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Vascular Sciences Research Group, Monash University Department of Medicine, Dandenong Hospital, Melbourne (H.J.T., D.G., F.S.D., B.P.M.)
Jean Hailes Research Group, Monash Institute Health Services Research, Clayton (H.J.T.)
Victoria, University of Western Australia, School of Medicine and Pharmacology, Perth, Western Australia (J.H.), AUSTRALIA
Address reprint requests to: Address correspondence to: Prof. Helena Teede, Jean Hailes Research Group, Monash Institute of Health Services Research, Level 1 Block E, Monash Medical Centre, 246 Clayton Road, Clayton 3168, Melbourne, AUSTRALIA. E-mail: helena.teede{at}med.monash.edu.au
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Design: In a 6 month double-blind, placebo controlled, cross-over trial, 41 hypertensive subjects (26 men, 15 postmenopausal women), 30–75 years, received soy cereal (40 g soy protein, 118 mg isoflavones) and gluten placebo cereal, each for 3 months.
Results: Thirty-eight subjects completed protocol with results expressed as mean or mean change (±SEM) with each intervention. Soy increased urinary isoflavones (daidzein: 8-fold; genistein: 8-fold; equol: 9-fold; ODMA: 18-fold) with no change during gluten placebo. There was no difference in the change in individual 24 hr ambulatory BP parameters (SBP: 2 ± 2 vs –1 ± 1 mmHg, p = 0.21; DBP: 1 ± 1 vs –1 ± 1 mmHg, p = 0.06) central BP (cSBP: –4 ± 2 vs 0 ± 2 mmHg, p = 0.2) or the change in arterial function (FMD: 0.3 ± 0.5 vs –0.2 ± 0.5%, p = NS; SAC: 0.02 ± 0.02 vs –0.02 ± 0.02 U/mmHg, p = NS; PWV central: –0.2 ± 0.2 vs 0.0 ± 0.2 m/sec, p = NS; PWV peripheral: 0.01 ± 0.3 vs –0.4 ± 0.4 m/sec, p = NS) noted between interventions. Analysis of the area under curve of 24 hr BP outputs demonstrated that soy protein compared to gluten protein resulted in higher 24 hr systolic BP by 2.3 mmHg (p = 0.003), a higher daytime systolic BP by 3.4 mmHg (p = 0.0002) and a higher daytime diastolic BP by 1.4 mmHg (p = 0.008). Overall 24 hr diastolic BP, night systolic BP and night diastolic BP were not significantly different between groups. Furthermore, soy protein compared to gluten protein resulted in higher 24 hr heart rates by 3.5 bpm (p < 0.0001).
Conclusions: In hypertensive subjects, compared to gluten placebo, soy dietary supplementation containing isoflavones had no effect on arterial function, on average 24 hr ambulatory blood pressure parameters or central blood pressure in men and women with hypertension. Area under the curve of 24 hr profiles demonstrated that daytime BP was higher after soy compared to gluten.
Key words: endothelial function, pulse wave velocity, arterial stiffness, soy protein, gluten, ambulatory blood pressure
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Arterial functional measures are useful surrogates of cardiovascular outcome [5,6]. PWV, an index of arterial stiffness, arguably the most robust of the non-invasive measures of arterial function, has been related to cardiovascular (CV) risk factors including hypertension and is predictive of CV events in subjects with hypertension and renal disease [5,6]. Human interventional data demonstrates a reduction in pulse wave velocity (PWV) after anti-hypertensive treatment [7]. Whilst a number of studies have suggested that isolated isoflavones may have beneficial effects on arterial function in healthy subjects [8,9], the effects of soy are inconsistent [2,3,10], have not been compared to vegetable protein sources and have not been adequately studied in hypertensive subjects.
In this double-blind, randomized, placebo-controlled, cross-over study of 6 months duration, we examined the effects of dietary soy protein supplementation in otherwise healthy hypertensive men and postmenopausal women. The primary endpoints were mean 24 h systolic and diastolic blood pressure and arterial functional assessment [systemic arterial compliance (SAC), pulse wave velocity (PWV) and brachial arterial flow-mediated vasodilation (FMD)] measured by non-invasive Doppler and tonometry ultrasound techniques.
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METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Postmenopausal status was defined as 12 months amenorrhea and FSH >20 IU/l. Exclusion criteria included moderate to severe menopausal symptoms, smoking (last 10 years), diabetes, alcohol consumption > 40 g/day, severe hypertension (>160/90), recent change in blood pressure or lipid lowering medications (these therapies had to be stable 3 months before and during the study period), abnormal uterine bleeding, cervical cytology or mammogram and co-existent major illness. The Southern Health Human Research and Ethics Committee approved the study and all participants gave written informed consent.
A medical assessment and cardiovascular risk assessment were completed at baseline 3 and 6 months [11]. Exercise information was based on specific physical activities not including household chores. Vascular functional assessments and ambulatory monitoring familiarization was completed at the screening visit. The subjects were then randomized (independently performed using computer generated random numbers) to either soy cereal or gluten placebo cereal for 3 months. Following this all 38 adherent participants were crossed over to the alternate cereal for 3 months with a total duration of 28 wks.
Participants were screened for dietary phytoestrogen intake at baseline based on history, dietary questionnaire and urinary phytoestrogen excretion. Supplements were presented in identical unmarked cereal packets and consumed daily for breakfast. Otherwise the participants were requested not to change their usual diet. The soy cereal was prepared by dehulling and defatting soybeans. All raw ingredients are passed through an extruder barrel (15–20 seconds) where chemical and physical changes occur. Water and heat are applied under high pressure to compress the material. Once the product exits the extruder, it is cut into small pieces and then dried from 12% to 3% moisture. The product was then tested prior to packaging to ensure content uniformity for protein and isoflavones and was provided in a single batch, by Specialty Cereals Pty Ltd (Mt Kuring-gai, NSW Australia). The soy cereal supplement contained 23.9% Sorghum (cereal grain), 4.4% Nutragen (soy isoflavone), 59.6% soy concentrate, 11.9% sugar and 0.18% salt. The alternate placebo equivalent contained 41.2% Sorghum, 46.2% gluten, 12.4% sugar and 0.18% salt. Each cereal packet contained powdered soy protein isolate of which 71% was protein. This product was tested for isoflavone content using HPLC with UV detection and determined to contain 2.1 mg total isoflavones per gram of protein. This was composed specifically of genestein 1.3, daidzein 0.7 and glycitein 0.1 (expressed as basic compound + respective glycosides in mg/g product). Overall this provided 40 gms of soy protein per day with a total of 118 mg isoflavones daily. This intake was selected as it provides a level of isoflavones between that of Pacific-rim nations (25–45 mg) and some areas in Japan (200 mg).
Dietary adherence was assessed by measurement of spot urine phytoestrogen concentrations at baseline and after each intervention (3 and 6 month time points). Urine isoflavones levels were normalised for creatinine, a standard methodology applied in previous work from both our group and that of other international groups involved in human interventional studies using isoflavones [2,12,13]. Height, weight, waist-hip ratio and heart rate were measured.
Assays
Isoflavone Assays.
Urinary isoflavones, including genistein and daidzein, and the daidzein metabolites O-desmethylangolensin (O-DMA) and equol were measured by HPLC [14] as previously described [2]. Samples were appropriately prepared then the HPLC separation of isoflavonoids was carried out on a Hewlett Packard 1050 Series fitted with an Alltech C18 reverse-phase column (5 µM, 250 x 4.6 mm). Isoflavones were monitored at 260 nm and 280 nm. Quantification was by comparing the area under the curve with reference standards. Samples for each subject were processed in one batch.
Hormone Assays.
Fasting morning blood samples were collected at baseline and centrifuged at 2500 gs for 12 minutes. Plasma was stored at –80°C and thawed immediately before analysis. Gonadotropins were measured on baseline bloods in women by radioimmunoassay performed on an automated MEIA using the ABBOTT AxSYM Immunoassay Analyzer (Abbott Diagnostics Division, IL 60064, USA). Standardization was against the WHO 2nd International Reference Preparation (78/549) and (80/552) for FSH and LH respectively.
Blood Pressure Measurements
24 hr Ambulatory Blood Pressure Monitoring.
The primary efficacy variables were the mean 24 hr ambulatory systolic (ASBP) and diastolic blood pressures (ADBP). Ambulatory blood pressure monitoring was performed using a portable lightweight device (Accutracker, Suntech Medical Instruments, Model II, Raleigh, NC, USA). The accuracy of the ABPM was confirmed in each subject by simultaneous auscultation and sphygmomanometry. Patients wore the device for 26 hrs with measurements every 30 minutes during the day and hourly overnight. All blood pressure readings and ambulatory monitor fittings and interpretation were completed by an experienced research assistant (DK). Subjects received verbal and written instructions on the monitors and completed a diary to record sleep, medication, posture activity and symptoms.
Central Blood Pressure.
Surrogate central blood pressure waveforms are obtained by applanation tonometry of the right common carotid artery. Waveforms are subsequently scaled to obtain an estimate of central systolic blood pressure by linear interpolation assuming a quality of mean and diastolic blood pressures.
Arterial Parameters
All arterial parameters were measured by an experienced research assistant (DK). Studies were performed after an 8 hour fast (including avoidance of caffeine-containing drinks), in a dark, quiet, air-conditioned clinical laboratory following 10 minutes rest in the supine position. Six brachial arterial blood pressure readings were recorded at 2 minute intervals using a Dinamap device (CRITIKON 1846 SX). The first reading was disregarded, the average of the subsequent 5 readings was used for analysis.
Total Systemic Arterial Compliance.
Non-invasive determination of blood flow and pressure waveforms were applied to determine SAC as previously described [2,15]. Aortic volumetric blood flow was measured from a hand held 3.5 MHz continuous wave Doppler flow velocimeter (Multidoplex MD1, Huntleigh Technology, Cardiff, UK) at the suprasternal notch. Simultaneous driving pressure was ascertained by applanation tonometry with a pressure transducer (Millar Mikro-tip, Millar Instruments Houston, Texas, USA) over the carotid artery, with pressures calibrated against Dinamap brachial artery pressures (CRITIKON 1846 SX). Given that SAC varies with changes in blood pressure, we corrected for this by standardizing SAC values at the mean blood pressure level for each individual. Compliance over the total systemic arterial tree was calculated by the following formula according to the method of Liu et al [16].
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Pulse Wave Velocity.
PWV was determined from recorded pressure waveforms over both the aorto-femoral (A-F) and the femoro-dorsalis (F-D) arterial segments [6,15]. Pulse transit time was defined as the time between the foot of simultaneously recorded pressure waves, occurring at the end of diastole and the beginning of systole, averaged over 10 cardiac cycles. Velocity was derived from computer generated pulse transit times and measured distances between the two recording sites, as previously described [6,15]. PWV was calculated based on the formula:
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t = time interval.
Brachial Artery Flow Mediated Vasodilation.
Brachial artery diameter was measured from B-mode ultrasound images captured on a Diasonics DRF-400 machine using a 10-MHz transducer, whilst an ECG trace was simultaneously recorded. Longitudinal scanning identified the clearest image of the brachial artery above the elbow, with continuous scanning held for 30 seconds prior and 4 minutes after ischaemia, induced via a pneumatic tourniquet inflated around the upper arm to 40 mmHg above systolic pressure for 4 minutes. Vessel diameter was measured during systole and diastole and averaged over 5 cardiac cycles. FMD was determined as the percentage change from baseline to 60 seconds post ischaemia, the point of maximal dilation [6,15].
Repeatability.
A repeatability study of these arterial parameters using identical techniques has been reported previously [15]. Bland-Altman plots showed satisfactory repeatability for SAC, PWV(A-F), PWV(F-D) and FMD with repeatability coefficients of 9.2%, 3.2%, 5.0% and 10.3% respectively [15]. From previous studies, this trial was powered to detect changes of 4% in SAC, 3% in PWV(A-F), 2% in PWV(F-D) and 10% in FMD. Greater changes than these have been reported in postmenopausal women in response to estrogen therapy [17].
Statistical Analysis
All data except for urinary isoflavones were normally distributed. Skewed data were analysed using non-parametric statistical methods including Wilcoxen rank text and Mann Whitney U tests and the data summarized as geometric means with 95% confidence intervals (CI). The other variables were summarized as arithmetic means ± 1 standard error (SE) or mean change ± 1 standard error (SE). Univariate analyses of variance (ANOVA) were completed for each of the independent variables with sex and order of treatment entered as covariates. Data from all 38 participants who completed the study protocol were included in the analyses. The levels of significance was accepted at the p 
0.05 level. Statistical calculations were performed using the SPSS statistical package version 9 (SPSS Inc, Chicago, USA). To analyse the area under the curve, over the 24 hour ambulatory monitoring period, BP was also analysed using repeated measures models. This also allowed for correlated error structures in the data and was completed using a pooled time series random effects model. The between group differences in ambulatory blood pressure and heart rate were analysed with random effects models using PROC MIXED (SAS 8.2 software, SAS Institute, Cary, NC, USA) [18].
A multivariate statistical analysis was performed incorporating descriptors of arterial function [SAC, PWV(A-F), PWV(F-D), FMD]. The outcome variables were entered as dependent variables in multivariate analysis of variance (MANOVA). The categorical, independent variables were treatment group (soy or gluten placebo) with sex and order of treatment entered as covariates.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Phytoestrogen Levels
Dietary phytoestrogen intake was low both at baseline and throughout the study, based on food frequency questionnaires and confirmed by urinary isoflavone excretion. Likewise, during gluten placebo there was no significant change of metabolite excretion. During soy cereals supplementation, isoflavone excretion rose significantly: genestein and daidzein excretion both increased 8 fold (p < 0.0005); equol excretion 9 fold (p < 0.0005) and ODMA excretion 18 fold (p < 0.0005).
Blood Pressure, Vascular Compliance and Endothelial Function
Fig. 1 demonstrates mean (± SEM) 24 hr ambulatory systolic (ASBP) and diastolic blood pressures (ADBP) as well as awake systolic and awake diastolic blood pressures. Ambulatory blood pressures did not change significantly during intervention periods with either intervention. Table 2 demonstrates mean (±SEM) clinic and central blood pressures before and after each intervention as well as change during interventions. Univariate ANOVA demonstrated no significant change in any individual blood pressure parameters during either active or placebo cereal supplementation (Table 2).
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Fig. 2 demonstrates mean (± SEM) systemic arterial compliance (SAC), pulse wave velocity [PWV(A-F) and PWV(F-D) and flow mediated vasodilation (FMD). Soy supplementation had no significant effect on vascular function overall, as assessed by MANOVA incorporating SAC, PWV(A-F), PWV(F-D) and FMD. On univariate ANOVA there was no significant with-in intervention group changes and no difference between change in vascular function between groups.
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DISCUSSION |
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A preliminary study in hypertensive individuals has suggested that soy combined with increased dietary fiber compared to low protein/fiber diet carbohydrate decreases BP [4].
The "Dietary Approaches to Stop Hypertension" (DASH) study showed that blood pressure levels fell with a healthy eating plan low in total fat, saturated fat, and cholesterol, and rich in fruits, vegetables, and low fat dairy products [1]. Yet it remains controversial as to whether specific dietary compounds can lower blood pressure. In healthy normotensive men and women, we have previously observed a reduction in systolic and diastolic blood pressures of 2.4, 3.9 mmHg respectively with 3 months of soy protein containing isoflavones, compared to gluten placebo [2]. These observed falls in BP were greater than those noted in the DASH study [1] and were consistent with the fall in BP reported with soy use in 51 normotensive women [19].
In this setting the current study focused on the effects of soy in a hypertensive population. We failed to demonstrate any beneficial effects of soy containing isoflavones on blood pressure in this important target group. The reasons for the discrepancies between the BP effects of soy in normotensive and hypertensive populations are unclear. They may include the fact that soy cannot overcome the neurohumoral abnormalities that occur in established hypertension or that soy effects are masked or blunted by conventional antihypertensive pharmacological agents used by 73% of the participants in the current study.
Soy protein contains vegetable protein as well as other components including isoflavones [20,21]. Soybeans are a rich source of isoflavones, which are nonsteroidal, plant-derived compounds, with binding affinity for the estrogen receptor [22] and a greater affinity demonstrated for ERß, the primary ER in the vessel wall [22,23]. Accumulating data suggests that the phytoestrogen component of soy may be responsible for specific observed vascular benefits [8,9,20,24]. Although, isoflavones in isolation appear to have little effect on blood pressure, consistent with the results of the current study. Hodgson et al reported that 8 weeks of concentrated isoflavones (55 mg/day) in tablet form, did not alter supine, erect or ambulatory blood pressure in normotensive subjects [25]. The current ambulatory blood pressure results were also analysed using repeated measures models to allow for correlated error structures in the data. This analysis took into account the repeated measures models throughout the day detecting smaller differences in BP (day, night, 24 hr) than using mean values. In this setting daytime BP was higher after soy intervention than after gluten.
The data on the effect of soy and isoflavones on arterial function is inconsistent to date [2,7,21,22]. PWV, an index of arterial stiffness, is arguably the most robust of the noninvasive measures of arterial function [6]. Non-invasive measurement of PWV with tonometry correlates with invasively obtained PWV (r = 0.85), is reproducible, accurate and is related to CV risk factors including hypertension [6,15]. It is predictive of CV events in subjects with hypertension [5,6] and appears to be independent risk factor for CVD with a direct role in the atherosclerotic process. Human interventional data demonstrates a reduction in PWV after treatment with anti-hypertensive therapy [7]. In the current study, no beneficial effect of soy on arterial compliance or central pulse wave velocity [PWV(A-F)] were observed in hypertensive individuals. In contrast two studies, using concentrated isoflavones, rather than soy, reported improved arterial function [8,24]. They involved peri and postmenopausal women and male subjects, although all were normotensive. In contrast, a larger trial in healthy men and women using soy, demonstrated only an improvement in peripheral pulse wave velocity [PWV(F-D)], possibly reflecting a reduced vasoconstriction in peripheral vessels, most likely related to the observed changes in blood pressure observed in these healthy individuals [2]. As little effect was observed on blood pressure in the current study, it is perhaps not surprising that arterial stiffness did not change. On the background of inconsistent results and given the current results further corroborative studies are needed.
Endothelial dysfunction, an early step in CVD [25], can be assessed in-vivo using brachial artery FMD. FMD, mediated by nitric oxide release, can be induced by sheer stress following transient ischaemia [26]. It has a 95% positive predictive value for coronary artery endothelial dysfunction [27]. Impaired FMD is associated with CV risk factor status [28] and is predictive of CV events [29]. An aim of the present study was to determine if dietary soy had effects on endothelial function, as assessed by brachial artery FMD. Compared to gluten placebo, soy/isoflavones had no effect on FMD in otherwise healthy hypertensive men and postmenopausal women. This is consistent with 2 recent double blind placebo controlled cross-over studies using isolated isoflavones, both noting no change in FMD [8,30]. Our previous soy protein intervention studies in healthy men and women suggested a small decline in endothelial function in men only with soy, a finding not noted in hypertensive men in the current study [2].
Conventionally soy protein has been compared to animal based casein as a protein source. In this study we selected gluten as an alternative non legume-based plant protein source to address concerns over potential differences in animal versus vegetable based proteins. Generally there was little difference in BP between the gluten and soy interventions, although on modeling overall, the area under the BP curve over 24 hrs was less with the gluten versus the soy interventions. There is no other data to suggest that gluten has specific anti-hypertensive actions.
Limitations of the current trial include that 73% of participants were on anti-hypertensive therapy, potentially interfering with mechanisms of action of soy/isoflavones. The trial included both men and women. Although no effect of sex was noted in statistical analysis, numbers in each group were small and gender effects cannot be excluded.
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CONCLUSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Funding: The study was funded by the High Blood pressure Research Council of Australia through the Australasian College of Physicians.
Authorship: HT and FD conceptualized and designed the trial, obtained ethics approval and commenced recruitment. DK recruited participants, collected data from end-points and analysed the data under the supervision of HT and BM. JH provided expertise in urine analysis of isoflavones and completed this work in his laboratory. HT, FD, JH and BM contributed to manuscript preparation.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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There is no conflict of interest to declare. The study was an independent investigator initiated and controlled trial and the company provided products only.
Received May 15, 2005. Accepted November 16, 2005.
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REFERENCES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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This article has been cited by other articles:
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  G. E. Mann, D. J. Rowlands, F. Y.L. Li, P. de Winter, and R. C.M. Siow Activation of endothelial nitric oxide synthase by dietary isoflavones: Role of NO in Nrf2-mediated antioxidant gene expression Cardiovasc Res, July 15, 2007; 75(2): 261 - 274. [Abstract] [Full Text] [PDF]
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