteries from postmenopausal CHD patients compared with normal subjects, with enhanced dilation in response to isoflavones (73) . Hodis et al. found that isoflavone supplementation failed to prevent the progression of subclinical atherosclerosis in healthy postmenopausal women overall; a subanalysis indicated, however, that healthy women within 5 years of becoming postmenopausal had a significantly reduced mean carotid artery intima-media thickness progression rate of 68 % compared with placebo (74) . Previous meta-analyses have also found lipid variables to be more positively affected by soya interventions in hypercholesterolaemic patients than in healthy subjects (12,75) . We identified thirty-five RCT that only used postmenopausal women; all of the studies that found a favourable association of equol producer status on risk factors of CHD had postmenopausal participants. There were a relatively equal number of RCT using healthy participants (n 20) v. participants with underlying health issues or a history of illness (n 22); of the fourteen studies that found equol producer status to improve risk factors for CHD, five had healthy participants (35,42,45,54,67) while nine had participants with underlying health issues related to CHD (27,30,39,50,55,56,59,61,66) .
In the present systematic review, electronic databases were extensively searched following our defined set of guidelines and used to extract relevant data. Our results may imply that equol is beneficial on cardiovascular health, yet the interpretation is limited largely because of the secondary analysis of equol producers in RCT of dietary sources of isoflavones. Recently, equol itself has become available as a dietary supplement. Orally administered equol has greater plasma accumulation than other dietary sources of isoflavones (76) and has the potential for enhanced therapeutic effects due to its more potent antioxidant properties and bioactivity among all isoflavones. In fact, one RCT of equol on risk factors of CHD has been conducted. Usui et al. (77) found a statistically significant improvement in LDL-C, glycated HbA1c levels, and cardio-ankle vascular index scores, a measure of vascular stiffness, in overweight and obese patients after dietary equol supplementation, particularly for non-equol producers. This study is limited by its small sample size and short duration of the intervention. We recommend additional RCT of equol itself as an intervention to directly assess its effects on CHD risk factors and potentially CHD.

Supplementary material
The supplementary material for this article can be found at http://dx.doi.org/10.1017/jns.2016. 18