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Original Article

Effect of soy protein and isoflavones onblood pressureand endothelial cytokines: a 6-month randomizedcontrolled trial among postmenopausal women

Zhao-Min Liua, Suzanne C. Hob, Yu-Ming Chenc, and Jean Wooa

Journal of Hypertension 2012, 30:000–000aDepartment of Medicine &Therapeutics, bDivision of Epidemiology, The Jackey Clubof School of Public Health and Primary Care, the Chinese University of Hong Kong,Hong Kong and cDepartment of Medical Statistics and Epidemiology, School of PublicHealth, Sun Yat-sen University, Guangzhou, PR, China

Correspondence to Professor Suzanne C. Ho, MPH, PhD, 4/F, Division of Epidemiol-ogy, The Jockey Club of School of Public Health and Primary Care, the ChineseUniversity of Hong Kong, Prince of Wales Hospital, Shatin, N.T. Tel: +852 26022925;fax: +852 26016050; e-mail: [email protected]

Received 28 June 2012 Revised 3 October 2012 Accepted 29 October 2012

Background: Despite data from animal models andobservational studies that are generally supportive for thesoy/isoflavones lowering blood pressure (BP) and improvingvascular function, the current findings from clinical trialsare still inconclusive.

Objectives: To examine whether soy protein withisoflavones or isoflavones alone reduce BP and endothelialcytokines, and whether the effects differed by baselineBP level.

Methods: A double-blind randomized, placebo-controlled trial was conducted among 180postmenopausal Chinese women with mild hyperglycemia.Participants were randomly assigned to one of the threearms to receive either 15 g soy protein and 100 mgisoflavones (Soy group), or 15 g milk protein and 100 mgisoflavones (Iso group), or 15 g milk protein (placebogroup) on a daily basis for 6 months.

Results: No significant difference was observed in thechange and %change of BP and endothelial cytokinelevels among the three study groups. However, asubgroup analysis among 130 pre and hypertensivewomen suggested that soy protein and isoflavonessignificantly reduced SBP [�4.25%, 95% confidenceinterval (CI) �7.9 to �0.6%, P¼0.02] and the level ofsoluble intercellular adhesion molecule (sICAM)-1(�22.6%, 95% CI �42.8 to �2.3%, P¼0.02) relativeto milk protein after 6-month intervention.

Conclusions: Our 6-month randomized controlled trial inprediabetic postmenopausal women indicated that soyprotein and isoflavones had no significant effect on BP andendothelial molecules; however, a favorable reduction onSBP, sICAM-1 and E-selectin was observed among womenwith initial elevated BP.

Keywords: adhesion molecules, blood pressure,isoflavones, soy protein

Abbreviations: ACE, angiotensin-converting enzyme;ANCOVA, analysis of covariance; ANOVA, analysis ofvariance; CVD, cardiovascular diseases; GLM, GeneralLinear Model; RCT, randomized controlled trial; SE,standard error; sICAM, soluble intercellular adhesionmolecule; sVCAM, soluble vascular adhesion molecule

Copyright © Lippincott Williams & Wilkins. UnauthJournal of Hypertension

INTRODUCTION

An estimated 1 billion individuals are hypertensiveworldwide. Elevated blood pressure (BP) is anindependent risk factor for cardiovascular diseases

(CVD) and renal impairment [1]. The risk of CVD will bedoubled with each increment of 20/10 mmHg (SBP/DBP)from 115/75 mmHg onwards [1]. Thus, the preventionduring the early stage of hypertension will have enormouspublic health implications.

Soybean is a versatile legume that contains high-qualityprotein, minimal saturated fat, and is an essential dietarysource of isoflavones. Data from in-vitro and animal modelssuggests that soy foods or isoflavones may be cardio-protective by mechanisms independent of blood lipids[2]. The nonlipid-related effects of soy, especially its influ-ence on BP and vascular function, have become recentfocus of research [3].

In-vitro and animal experiments showed that soy/iso-flavones stimulate nitric oxide production [4,5], improvesystemic arterial compliance [4,6], and favorably affectsalt and water balance [7], which suggests a preventive rolein the progression of hypertension. Several observationalstudies also found a greater soy foods or isoflavones intakeis associated with lower BP [8,9] and stroke mortality[10]. However, the results of randomized trials have beeninconclusive; some trials have found improvements inBP or endothelial function with soy protein or isoflavones[11–15], whereas others did not [16,17]. The discrepancycould be due to the dosage of soy products, varieties ofcontrols, differing baseline status, outcome measures, etc.Most of these trials investigated the effects of specific soycomponents for a short duration or enrolled small numbers

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Liu et al.

of participants. The mechanisms involved in the alterationof BP by soy consumption have not yet been established.

Recent data have demonstrated a strong associationbetween hypertension and inflammatory process [18].Adhesion molecules such as selectin, and soluble vascularand intercellular adhesion molecules (sVCAM, sICAM) medi-ate the adhesion of leukocytes to the vascular endothelium,thereby initiating the atherosclerotic process [19]. Since thevascular adhesion molecules can be modulated by estrogen[20], it is speculated that soy isoflavones (one importantphytoestrogen) may affect the pro-inflammatory andadhesion cytokines as well. In-vitro and some animal datasuggest isoflavones decrease the levels of adhesion mole-cules and pro-inflammatory cytokines [21–23]. However,findings from human intervention studies are inconsistent.

The article reports the effects of soy protein withisoflavones and isoflavone extract on BP and adhesionmolecules in a 6-month randomized controlled trial (RCT)among 180 prediabetic postmenopausal Chinese women.We hypothesized that soy protein with isoflavones maylower BP by improving vascular inflammation with pro-nounced effect observed in pre and hypertensive women.

METHODS

Participant recruitmentParticipants were recruited from the local communitymainly by advertisements in newspaper. A total of 180postmenopausal women with pre or early diabetes wereenrolled to participate in a 6-month RCT designed to assessthe effect of soy protein and isoflavones on glycemiccontrol and cardiovascular risks. The detailed study proto-col has been reported previously [24]. In brief, participantswere Hong Kong Chinese menopausal women aged 48–70 years without menstrual cycle for at least 1 year.Individuals were excluded if they had a history of breast,uterine or ovarian cancer, or stroke, coronary heart disease(CHD), and thyroid disease in recent 5 years; were currentlyor in the preceding 3 months under medication treatmentfor weight, glucose, or lipids reduction, on hormonetherapy; or had a known allergy to soy or milk. EthicalCommittee of the Chinese University of Hong Kongapproved the study and all participants gave writteninformed consent prior to their enrollment.

Study design and intervention regimesThis is a 6-month double-blind, randomized, placebo-controlled trial. Prior to the proper study, each participanthad a 2-week adaptation period by intake of 15 g milkprotein, the placebo (Pacific Dairy Ingredients Co., Ltd,Shanghai, China). Women with good compliance and noadverse event during the run-in were randomly assignedto one of the three groups: 15 g soy proteinþ 100 mgisoflavones (Soy group), or 15 g milk proteinþ 100 mgisoflavones (Iso group), or 15 g milk protein (placebogroup). Soy protein (Solpro 931) and isoflavones (Solgen40/s) were provided by Solbar Industries Ltd, Israel. Base-line measurements were performed before randomization.The compliance was assessed mainly by counting theempty and unused sachets, and estimating the percentageof supplements consumed.

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Randomization and blindingThe study applied block randomization in block intervalof 15 for treatment assignments. In each block, threetreatments were randomly allocated for each serial numberbased on the sequence of computer-generated randomnumbers. The 180 serial numbers were labeled on thecorresponding supplements. The supplements wereassigned to participants according to the sequence of theirvisits after run-in. Participants, investigators, and laboratorytechnicians were blinded to the treatment assignmentuntil the conclusion of the trial. To assess the efficacy ofblinding, the participants were asked, at the end of theintervention, which group they thought they had beenassigned to. The proportion of participants thought beingassigned to either one of the treatments was similaramong the treatment groups, indicating a successful blind-ing efficacy (data not shown).

Supplement preparation and deliverySupplements were processed by a local pharmaceuticalcompany. The three kinds of preparation were formulatedinto iso-caloric powder with similar color, flavor, andnutrient profile (protein, fat, carbohydrate, magnesium,and calcium) by extra addition of vitamins, minerals, andother additives. The daily dose was filled into identicallooking unmarked packets. The product was tested beforepackaging by random sampling to ensure content uni-formity for protein and isoflavones. Each sachet contained25 g powdered supplements, of which 60% was protein.Isoflavones was tested using high performance liquidcromatography. Each 100 mg isoflavones, expressed inaglycone form, contain 35mg daidzin, 59mg genistin,and 4mg glycitin, with 98.5% in conjugated form. Supple-ments were suggested to be mixed with 300 ml of water,juice, milk, or adding to soft foods such as oatmeal. Womenwere asked to consume one packet daily, preferablyat breakfast.

Power of studyWith a planed number of participants of 60 in each arm(180 in total), we would have 90% power to find a4.0 mmHg reduction in SBP with the SD 10.0 mmHgfor the change in SBP, based on a convention assumptionof a level 0.05 (for a two-side test).

Data collectionData were collected by trained interviewers through face-to-face interview based on a structured and previouslyvalidated questionnaire on socio-demographic data, medi-cal history, medication treatment, dietary habits, smokingand alcohol drinking, and physical activities.

Dietary intake and physical activity assessmentDietary intake and physical activity were assessed by pre-viously validated questionnaires, as reported previously[24]. The nutrients intake was calculated based on the ChinaFood Composition Table (2004). Participants were askednot to take supplements containing phytoestrogen or otherextracts known to affect outcome measures. Participants’vitamin and mineral supplement usage were recorded at

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Effect of soy protein and isoflavones on BP and endothelial cytokines

the baseline and they were instructed to make no change inthe doses during the study period, and maintain theirhabitual diet and level of physical activity during the study.

Blood pressure measurementsBlood pressure was measured using mercury sphygmo-manometer after the participants had been seated quietlyfor 5min and the right arm supported at heart level. A cuffbladder encircling at least 80% of the arm was used toensure accuracy. Two readings were obtained with a 1-mininterval. A third BP was measured if more than 5mmHgdifference in SBP between the two readings was noted, andthe mean of the two closest was taken as the valid BP.Participants were asked not to take hypotensive agents 24 hprior to the visit for BP measurement. The prehypertensionwas defined as 120� SBP< 140 or 80�DBP< 90 andhypertension was SBP at least 140 or DBP at least90 (mmHg).

Biomarker assaysParticipants were asked to avoid nonsteroidal anti-inflammatory agents (including aspirin) 1 week beforeblood taking. sICAM-1, sVCAM-1, and E-selectin weremeasured by an ELISA assay with reagent kits purchasedfrom Raybiotech, Inc. Serum samples were diluted20-fold for measurement of sICAM-1 and E-selectin,and 50-fold for sVCAM-1. Assays for all three cytokineswere done concurrently to minimize any effects ofrepeated freeze–thaw cycles. The intra-assay coefficientsof variations for ICAM-1, VCAM-1, and E-selectin were8.7, 10.2, and 8.3%, respectively. Dilution curves of samplesparalleled those of the standards.

Anthropometric measurementsBody weight and height were measured by standardmethods. The measures for waist and hip circumferences,BMI, and waist-to-hip ratio (WHR) were reported pre-viously [24]. All measurements were performed twice andthe average was recorded.

Statistical analysisStatistical analysis was performed with the use of SPSS 13.0software (SPSS Inc., Chicago, Illinois, USA) and the signifi-cance level set at 0.05 for two-tailed P value. For skewedvariables (all markers of endothelial cytokines), we usedlog-transformed values in all analyses and reported arith-metic means and SD. Participants’ baseline characteristics,dietary intake, and physical activity were comparedamong the three study groups in both normotensive andpre or hypertensive women. Data were analyzed accordingto an intention-to-treat principle including all 180 partici-pants. The %changes and changes in outcome measureswere calculated as (follow-up value�baseline value)�100%/baseline value. Bonferroni test was used for post-hoc multiple comparisons. To examine whether the effectswere modified by the baseline BP level, we added inter-action terms between intervention and baseline BP status tothe General Linear Model (GLM). A subgroup analysis wasthen conducted to test the effect modification by baselineBP. The results were also analyzed without inclusion


of women who were taking antihypertensive medicationsand women who have poor compliance to the intervention(defined as consuming <80% of provided supplements orabsent for data collection).

RESULTSFrom November 2007 to April 2008, a total of 180 womenwere randomized into the three groups.

Drop-out and complianceEighteen women withdrew after the run-in due to loss ofinterest or time constraint, and 14 women (7.8%) withdrewduring the study after randomization [24]. The majorreasons of dropout included gastrointestinal tract dis-comfort (n¼ 5) and too busy to return for follow-up visits(n¼ 2). There were nine dropouts observed in the placebogroup, three in the Soy group, and two in the Iso group.Adherence did not differ among the three groups with morethan 90% participants using 95.9% of the supplements.

Baseline characteristicsAt baseline, the prehypertensive and hypertensive women(n¼ 130) experienced menopause for a longer time (6.5 vs.4.6 years; P¼ 0.021), had lower total physical activities(1075.9 vs. 1323.6 mets-min/day; P¼ 0.006), were heavier(BMI 24.9 vs. 23.4 kg/cm2; P¼ 0.013), larger waist cir-cumference (84.5 vs. 80.2 cm; P¼ 0.004), higher body fatpercentage (33.0 vs. 30.0; P¼ 0.01), and higher fastingglucose (6.5 vs. 6.1 mmol/l; P¼ 0.036) compared withthe normotensive group (n¼ 50) (data not shown). Among130 prehypertensive and hypertensive women (Table 1),their baseline characteristics were comparable among thethree study groups in terms of age, years since menopause(YSM), education, family and medical history, smoking, anddietary intake. No significant change in dietary nutrientsintake and physical activities occurred in either group ofwomen during the course of the study (Table 1). Baselinelevels of BP, sVCAM-1, sICAM-1, and E-selectin were com-parable between the three groups either in whole (Table 2)or hypertensive subgroup participants (Table 3). Womenlost to follow-up (n¼ 14) or with poor compliance (n¼ 2)had similar baseline characteristics to women retained inthe study (data not shown).

Effects on blood pressure and biomarkers ofendothelial functionNo significant difference was observed in the change or%change of BP and endothelial cytokine levels amongthe three study groups at follow-up visits by analysisof variance (ANOVA) test (Table 2). To examine the effectmodification of baseline BP, we included the interactiveterms of treatment and baseline BP status into the GLM.There were marginal or significant interactions betweenbaseline BP and treatment for the 6-month change% of SBP(P< 0.01), DBP (P¼ 0.065), sICAM-1 (P¼ 0.041), sVCAM-1(P¼ 0.108), and E-selectin (P¼ 0.074).

A further subgroupanalysis inpreorhypertensivewomenindicated that the average changes in SBP at 6 months were�7.0� 7.3, �4.3� 9.1, and �2.7� 8.8mmHg, respectively,

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TABLE 1. Baseline and follow-up characteristics of normotensive and pre and hypertensive women by three treatment groupsa

Normotensive group Prehypertensive and hypertensive group

Soy group Iso groupPlacebogroup P Soy group Iso group

Placebogroup P

n 17 17 16 43 43 44

Age (years) 54.3�4.4 56.3�5.1 54.8�3.5 0.387 57.2�4.7 55.9�4.1 56.4�3.8 0.329

Menopause year (years) 3.8�3.0 6.2�5.2 3.8�3.4 0.169 6.9�5.4 6.2�5.6 6.5�4.5 0.852

Bs BMI (kg/m2) 23.6�3.8 23.9�3.4 22.6�3.2 0.524 24.3�3.8 25.1�4.0 25.3�3.2 0.394

Bs WC (cm) 80.3�8.7 81.9�9.0 78.2�9.6 0.496 84.0�9.1 84.6�10.1 84.9�7.4 0.885

Bs body fat% 30.9�7.6 30.5�6.0 28.5�7.1 0.569 31.8�6.3 33.9�8.7 33.2�5.2 0.359

Bs fasting glucose 6.3�0.6 6.1�1.2 5.9�0.7 0.407 6.5�0.8 6.4�1.2 6.5�0.9 0.990

Bs DBP (mmHg) 67.3�7.2 69.0�4.8 72.0�8.0 0.137 80.9�10.0 81.9�8.7 81.9�8.7 0.840

Bs SBP (mmHg) 107.4�6.9 107.3�7.6 109.9�9.0 0.563 136.0�14.5 133.3�10.1 131.6�14.3 0.283

Bs weight (kg) 56.1�10.2 56.8�9.3 54.4�8.8 0.750 59.5�10.1 61.6�10.4 62.1�8.7 0.439

Weight change at6 months (kg)

�0.73�1.83 �0.11�1.44 �0.54�1.26 0.486 �0.54�1.58 0.01�1.77 0.04�1.63 0.188

Baseline dietary intakeEnergy (kcal/day) 1882�681 1689�498 1895�605 0.645 1936�617 1841�670 1812�643 0.699

Protein (g/day) 88.2�53.1 64.6�24.0 85.0�50.9 0.258 88.1�34.0 81.0�27.4 81.8�41.1 0.585

Fat (g/day) 50.3�27.2 41.3�21.9 53.7�40.1 0.483 50.0�24.4 53.3�28.0 50.4�35.2 0.854

% kcal from fat 23.2�5. 5 21.4�7.2 23. 6�7.6 0.614 22.6�6.1 25.3�8.1 23. 8�6.9 0.204

Soy protein (g/day) 13.4�8.3 9.4�7.9 14.2�13.3 0.340 15.4�13.9 12.2�8.8 13.4�9.6 0.396

Isoflavones (mg/day) 30.6�17.7 21.7�17.6 32.2�32.3 0.383 36.2�30.3 28.2�20.1 31.5�21.1 0.309

Sodium (mg/day) 676.1�307.1 540.6�303.7 754.6�533.4 0.293 699.1�391.4 663.3�316.0 720.7�478.9 0.798

Dietary intake atfollow-ups in averageEnergy (kcal/day) 2105�685 2018�485 1902�589 0.425 2251�648 2267�524 1987�641 0.215

Protein (g/day) 94.6�35.8 86.2�26.2 84.3�17.5 0.312 95.4�36.2 90.5�28.6 86.3�25.9 0.310

Total fat (g/day) 70.5�28.0 71.2�22.8 67.1�20.1 0.466 71.9�29.3 72.4�28.5 67.7�19.4 0.365

Soy protein (g/day) 12.6�10.9 11.0�5.9 12.3�8.1 0.562 12.5�12.3 12.7�6.9 13.6�9.0 0.752

Energy expenditure on total physical activity (TPA) level (mets-min/day)b

Bs TPA 1385�662 1231�627 1361�535 0.736 1154�439 1058�575 1016�500 0.436

TPA at follow-up 1266�670 1228�540 1284�570 0.963 978�424 987�483 993�488 0.989

Sports (>3/week) 7 (43.7%) 8 (47.0%) 9 (56.2%) 0.706 12 (27.9%) 20 (46.5%) 12 (27.3%) 0.583

Current use of hypotensivemedication (%)

1 (5.9%) 1 (5.9%) 0 0.613 15 (34.9%) 13 (30.2%) 13 (30.2%) 0.845

Family history of hypertension 11 (64.7%) 9 (52.9%) 11 (64.7%) 0.621 29 (67.4%) 32 (74.4%) 34 (77.3%) 0.569

Ever HRT use 2 (12.5%) 6 (35.3%) 1 (6.2%) 0.075 11 (25.6%) 6 (14.0%) 10 (22.7%) 0.383

Smoking 0 0 0 0 0 0

Passive smoking 3 (18.8%) 3 (17.6%) 2 (12.5%) 0.877 9 (20.9%) 10 (23.3%) 14 (31.8%) 0.469

Regular alcohol drinking 2 (12.5%) 3 (17.6%) 1 (6.2%) 0.607 3 (7.0%) 2 (4.7%) 2 (4.5%) 0.852

Regular coffee drinking 8 (50.0%) 3 (17.6%) 5 (31.2%) 0.139 11 (25.6%) 14 (32.6%) 11 (25.0%) 0.683

Education 0.615 0.747

Primary 4 (25.0%) 5 (29.4%) 3 (18.8%) 7 (16.3%) 11 (25.6%) 12 (27.9%)

Middle school 12 (75.0%) 10 (58.8%) 12 (75.0%) 29 (67.4%) 25 (58.1%) 24 (55.8%)

University 0 (0%) 2 (11.8%) 1 (6.2%) 7 (16.3%) 7 (16.3%) 7 (16.3%)

Bs, baseline; HRT, hormone replacement therapy; TPA, total physical activity; WC, waist circumference. Regular alcohol/coffee drinking denotes alcohol/coffee consumption more thanonce per week (150 ml per time).aData are presented as mean� SD for continuous variables, or number (%) for categorical variables. ANOVA test for continuous variables and chi-squared test for categorical variables.bEnergy expenditure on physical activity (mets-min/day) was calculated by [(the amount of time spent in each activity)� (the average metabolic equivalent (METs or kcal/kg/h) of eachcategory].

Liu et al.

in the Soy, Iso, and placebo groups (Table 3). The corre-sponding net change and %change (placebo corrected) at6 months were �4.4 mmHg [95% confidence interval (CI)�8.8 to 0.01, P¼ 0.05] and�4.25% (95% CI�7.9% to�0.6%,P¼ 0.02), respectively, for the Soy compared with theplacebo group. Baseline SBP and DBP were significantlyinversely correlated with the changes of SBP and DBP atfollow-up visits by soy treatment (Table 4).

The effects of the three treatments on endothelial cyto-kines are presented in Table 5. Among pre or hypertensivewomen, sICAM-1 and E-selectin levels were significantlydecreased in the Soy group compared with the placebogroup. The %change of sICAM-1 at 6 months were�9.4� 31.6, 5.1� 39.2, and 13.1� 43.7, respectively, for


the Soy, Iso, and placebo groups (P¼ 0.026). The corre-spondingnet%change in ICAM-1was�22.6% (95% CI�42.8to �2.3%, P¼ 0.02) between the Soy and placebo groups.The%changes of E-selectinwere�9.1� 16.9, 2.2� 26.0, and1.9� 19.7, respectively, in the Soy, Iso, and placebo groups(P¼ 0.029). For pre or hypertensive women, similar findingswere observed by using analysis of covariance (ANCOVA)after adjustment for basal fasting glucose, body weightchange, dietary energy, and soy intake at follow-up visits.The results also remained unaffected after excluding41 women who were taking BP-lowering agents (data notshown). The adverse events have been reported previously[24]. The number and types of adverse events did not differsignificantly among the three groups.



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TABLE 2. Blood pressure and adhesion molecules level at baseline, follow-ups and their changes or %change among 180 postmenopausalwomen by intention-to-treat analysis

Soy group (n¼60) Iso group (n¼60) Placebo group (n¼60) P

DBP (mmHg)Baseline 77.0�11.1 78.2�9.7 79.2�9.52 0.48

3-month 73.5�8.8 75.9�8.9 76.4�8.5 0.16

6-month 72.5�8.4 74.6�8.8 75.1�7.6 0.21

0–3-month change �3.6�7.1 �2.3�7.6 �2.8�6.8 0.58

0–3-month %change �3.9�9.7 �2.3�10.6 �2.7�9.6 0.65

0–6-month change �4.9�8.3 �3.7�7.8 �3.8�7.0 0.60

0–6 %change �5.5�11.5 �4.1�10.6 �4.3�9.3 0.71

SBP (mmHg)Baseline 127.9�18.2 125.9�15.1 125.8�16.2 0.74

3-month 124.8�15.7 124.5�15.0 124.0�15.4 0.96

6-month 123.4�16.7 123.1�14.9 124.3�16.5 0.91

0–3-month change �3.7�9.4 �1.4�8.9 �1.4�6.6 0.23

0–3-month %change �2.4�7.0 �0.9�6.8 �0.9�5.6 0.35

0–6-month change �4.6�8.3 �2.8�8.7 �1.8�8.6 0.21

0–6-month %change �3.7�7.3 �2.0�7.3 �1.0�7.4 0.12

sICAM-1 (ng/ml)a

Baseline 408.6�246.0 396.7�217.9 371.9�207.2 0.66

6-month 364.2�215.0 398.3�225.4 388.8�222.0 0.69

0–6 %change �6.5�33.2 3.6�38.1 7.2�42.7 0.13

sVCAM-1 (ng/ml)a

Baseline 543.7�243.8 556.0�208.5 551.8�196.1 0.95

6-month 532.7�262.0 540.9�207.7 519.6�224.7 0.88

0–6 %change 1.0�38.7 2.9�36.0 �1.1�40.0 0.86

E-selectin (ng/ml)a

Baseline 30.3�9.2 28.3�9.3 29.2�9.6 0.52

6-month 27.3�9.2 27.9�10.3 28.6�9.8 0.75

0–6 %change �7.3�21.3 0.72�24.0 0.22�21.9 0.11

sICAM, soluble intercellular adhesion molecule; sVCAM, soluble vascular adhesion molecule.aThe levels of sICAM-1, sVCAM-1, and E-selectin are log-transformed in ANOVA analyses and reported arithmetic means and SD; P value by ANOVA analysis.


DISCUSSION

This 6-month RCT in prediabetic postmenopausal womenindicated that soy protein and isoflavones had no signifi-cant effect on BP and endothelial molecules; however,

Copyright © Lippincott Williams & Wilkins. Unauth

TABLE 3. Blood pressures at baseline, 3 and 6-month follow-ups andnormotensive and pre or hypertensive women

Normotensive group

Soygroup

(n¼17)

Isogroup

(n¼17)

Placebogroup

(n¼16)

DBP (mmHg)Baseline 67.3�7.2 69.0�4.8 72.0�8.0

3-month 67.1�7.4 70.8�6.0 70.2�6.8

6-month 65.4�5.3 68.8�7.6 69.9�4.3

0–3-month change �0.1�7.1 1.8�8.2 �1.8�6.7

0–3-month %change 0.4�11.1 3.3�13.6 �1.8�10.2

0–6-month change �1.9�5.3 �0.2�8.7 �2.1�6.7

0–6 %change �2.2�8.8 0.2�14.5 �2.0�10.0

SBP (mmHg)Baseline 107.4�6.9 107.3�7.6 109.9�9.0

3-month 109.6�10.2 109.6�8.8 109.9�8.3

6-month 109.1�9.7 108.3�8.6 110.3�7.8

0–3-month change 2.2�7.7 2.3�5.8 0.0�6.8

0–3-month %change 2.1�6.9 2.2�5.5 0.3�6.3

0–6-month change 1.7�7.6 1.0�6.3 0.4�7.6

0–6-month %change 1.6�7.0 1.0�5.9 0.7�6.9

P value by ANOVA analysis and post-hoc multiple comparison by Bonferroni test.aRepresent P<0.05 by comparing with placebo group.

Journal of Hypertension

a favorable reduction on SBP and serum endothelial cyto-kines sICAM-1 and E-selectin was observed amongwomen with initial elevated BP compared with milkprotein. The subgroup findings implicated that soyprotein and isoflavones may attenuate the development


their changes and %change by three treatment groups among

Prehypertensive and hypertensive group

P

Soygroup

(n¼43)

Isogroup

(n¼43)

Placebogroup

(n¼44) P

0.14 80.9�10.0 81.9�8.7 81.9�8.7 0.84

0.25 76.1�8.0 78.0�9.1 78.6�8.0 0.34

0.08 75.4�7.7 76.8�8.2 76.9�7.7 0.58

0.37 �5.0�6.7 �3.9�6.8 �3.2�6.9 0.43

0.46 �5.7�8.6 �4.6�8.3 �3.0�9.5 0.37

0.69 �6.2�9.0 �5.0�7.1 �4.5�7.1 0.56

0.79 �6.9�12.3 �5.8�8.6 �5.1�9.3 0.70

0.56 136.0�14.5 133.3�10.1 131.6�14.3 0.28

0.99 130.8�13.3 130.4�12.6 129.1�14.2 0.84

0.80 129.0�15.5 129.0�12.6 129.4�15.9 0.99

0.56 �6.0�9.0a �2.9�9.5 �1.9�6.6 0.06

0.62 �4.1�6.3a �2.1�6.9 �1.3�5.3 0.09

0.87 �7.0�7.3a �4.3�9.1 �2.7�8.8 0.05

0.92 �5.9�6.2a �3.1�7.5 �1.6�7.5 0.02

www.jhypertension.com 5


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TABLE 4. The correlation of baseline SBP and DBP with the changes of SBP and DBP at 3 and 6-months

0–3 month change 0–6 month change

SBP DBP SBP DBP

Baseline DBPPearson correlation �0.238 �0.523 �0.221 �0.562

P 0.001 0.000 0.003 0.000

Baseline SBPPearson correlation �0.389 �0.224 �0.411 �0.220

P 0.000 0.002 0.000 0.003

Change of BP¼3 or 6-month BP – Baseline BP.

Liu et al.

of hypertension by improving vascular inflammation andendothelial function.

The study has several strengths. Whereas several studiesexamined the effect of soy or isoflavones on BP andendothelial function, this effect has not been studied inprediabetic population. This study was a strictly conductedRCT with a relatively large sample size and successfulblindness efficacy. The three kinds of supplements wereformulated into iso-caloric powder with similar nutrientsprofile, which allows the comparison of supplementationeffects only between protein source (soy or milk) andisoflavones content (with or without). Participants’ com-pliance was quite good with an attrition rate of 7.8%.Dietary intake and physical activity, the two most importantmodifiers of treatment effect, were steady across the inter-vention course, and were unlikely to confound the effectsof soy on the study outcomes. The habitual soy intake hadno significant association with the changes of outcomes in amultivariate model as discussed in our previously publishedpaper [24].

In this study, soy protein with isoflavones tended todecrease BP and vascular endothelial biomarkers in womenwith pre or hypertension, but not in normotensive individ-uals, implying that basal BP level influences the effect of soyon vascular function. We hypothesized that individuals with

Copyright © Lippincott Williams & Wilkins. Unauth

TABLE 5. The concentrations of endothelial cytokines at baseline and 6normotensive and prehypertensive womena

Normotensive group

Soygroup

(n¼17)

Isogroup

(n¼17)

Placebogroup

(n¼16)

ICAM-1 (ng/ml)Baseline 397.8�303.4 363.3�230.9 407.4�230.9 0

6-month 365.5�236.1 328.2�177.6 355.3�231.0 0

0–6 %change 1.3�37.1 0.05�36.2 �2.8�36.9 0

VCAM-1 (ng/ml)Baseline 601.2�297.7 536.7�208.1 565.1�175.5 0

6-month 480.6�255. 6 499.6�242.9 523.1�213.9 0

0–6 %change �16.8�32.4 �5.7�29.4 �7.0�27.3 0

E-selectin (ng/ml)Baseline 28.5�8.9 27.9�7.3 27.9�11.6 0

6-month 27.0�9.9 27.7�11.1 26.0�11.3 0

0–6 %change �3.1�29.0 �3.1�17.9 �4.5�27.2 0

sICAM, soluble intercellular adhesion molecule; sVCAM, soluble vascular adhesion molecule.aThe levels of sICAM-1, sVCAM-1, and E-selectin were log-transformed in ANOVA analyses andcomparison by Bonferroni test.�Represent P<0.05 by comparing with placebo group.��Represent P<0.05 by comparing with Iso group.

6 www.jhypertension.com

elevated BP and inflammatory status were more sensitiveand suitable to detect a lower BP and inflammatory effect ofsoy than those with normal BP. Our findings are consistentwith a recent meta-analysis [25] which revealed that largerreductions were identified in hypertensive women than innormotensive individuals after soy/isoflavone treatment,with a reduction in SBP of 5.9 mmHg (95% CI �10.55 to�1.34, P¼ 0.01) and in DBP of 3.4 mmHg (95% CI �6.52 to�0.19, P¼ 0.04), whereas no significant changes wereidentified in the normotensive subgroup. We further con-firmed that the changes of SBP and DBP were inverselycorrelated with baseline BP levels, suggesting that futureclinical trials focusing on patients with initially elevated BPwould have sufficient scope for a detectable improvementby soy treatment.

In this study, pre and hypertensive women had a4.2 mmHg lower SBP in the soy protein and isoflavonesgroup in comparison with the placebo group. Although themagnitude of the decrease in SBP is less than that of theantihypertensive agents (i.e. 15mmHg by calcium-blockingdrugs, 8 mmHg by angiotensin-converting enzyme inhibi-tors, or 5mmHg by b-blockers) [26], the modest decreasesin BP can also substantially reduce cardiovascular risk ifapplied to an entire population. Perspective studies havedemonstrated that a reduction in SBP of only 2mmHg may


-month visit and their %change by three treatment groups among

Prehypertensive and hypertensive group

P

Soygroup

(n¼43)

Isogroup

(n¼43)

Placebogroup

(n¼44) P

.874 412.9�223.0 410.2�213.7 358.3�198.7 0.414

.876 363.7�209.6 426.7�238.1 401.0�220.1 0.426

.728 �9.4�31.6� 5.1�39.2 13.1�43.7 0.026

.724 521.0�218.8 563.8�210.6 547.0�204.8 0.643

.880 552.5�264.7 558.1�191.99 518.3�231.2 0.697

.511 7. 8�39.1 6.5�38.2 1.1�44.0 0.728

.980 31.0�9.3 28. 5�10.0 29.7�8.7 0.465

.892 27.4�9.0 28.0�10.1 29.6�9.1 0.527

.983 �9.1�16.9�,�� 2.2�26.0 1.9�19.7 0.029

reported arithmetic means and SD. P value by ANOVA analysis, post-hoc multiple

Volume 30 � Number 00 � Month 2012


HJH 202859


already result in a 6% reduction in fatal stroke, and a 4%reduction in fatal CHD [27]. Thus, our findings on soyprotein and isoflavones may have enormous healthimplications in reduction of CVD risk if applied to thehypertensive population.

Currently, a total of eight trials addressed the relation-ship of soy/isoflavone intake and adhesion molecules,and reported inconsistent results [28]. Three of the eightstudies found statistically significant reductions in atleast one of the adhesion molecules [29–31]. One study[29] used isolated genistein and the other two used mixedisoflavones [30] and soy nuts [31]. The study by Colacurciet al. showed that Italian women taking 120 mg isoflavonesfor 6 months experienced near 20, 10, and 40% reductionsin concentration of sICAM-1, sVCAM-1, and E-selectin,respectively. Nikander et al. [32] reported that the effecton adhesion molecule was related to the isoflavonesdosage. In Hall et al.’s study [33] using 50 mg isoflavonesdaily for 3 months, sVCAM-1 concentrations were signifi-cantly decreased in participants with the estrogen receptorbALuI genotype but not in other genotypes, suggestingthat the variation of estrogen receptor may influencethe expression of adhesion molecule in response toisoflavones.

The mechanisms through which soy protein may reduceBP and circulating adhesion molecules are not fully known.One plausible hypothesis is that soy protein containshigher levels of arginine, the metabolic precursor of thepotent vasodilator nitric oxide than milk protein diets [34].Additionally, dietary soybean protein may increase insulin-receptor gene expression and reduce insulin resistancedue to a defect of insulin-receptor gene expression [35].Insulin resistance and the concomitant compensatoryhyperinsulinemia may be a major underlying pathogeneticmechanism of hypertension [35,36]. Our finding of a nileffect of purified isoflavones without soy protein on BP andadhesion molecules implied that the hypotensive effectof soy might be due to the synergistic interaction of soyprotein and isoflavones or soy protein alone [37].

Our study has several limitations. Firstly, the positivefindings of soy effects on BP and endothelial cytokinesamong pre and hypertensive women were from a non-prespecified subgroup analysis. Women in this subgroupwere not randomized accordingly and the positive effect ofsoy may be ascribed to a chance finding. However, thebaseline characteristics in pre or hypertensive women werenot significantly different among the three study groupssuggesting a good randomization balance among pre andhypertensive subgroup, which we believe may have limitedinfluence on the validity of the results of this study. Furthercontrolling of possible baseline confounders by ANCOVAmade little impact on the overall effect on BP and adhesionmolecules. With a total of 130 hypertensive women and46 or 47 women in each group, we have more than 75%power to detect a 4.0 mmHg reduction in SBP. However, weonly have less than 40% power to find a 2mmHg change inDBP. Whether the nonsignificant finding on DBP be due tothe larger standard error of the net change in DBP (2.15)than that of SBP (1.55) is still unknown. Future studieswith prespecified subgroup analysis and larger sample sizeamong hypertensive women are warranted.


Secondly, 41 of the 130 pre or hypertensive womenwere on antihypertensive agents and these may influencethe effect of soy on BP and vascular inflammation. Therelatively weak effects of soy may be masked or attenuatedby the pharmacological agents. However, the reducedscope of improvement could only increase the false-negative effect of soy treatment and would not affect thecurrent positive findings. In this study, the number (15, 13,and 13 for the Soy, Iso, and placebo groups, respectively, asshown in Table 1) and baseline characteristics of the userson antihypertensive agents were similar among the threestudy groups (data not shown). In addition, the dailydosage of hypotensive agents remained unchanged duringthe study course. We also reanalyzed the data by excludingthe 41 users in the ANOVA analysis, but little changewas observed in the outcome measures. Thus, the usageof antihypertensive medication made little influence onthe overall validity of the results.

Third, the BP measurement on a single occasion isanother limitation but even a single reading has beenshown to be a strong predictor of future CVD events[38]. Another limitation is that VCAM-1 had a marginalcoefficient of variation of 10.2% which may affect power;however, based on the current change and correspondingSD in VCAM-1 at 6 months, we had 85% power for thenonsignificant results. Finally, equol production status hasnot been assessed in our trial. There is increasing evidencethat clinical efficacy of isoflavones in humans dependson the ability to produce equol, a microbial metaboliteof daizein and is more estrogenic than daizein [39]. Equolmay be an important modifier of the effects of soy. Inaddition, the variation of the estrogen receptor genotypewould result in various efficacies from soy supplement.

In conclusion, our 6-month RCT in prediabetic post-menopausal women indicated that soy protein and iso-flavones had no significant effect on BP and endothelialmolecules; however, a favorable reduction on SBP, sICAM-1, and E-selectin was observed among women with initialpre or hypertension relative to milk protein. Future studieswith prespecified stratification analysis, observing 24 hambulatory blood pressure as primary outcome, and inconsideration of isoflavone metabolizing and estrogenreceptors phenotypes may provide better insight into therole of dietary protein in BP.

ACKNOWLEDGEMENTSWe thank all the participants and research staff of the Centerof Research and Promotion of Women’s Health for theircontribution to the study. S.C.O., Y.-M.C. and J.W. designedthe study and applied the grant; Z.-M.L. collected andanalyzed the data; and all authors contributed to manuscriptpreparation.

The authors acknowledge University Grant Council ofHong Kong for the funding support (grant ID CUHK4450/06 M) and Solbar industries Ltd (Israel) for supplying theraw materials of isoflavones and isolated soy protein forthis study.

Disclosure: All authors have no relevant conflict ofinterest to disclose, and no previous presentation of themanuscript.



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Liu et al.

Conflicts of interestThere are no personal or financial conflicts of interest.

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Reviewers’ Summary Evaluations

Referee 1This study has been carefully designed and conductedand has avoided many of the design flaws of pre-vious dietary intervention studies with soy and/orisoflavones. The report focuses on a post hoc subgroupanalysis of participants with elevated blood pressureas there were no significant changes in the primaryanalysis of blood pressure and inflammatory markers inthe cohort as a whole. As pointed out by the authorsthe results of previous studies with these interventionshave been inconsistent so this carefully designed, wellblinded study showing a negative result is a usefuladdition to the literature. The fact that there appearsto be a fall in systolic blood pressure in those who


had higher blood pressures at baseline is hypothesisgenerating.

Referee 2The current randomized controlled trial of 180 postmeno-pausal women with mild hyperglycemia identified no effectof soy protein and isoflavones on BP and endothelialmolecules compared to soy protein alone. However, sub-group analysis suggested that soy protein and isoflavonesmay lower blood pressure and sICAM-1 among women withelevated blood pressure. Trial strengths include the relativelylarge sample size, successful employment of randomizationand blinding, intention-to-treat analyses, and high compli-ance to the supplements. The post-hoc nature and limitedsample size of the subgroup analysis limit its interpretability,providing only a direction for future research efforts.
