bmj open · for peer review only 1 a randomized controlled trial of whole soy diet in place of...

For peer review only

A randomized controlled trial of whole soy diet in place of red/processed meat and high fat dairy products on

metabolic features in postmenopausal women - study protocol

Journal: BMJ Open

Manuscript ID bmjopen-2016-012741

Article Type: Protocol

Date Submitted by the Author: 23-May-2016

Complete List of Authors: Liu, Zhao-min; The Chinese University of Hong Kong , Jockey Club School of Public Health and Primary Care Ho, Suzanne; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Hao, Yuantao; Sun Yat-Sen University, School of Public Health Chen, Yu-ming Woo, J; The Chinese University of Hong Kong, Department of Medicine and Therapeutics Wong, Samuel; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care

He, Qiqiang; Wuhan University, School of Public Health Xie, Yao Jie; The Hong Kong Polytechnic University, School of Nursing Tse, LA; the Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Chen, Bailing; Sun Yat-sen University First Affiliated Hospital, Department of Spine Surgery Lao, XQ; The Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Su, Xuefen; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Chan, Ruth; The Chinese University of Hong Kong, Medicine & Therapeutics Ling, Wenhua; Sun Yat-Sen University, School of Public Health

<b>Primary Subject Heading</b>:

Nutrition and metabolism

Secondary Subject Heading: Public health

Keywords: whole soy, replacement diet, metabolic syndromes, randomized controlled trial

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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A randomized controlled trial of whole soy diet in place of

red/processed meat and high fat dairy products on metabolic

features in postmenopausal women: Study protocol

Zhao-min Liu1*, Suzanne Ho1, Yuan-tao Hao2, Yu-ming Chen2, Jean Woo3, Samuel

Yeung-shan Wong1, Qiqiang He4, Yao Jie Xie5, Lap Ah Tse1, Bailing Chen6*, Xiang-qian

Lao1, Xue-fen Su1, Ruth Chan3, Wen-hua Ling2

1 Jockey Club School of Public Health and Primary Care, the Chinese University of Hong Kong,

Hong Kong SAR ; [email protected]; [email protected];

[email protected]; [email protected]; [email protected]; [email protected];

2 School of Public Health, Sun Yat-Sen University, Guangzhou, PR, China; [email protected];

[email protected]; [email protected];

3 Department of Medicine and Therapeutics, the Chinese University of Hong Kong, Hong Kong

SAR ; [email protected]; [email protected];

4 School of Public Health, Wuhan University, Wuhan, PR, China; [email protected];

5 School of Nursing, The Hong Kong Polytechnic University, Hong Kong SAR ;

[email protected];

6 Department of Spine Surgery, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou,

PR, China; [email protected]

Correspondence:

Dr. Zhao-min Liu, Research Assistant Professor, The Jockey Club School of Public

Health and Primary Care, the Chinese University of Hong Kong, Hong Kong SAR. E-

mail: [email protected]; Tel: 852 22528750 Fax: 852 2606 3500.

Dr. Bailing Chen, Professor, Department of Spine Surgery, The First Affiliated Hospital

of Sun Yat-sen University, Guangzhou, PR, China. E-mail: [email protected]; Tel :

86 20 87755766

Word count for abstract and text: 264 words for abstract and 3079 words for text.

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Abstract

Introduction

Metabolic syndrome (MetS) is an essential public health problem in

postmenopausal women. Whole soy foods are rich in unsaturated fats, high quality plant

protein and various bioactive phytochemicals that could benefit on MetS. The aim of the

study is to examine the effect of whole soy replacement diet on the features of MetS

among postmenopausal women.

Methods and analysis

This will be a 12-month randomized, single-blind, parallel controlled trial

among 208 postmenopausal women with high risk or early MetS. After 4 weeks’ run-in,

subjects will be randomly allocated to either of two intervention groups, whole soy

replacement group or control group, each for 12 months. Subjects in whole soy group

will be required to include 4 servings of whole soy foods (containing 25g soy protein)

into their daily diet iso-calorically replacing red or processed meat and high fat dairy

products. Subjects in the control group will remain a usual diet. The outcome measures

will include metabolic parameters as well as a 10-year risk for ischemic cardiovascular

disease. We hypothesize that whole soy substitution diet will notably decrease the risk of

MetS. The study will have both theoretical and practical significance. If proven effective,

the application of whole soy replacement diet model will be a safe, practical and

economical strategy for Mets prevention.

Ethics and dissemination

Ethics approval has been obtained from the Ethics Committee of the Chinese

University of Hong Kong. The results will be disseminated via conference presentations

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and papers in academic peer reviewed journals. Data files will be deposited in an

accessible repository.

Trial registration no: NCT02610322

Ethical approval no: CRE2013.121

Keywords:

Whole soy; Replacement diet; Metabolic Syndromes; Randomized Controlled Trial.

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Strengths and limitations of this study

� The first randomized controlled trial to investigate the effect of whole soy

replacement diet on metabolic syndromes among Chinese postmenopausal women;

� Pragmatic design to facilitate implementation in daily life;

� Single-center and non-blinded design.

Authors contribution

ZML conceived and developed the idea for the study protocol. All authors

critically commented and revised the protocol.

Competing interests:

None declared.

Acknowledgement

The cost of manuscript publication in open access is supported by the Direct

Grant of the Chinese University of Hong Kong (No. 4054150).

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[Insert Running title of <72 characters]

Introduction

Epidemiology of metabolic syndromes and their prevention

Metabolic syndrome (MetS) is a constellation of interrelated metabolic risk factors

including abdominal obesity, raised fasting glucose, dyslipidemia and hypertension, which

predispose an individual to an increased risk of cardiovascular morbidity and mortality1. The

cardiovascular risk conferred by the MetS was 3 folds higher in women than it was in men2.

Menopause is a predictor of MetS independent of age3. The Guangdong (South China) Health

Survey 20104 reported the age-standardized prevalence of MetS increased 4-fold than that of

2002. Urban midlife women had the highest prevalence of MetS (33.7% in women aged 40-59

and 42.9% in women aged ≥60) according to the International Diabetes Federation (IDF)

criteria4. More than 60% of the adults had at least one component of the MetS

5. Thus, there is

urgent need to develop a population level strategy for the prevention of MetS especially among

post-menopausal women.

Non-pharmacologic approaches are the major efforts to reduce the prevalence of MetS6.

Studies have shown a diet that includes more unsaturated fats7 , dietary fiber

8 and low-fat dairy

products9 will benefit patients with MetS

10. Soy is a traditional Asia diet and a valuable source

of nutrients and phytochemicals. Soybeans contain a high-quality plant protein, a healthy

unsaturated fatty acid profile, and a good source of insoluble polysaccharides, appreciable

amounts of B vitamins and the minerals. Soybeans are also rich in various types of bioactive

phytochemicals such as isoflavones, saponin and sterol which are beneficial to human health11

.

Several large-scale observational studies suggest higher habitual soy foods intake is associated

with lower lipids profile12

, blood pressure13

and risk of type 2 diabetes14

. In vitro and animal

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experiments also suggest that soy intake has selective effects on up-regulation of genes involved

in glucose and lipid metabolism, enhances insulin sensitivity, and promotes a select loss of

visceral adipose tissue15

. However, in clinical trials, soy products are often provided as dietary

supplements, and the benefits have typically been small and inconsistent 16 17

. Soy protein isolate

and isoflavones are the mostly studied soy components in human trials. The discrepancies

between the inconsistent results of clinical trials that using individual soy components and the

generally positive results of habitual soy foods in observational studies suggest that nutrients in

supplements may not reduce risk to the same extent as the nutrients in foods. Most of traditional

Asian soy foods such as tofu, soy flour soy milk, soy nuts and dried bean curd etc. are minimally

processed and belong to whole soy foods/diet. The more noted health effect of whole soy than a

selected soy component(s) could be due to the alteration of amino acid or phytochemical

compositions as a result of complex methods for extraction of soy components thus influence the

nutritional value16

. Application of single nutrients rather than the whole food may ignore

interactions between dietary components. Recent researches on diet and chronic disease risks

have also been focused on whole foods or complete diets rather than on individual dietary

components 16

.

The evidence gap of researches on whole soy replacement diet

Whole soy diet are not only healthy food choice, but can also act indirectly on health

through the displacement of foods of lower nutritional density and quality18

. The displacement

value are unique to soy since other foods or food components are added to the diet rather than

exchanging for suboptimal foods, which makes soy a particularly valuable tool in the dietary

armamentarium to reduce cardiovascular risks16

. A recent systemic review18

using a predictive

model in NHANES Ⅲ survey data suggested that the soy consumption (13~58 g/d) had an

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additional 3.6 to 6.6% LDL-c reduction due to displacement of saturated fat and cholesterol from

animal foods. Thus, the combined intrinsic (4.3%) and extrinsic effects of soy foods on LDL-c

ranged from 7.9 to 10.3%. The American Heart Association (AHA) review also suggested that

although soy protein with isoflavones has minor effects on LDL-C (3~5%), the whole soy foods

may be beneficial to cardiovascular and overall health if used to replace fatty foods19

. Our recent

6-m randomized controlled trial among 270 pre-hypertensive equol-producing postmenopausal

women also indicated that whole soy, but not purified daidzein, had a beneficial effect on

reduction of LDL-C and inflammatory marker20

. However, the replacement effect of soy has not

been specifically examined18

. In literature, several trials21-24

used one kind of whole soy food

(tofu, soy milk or soy nuts) in substitution of one animal food (cheese, cow milk or red meat) as

treatment and all reported beneficial effects of soy on lipids. However, all the studies had

relatively small sample size (10~50) and short duration (3~8 weeks). In addition, most of the

findings are from laboratory studies, not in free-living conditions. Positive findings from a

laboratory conditions, however, provide only a theoretical maximum effect that may or may not

be achievable under free-living status. Thus, clinical trials exploring the potential displacement

value of whole soy diet and the longer-term effect are warranted 18

.

Soy foods, red or processed meat and dairy products consumption in Hong Kong

Historically, soy was deemed as ‘the meat without bones’ and ‘the meat of the field’

among the less affluent people of Asia. As Hong Kong (South China) increases its rate of

modernization and westernization, more people are likely to consume energy-dense western-

style diets (inadequate plant food and increase in red-meat and high fat dairy product etc.) while

the traditional soy foods consumption is notably reduced. A health behavior survey

commissioned by Hong Kong Health Department in 2007 indicated more than 94% Hong Kong

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women consume less than 1 serving of soy foods (tofu or soy milk etc) per day and 34.8% had

no intake of any soy foods in recent week before interview25

. The average daily intakes of soy

protein are 30g in Japan, 20g in Korea, but only 7g in Hong Kong26

. Soy consumption is even

less in elderly women27

. Our previous population-based study indicated women aged 50y above

had significant lower soy intake (29.2g/wk soy protein) than man (36.5 g/wk) in the same age or

women less than 50y (46.4g/wk)27

. A survey in 200928

reported that the average amount of red

meat intake in Hong Kong was 130g/d, 57.8% consume processed meat at least once per week,

and 65% choose full fat milk rather than low fat or skim milk. The figures fall short of the

recommendation of the Chinese Dietary Guideline29

, suggesting room for improvement.

Study aim

Thus, we propose a 12-month randomized controlled trial (RCT) among Hong Kong

postmenopausal women at risk of MetS or early MetS to examine the effect of whole soy foods

in place of red or processed meat and high fat dairy products on metabolic risk factors (central

obesity, serum lipids profile, glucose and BP etc.). We hypothesize that whole soy replacement

diet will significantly improve metabolic features in postmenopausal women with risk of MetS.


Participants

Participants will be recruited by advertisements in newspaper, health talk or referrals.

They will be initially screened via telephone or in person using a prescreening questionnaire, in

which a detailed medical history and medications will be reviewed and the risk of MetS will be

evaluated based on established risk factors of MetS (obesity, elevated BP, fasting glucose and

lipids, family history of hypertension, diabetes or hyperlipidemia, as well as physical inactivity

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etc.). Subjects who meet the initial criteria will be invited for a clinic visit to determine their

eligibility by assessment of central obesity, blood pressure, glucose and lipids etc.

Inclusion and exclusion criteria

Participants will be recruited if they are aged 45~70y within 15 years after menopause;

women at risk of MetS will be identified based on a modified National Cholesterol Education

Program Adult Treatment Panel (NCEP ATP III) criteria30

, which have considered the ethnic

difference in the definition of central obesity and the modified American Diabetes Association

criteria for impaired fasting glucose. Participants who meet 2 or more of the following items will

be enrolled (i) waist circumference (WC) ≥80 cm; (ii) triglyceride concentration ≥1.7 mmol/l; (iii)

HDL-c <50 mg/dl (1.29 mmol/l); (iv) SBP/DBP ≥130/85 mm Hg; (v) fasting glucose ≥5.6

mmol/l.

Women will be excluded if they are on use of medications known to affect body weight,

lipids and glucose within past 3-month such as hypoglycemic or hypocholestrolemic or weigh

reduction agents or hormone therapy; medical history or presence of severe systemic or

endocrine diseases such as stroke, cardiac infarction, severe liver and renal dysfunction, gout;

present or history of breast, endometrial or ovarian cancer, abnormal uterine bleeding after

menopause; on prescribed or vegetarian diet and known soy allergy.

Study design (the study flow chart please see Figure 1)

This will be a 12-month, randomized, single-blind, controlled, parallel trial. Before

randomization, a 4-week run-in exercise will be performed to make subjects familiar with the

study requirements. Subjects will take training on estimation of foods amount and fulfillment of

a 7-day dietary record, as well as 24h urine collection for the study adherence assessment.

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Subjects who are qualified in dietary record and urine collection and willing to continue after

run-in will be randomly assign to either one of two intervention groups, whole soy replacement

diet group (Whole soy group) and usual diet group (Control group), each for 12 months.

Measurements will be obtained at baseline, 6 and 12 months. Participants are free-living

during the study and prepare their own meals. The participants will be asked to maintain their

habitual physical activity and record their dietary intake for consecutive 3-day in each month

during the study. The completed dietary records will be mailed back to research center in the pre-

paid envelopes. All the participants will be in touch with the nutritionist or research staff every

month via telephone. The preceding dietary record will be assessed by the nutritionist and

discussed with the participant during the interview. Participants, who completed the study

according to the protocol, will be given a one year membership in health center as incentive.

Intervention

Participants who are assigned to the usual diet group will receive a 5~10 min

conventional lifestyle education on MetS by a research staff in which the general

recommendation for macronutrient composition of the control diet will be 50–60% of energy as

carbohydrate, 15–20% of energy as protein, and <30% of energy as total fat.

Participants who are allocated to whole soy group will receive a 30~40 min counseling

session by an experienced nutritionist on: 1) conventional lifestyle education on MetS; 2) The

benefits of whole soy diet; 2) Practical techniques to incorporate of 4 servings of whole soy

foods (equivalent to 25g soy protein) into their daily diet and reduce high saturated fat and

cholesterol rich animal foods based on their prior 7-day dietary record during run-in; (3)

Participants will also receive a pamphlet and an 30 min DVD which will provide practical

cooking recipes with both illustration and demonstration on how to prepare whole soy foods/diet

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in an easy and fun way to replace fatty animal meat and dairy products; the food composition

table with major nutrients in common soy and animal foods/products; the updated knowledge on

soy intake and women’s health; the tips on how to estimate food amounts, utensil size and record

of dietary intake; as well as a soy food exchange/replacement list applied in daily meal schedules.

Rationale of whole soy dosage

The rationale for the suggested daily whole soy amount is based on existing

epidemiologic studies and clinical trials on soy and health in which the optimal adult soy intake

is two to four servings per day31

. Participants are required to consume 4 servings of whole soy

foods (tofu, soy milk, soy flour, bean curb or soy nuts etc) per day. One serving of soy foods

contains 6.25 g soy protein. Twenty-five gram soy protein is equivalent to 300g tofu, or 600ml

soy milk, or 120g dried bean curd or 65g dried soy nuts, which will iso-calorically substitute for

high saturated fat/cholesterol animal sources foods such as red meat (pork, beef and mutton),

processed meat (bacon, sausage, roast etc.), full-fat dairy products (cow milk, cheese or yogurt

etc). Each 30 g soy nut would be exchanged as one serving of red meat32

. Dietary records will

then be coded according to the prescribed protocol and analyzed for content of energy and the

other nutrients based on Chinese Food Composition Table 2007.

Sample size planning

Based on our previous RCTs on soy, we assume whole soy replacement diet will result

in a 5 mmHg (SD of change, 10 mmHg) reduction in SBP33

, 5mg/dl (SD of 12mg/dl) reduction

in fasting glucose17

, and 2% (SD of 4%) in body weight 34

and 5% (SD of 12%) in LDL-c 35

.

Based on the below formula, 90 subjects per group will yield at least 80% power at 5% level of

significance (2-side) to detect a difference in above four metabolic components. Our previous

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RCTs on soy reported a 7.8% (RGC-CUHK 4450/06M)33

and 6.3% (RGC-GRF 465810)36

drop-

out rates and more than 90% good compliance (>80% required amount) with soy

supplementation in Chinese postmenopausal women. Thus, assuming 15% non-compliance

including drop-outs, 104 subjects per group and total 208 participants will be appropriate for the

project.

Randomization and blindness

A block randomization procedure will be used for subject allocation in a block size of 8

using the SPSS (21.0, SPSS Inc., Chicago, USA) procedure. In brief, 208 continuous serial

numbers (1-208) will be divided into 26 sub-blocks. Two treatments will be randomly allocated

to the two groups. A total 208 numbers will be assigned to eligible participants according to the

sequence of their visits after run-in. The allocation code will be placed in numbered envelopes to

be opened by the nutritionist or research staff in the presence of the participants. The statistician,

investigators, and laboratory staff who analyze the samples or conduct data collection and

analyses will be unaware of participant allocation.

Adherence assessment

Adherence will be estimated mainly based on the monthly 3-day dietary records and

24h urinary isoflavones concentration at basal and final term visits. The good compliance of

whole soy diet is defined as daily average soy protein intake more than 20 g (80% of target

amount of 25g) and urinary total isoflavones levels notably exceeding baseline level in whole

soy group.

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Code breaking and conditions for withdrawal

The principal investigator will be responsible for breaking the randomization code after

the completion of data analyses or in emergency situations (if subjects have adverse reaction

/side effects to the treatments). Conditions for withdrawal include any situation where, in the

opinion of the investigator, continuation of the study would not be in the best interest of the

subject, including but not limited to reaction or discomfort from the treatments; subjects

developed conditions or on medications as specified under exclusion criteria; or by subject

request.

Data collection/outcome measures

The primary outcome of the study is to examine the effect of whole soy replacement

diet on the metabolic components of MetS. Data collection will be performed at baseline, 6 and

12-month after treatment. Twenty-four hours urine and overnight fasting (10-12h) blood sample

will be collected at baseline and at 12-month after intervention. Plasma/serum will be isolated

within 2h after collection. Specimens will be stored at -80°C freezer until analyses. All samples

from each subject will be run in the same batch to avoid inter-assay variability. Structured

questionnaire interview and anthropometric measurements will be performed at baseline, 6 and

12-month.

1) Anthropometric measures:

Body weight, height, waist and hip circumferences will be measured according to

standard procedures. Body mass index (BMI) and waist to hip ratio (WHR) will be calculated.

Body fat percentage (BF%), fat mass (FM) and free fat mass (FFM) will be measured by a bio-

electrical impedance analyzer (TBF-410-GS Tanita Body Composition Analyzer, Japan).

2) Blood pressure (BP):

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BP will be measured twice on a standardized procedure after the participants sit for 15

min using cycling Dinamaps (GE Medical System Information Technologies, Inc, Milwaukee,

Wis) at the baseline, 6 and 12-month. Two readings will be obtained at least 1 min apart. If there

is more than 5 mm Hg difference in systolic BP between the 2 readings, a third reading will be

obtained.

3) Serum lipids and glucose levels:

A fasting blood samples (10~12h) will be collected into plain tubes and centrifuged at 4

°C and 3000 × g for 10 min to separate the serum. Fasting serum total cholesterol and

triglyceride will be measured by standardized enzymatic colorimetric methods. Serum HDL-C

and LDL-C will be measured by enzymatic clearance assay. All analyses will be performed on

automated analyzer at a certified clinical laboratory.

4) Estimation of 10-year risk for ischemic cardiovascular disease (ICVD):

The 10-year ICVD risk score will be estimated based on an established equation model

recalibrated in Chinese population37

.

Covariates and biomarkers for compliance

1) Socio-demographic data: collected by face-to-face interview based on structured and

previously validated questionnaire;

2) Habitual physical activities: collected by a modified Baecke questionnaire validated in Hong

Kong population;

3) Urinary isoflavones: will be determined by high performance liquid chromatography

(HPLC).

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Statistical analysis

The primary analysis will be an intention to treat analysis that included all subjects

who are randomized. A secondary per protocol analysis will be performed including subjects

with good compliance (defined as subjects who consumed 80% of required amounts and

completed all assessments and sample collections). The non-compliant subjects will be described

and compared to the compliant subjects. Skewed variables or variables with significant

heterogeneity will be log-transformed first. Relevant parametric and non-parametric tests will be

used for test of differences in the baseline characteristics of the two study groups. Comparisons

of changes and the percentage of change of outcome measures (MetS components) at 6 and 12

months will be made in both repeated-measures analysis of variance and paired t test analyses. If

necessary, analysis of covariance will be applied for adjusting potential confounders. All results

will be considered significant if the two-tailed P value is less than 0.05. Statistical analysis will

be performed using SPSS 21.0 software.

Discussion

The study is specifically designed among postmenopausal women at risk of MetS and

explores the effect of whole soy diet in place of high saturated fat and cholesterol rich animal

foods on metabolic features. Application of whole soy replacement diet model would be a safe,

practical, and economical diet strategy to improve metabolic diseases and cardiovascular health.

The modality may obtain more effective compliance than other dietary restrictions. If proven

effective, this diet strategy will offer an additional or alternative nutritional approach to the

prevention and management of MetS. The study will have important public health implications

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when the findings are disseminated in communities. With the increasing prevalence of MetS and

its complications in postmenopausal women, this study will explore an area with important

public health implications both locally and internationally.

Ethics and dissemination:

Written informed consent will be obtained from all participants before the intervention.

Ethics approval has been obtained from the Ethics Committee of the Chinese University of Hong

Kong (CRE2013.121). The results will be disseminated via conference presentations and papers

in academic peer reviewed journals. The protocol will be performed in accordance with the

Declaration of Helsinki. A report will be submitted to the ethics committee yearly. The scientific

committee does not require auditing for this study. Data files will be deposited in an accessible

repository.

Conflicts of Interest:

All the authors declared no conflict of interest.

Abbreviations

The following abbreviations have been used in this manuscript:

MetS: Metabolic syndromes

IDF: International Diabetes Federation

AHA: American Heart Association

BMI: Body mass index

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WHR: Waist to hip ratio

BF%: Body fat percentage

FFM: Free fat mass

BP: Blood pressure

WC: Waist circumference

NCEP ATP: National Cholesterol Education Program Adult Treatment Panel

RCT: Randomized controlled trial

ICVD: Ischemic cardiovascular disease

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and endothelial cytokines: a 6-month randomized controlled trial among postmenopausal

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34. Liu ZM, Ho SC, Chen YM, Ho YP. A mild favorable effect of soy protein with isoflavones

on body composition--a 6-month double-blind randomized placebo-controlled trial

among Chinese postmenopausal women. Int J Obes (Lond) 2010;34(2):309-18.

35. Liu ZM, Ho SC, Chen YM, Ho YP. The effects of isoflavones combined with soy protein on

lipid profiles, C-reactive protein and cardiovascular risk among postmenopausal Chinese

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36. Liu ZM, Ho SC, Chen YM, Woo J. A six-month randomized controlled trial of whole soy

and isoflavones daidzein on body composition in equol-producing postmenopausal

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37. Wu Y, Liu X, Li X, Li Y, Zhao L, Chen Z, et al. Estimation of 10-year risk of fatal and

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FormB (Rev. June/12) Page 1 of 1

Study flow of chart based on CONSORT guideline.

Subjects recruitment by advertisement, health talks and others

Subjects prescreening by questionnarie

(medical history, medication and risk factors of MetS)

Determination of subjects’ final eligibility by MetS

4-week run-in exercise

Baseline data collection and randomization

Whole soy replacement group (n=104) Control diet group (n=104)

1)Conventional education on MetS;

2)Four servings of whole soy/day;

3)Pamphlet on food recipes.

Conventional education on MetS

prevention;

Mid-trial evaluation (6-month)

Final evaluation (12-month)

Data collection, analysis and report writing

Subjects recruitment by advertisement, health talks and others

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SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*

Section/item Item No

Description Addressed on page number

Administrative information

Title 1 Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym __1___________

Trial registration 2a Trial identifier and registry name. If not yet registered, name of intended registry ___3__________

2b All items from the World Health Organization Trial Registration Data Set ___3__________

Protocol version 3 Date and version identifier In documents for

ethical approval_

Funding 4 Sources and types of financial, material, and other support __4___________

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors __4__________

5b Name and contact information for the trial sponsor __4__________

5c Role of study sponsor and funders, if any, in study design; collection, management, analysis, and

interpretation of data; writing of the report; and the decision to submit the report for publication, including

whether they will have ultimate authority over any of these activities

NA

_____________

5d Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint

adjudication committee, data management team, and other individuals or groups overseeing the trial, if

applicable (see Item 21a for data monitoring committee)

_15-6_______

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2

Introduction

Background and

rationale

6a Description of research question and justification for undertaking the trial, including summary of relevant

studies (published and unpublished) examining benefits and harms for each intervention

___5-8______

6b Explanation for choice of comparators ___5-8______

Objectives 7 Specific objectives or hypotheses ____8_______

Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group),

allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory)

___9-10______

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will

be collected. Reference to where list of study sites can be obtained

_8-9_______

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and

individuals who will perform the interventions (eg, surgeons, psychotherapists)

_9________

Interventions 11a Interventions for each group with sufficient detail to allow replication, including how and when they will be

administered

_9-11________

11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose

change in response to harms, participant request, or improving/worsening disease)

_NA_________

11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence

(eg, drug tablet return, laboratory tests)

__10________

11d Relevant concomitant care and interventions that are permitted or prohibited during the trial __NA_______

Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood

pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation

(eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen

efficacy and harm outcomes is strongly recommended

__13-15______

Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits

for participants. A schematic diagram is highly recommended (see Figure)

___NA_________

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Sample size 14 Estimated number of participants needed to achieve study objectives and how it was determined,

including clinical and statistical assumptions supporting any sample size calculations

___11-2_____

Recruitment 15 Strategies for achieving adequate participant enrolment to reach target sample size ___8-10______

Methods: Assignment of interventions (for controlled trials)

Allocation:

Sequence

generation

16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any

factors for stratification. To reduce predictability of a random sequence, details of any planned restriction

(eg, blocking) should be provided in a separate document that is unavailable to those who enrol

participants or assign interventions

__12______

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered,

opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned

__12________

Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to

interventions

_12________

Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome

assessors, data analysts), and how

__12________

17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s

allocated intervention during the trial

_13_______

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related

processes to promote data quality (eg, duplicate measurements, training of assessors) and a description

of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known.

Reference to where data collection forms can be found, if not in the protocol

__13-14________

18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be

collected for participants who discontinue or deviate from intervention protocols

___12______

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Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality

(eg, double data entry; range checks for data values). Reference to where details of data management

procedures can be found, if not in the protocol

____15___

Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of

the statistical analysis plan can be found, if not in the protocol

____15_____

20b Methods for any additional analyses (eg, subgroup and adjusted analyses) ____15______

20c Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any

statistical methods to handle missing data (eg, multiple imputation)

____15______

Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement

of whether it is independent from the sponsor and competing interests; and reference to where further

details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is

not needed

_____16_____

21b Description of any interim analyses and stopping guidelines, including who will have access to these

interim results and make the final decision to terminate the trial

_16__________

Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse

events and other unintended effects of trial interventions or trial conduct

___13__________

Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent

from investigators and the sponsor

__16______


Research ethics

approval

24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval ___16___

Protocol

amendments

25 Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes,

analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals,

regulators)

___16_______

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Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and

how (see Item 32)

___16____

26b Additional consent provisions for collection and use of participant data and biological specimens in

ancillary studies, if applicable

In documents for

ethical approval_

Confidentiality 27 How personal information about potential and enrolled participants will be collected, shared, and

maintained in order to protect confidentiality before, during, and after the trial

____16_______

Declaration of

interests

28 Financial and other competing interests for principal investigators for the overall trial and each study site ___16_______

Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that

limit such access for investigators

In documents for

ethical approval_

Ancillary and post-

trial care

30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from

trial participation

In documents for

ethical approval_

Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals,

the public, and other relevant groups (eg, via publication, reporting in results databases, or other data

sharing arrangements), including any publication restrictions

____16______

31b Authorship eligibility guidelines and any intended use of professional writers ___4_______

31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code In documents for

ethical approval_

Appendices

Informed consent

materials

32 Model consent form and other related documentation given to participants and authorised surrogates In documents for

ethical approval_

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular

analysis in the current trial and for future use in ancillary studies, if applicable

_In documents for

ethical approval__

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items.

Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons

“Attribution-NonCommercial-NoDerivs 3.0 Unported” license.

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A randomized controlled trial of whole soy diet in place of red/processed meat and high fat dairy products on features

of metabolic syndrome in postmenopausal women: Study protocol

Journal: BMJ Open

Manuscript ID bmjopen-2016-012741.R1

Article Type: Protocol

Date Submitted by the Author: 19-Aug-2016

Complete List of Authors: Liu, Zhao-min; The Chinese University of Hong Kong , Jockey Club School of Public Health and Primary Care Ho, Suzanne; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Hao, Yuantao; Sun Yat-Sen University, School of Public Health Chen, Yu-ming Woo, J; The Chinese University of Hong Kong, Department of Medicine and Therapeutics Wong, Samuel; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care

He, Qiqiang; Wuhan University, School of Public Health Xie, Yao Jie; The Hong Kong Polytechnic University, School of Nursing Tse, LA; the Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Chen, Bailing; Sun Yat-sen University First Affiliated Hospital, Department of Spine Surgery Su, Xuefen; Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Lao, XQ; The Chinese University of Hong Kong, Jockey Club School of Public Health and Primary Care Wong, Carmen; The Chinese University of Hong Kong , Jockey Club School of Public Health and Primary Care

Chan, Ruth; The Chinese University of Hong Kong, Medicine & Therapeutics Ling, Wenhua; Sun Yat-Sen University, School of Public Health

<b>Primary Subject Heading</b>:

Nutrition and metabolism

Secondary Subject Heading: Public health

Keywords: whole soy, replacement diet, metabolic syndromes, randomized controlled trial


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A randomized controlled trial of whole soy diet in place of

red/processed meat and high fat dairy products on features

of metabolic syndrome in postmenopausal women: Study

protocol

Zhao-min Liu1,2*, Suzanne Ho1, Yuan-tao Hao2, Yu-ming Chen2, Jean Woo3, Samuel

Yeung-shan Wong1, Qiqiang He4, Yao Jie Xie5, Lap Ah Tse1, Bailing Chen6*, Xue-fen

Su1, Xiang-qian Lao1, Carmen Wong1, Ruth Chan3, Wen-hua Ling2

1 Jockey Club School of Public Health and Primary Care, the Chinese University of Hong Kong,

Hong Kong SAR ; [email protected]; [email protected]; [email protected];

[email protected]; [email protected]; [email protected]; [email protected];

2 School of Public Health, Sun Yat-Sen University, Guangzhou, PR, China; [email protected];

[email protected]; [email protected];

3 Department of Medicine and Therapeutics, the Chinese University of Hong Kong, Hong Kong

SAR ; [email protected]; [email protected];

4 School of Public Health, Wuhan University, Wuhan, PR, China; [email protected];

5 School of Nursing, The Hong Kong Polytechnic University, Hong Kong SAR ;

[email protected];

6 Department of Spine Surgery, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou,

PR, China; [email protected]

Correspondence:

Dr. Zhao-min Liu, Research Assistant Professor, The Jockey Club School of Public

Health and Primary Care, the Chinese University of Hong Kong, Hong Kong SAR. E-

mail: [email protected]; Tel: 852 22528750 Fax: 852 2606 3500.

Dr. Bailing Chen, Professor, Department of Spine Surgery, The First Affiliated Hospital

of Sun Yat-sen University, Guangzhou, PR, China. E-mail: [email protected]; Tel :

86 20 87755766

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Word count for abstract and text: 264 words for abstract and 3426 words for text.

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Abstract

Introduction

Metabolic syndrome (MetS) is an essential public health problem in

postmenopausal women. Whole soy foods are rich in unsaturated fats, high quality plant

protein and various bioactive phytochemicals that could benefit on MetS. The aim of the

study is to examine the effect of whole soy replacement diet on the features of MetS

among postmenopausal women.


This will be a 12-month randomized, single-blind, parallel controlled trial

among 208 postmenopausal women with high risk or early MetS. After 4 weeks’ run-in,

subjects will be randomly allocated to either of two intervention groups, whole soy

replacement group or control group, each for 12 months. Subjects in whole soy group

will be required to include 4 servings of whole soy foods (containing 25g soy protein)

into their daily diet iso-calorically replacing red or processed meat and high fat dairy

products. Subjects in the control group will remain a usual diet. The outcome measures

will include metabolic parameters as well as a 10-year risk for ischemic cardiovascular

disease. We hypothesize that whole soy substitution diet will notably improve features of

MetS in postmenopausal women with risk of MetS or early MetS. The study will have

both theoretical and practical significance. If proven effective, the application of whole

soy replacement diet model will be a safe, practical and economical strategy for Mets

prevention and treatment.


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Ethics approval has been obtained from the Ethics Committee of the Chinese

University of Hong Kong. The results will be disseminated via conference presentations

and papers in academic peer reviewed journals. Data files will be deposited in an

accessible repository.

Trial registration no: NCT02610322

Ethical approval no: CRE2013.121

Keywords:

Whole soy; Replacement diet; Metabolic Syndrome; Randomized Controlled Trial.

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Strengths and limitations of this study

� The first randomized controlled trial to investigate the effect of whole soy

replacement diet on metabolic syndrome among Chinese postmenopausal women;

� Pragmatic design to facilitate implementation in daily life;

� Single-center and non-blinded design.

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Liu et al. Page 6


Introduction

Epidemiology of metabolic syndromes and their prevention

Metabolic syndrome (MetS) is a constellation of interrelated metabolic risk factors

including abdominal obesity, raised fasting glucose, dyslipidemia and hypertension, which

predispose an individual to an increased risk of cardiovascular morbidity and mortality1. The

cardiovascular risk conferred by the MetS was 3 folds higher in women than it was in men2.

Menopause is a predictor of MetS independent of age3. The Guangdong (South China) Health

Survey 20104 reported the age-standardized prevalence of MetS increased 4-fold than that of

2002. Urban midlife women had the highest prevalence of MetS (33.7% in women aged 40-59

and 42.9% in women aged ≥60) according to the International Diabetes Federation (IDF)

criteria4. More than 60% of the adults had at least one component of the MetS

5. Thus, there is

urgent need to develop a population level strategy for the prevention of MetS especially among

post-menopausal women.

Non-pharmacologic approaches are the major efforts to reduce the prevalence of MetS6.

Studies have shown a diet that includes less saturated fats7, but more unsaturated fats

8 , dietary

fiber9 and low-fat dairy products

10 will benefit patients with MetS

11. Red meat and full-fat dairy

products are among the main sources of saturated fat in diets. Most of epidemiological studies

reported that the consumption of low-fat instead of high-fat dairy products are favorably in

improving glycemic control12 13 and decreasing the risk of MetS 11 13 14 and diabetes15-17. Soy is a

traditional Asia diet and a valuable source of nutrients and phytochemicals. Soybeans contain a

high-quality plant protein, a healthy unsaturated fatty acid profile, and a good source of insoluble

polysaccharides, appreciable amounts of B vitamins and the minerals. Soybeans are also rich in

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various types of bioactive phytochemicals such as isoflavones, saponin and sterol which are

beneficial to human health18

.

Several large-scale observational studies suggest higher habitual soy foods intake is

associated with lower lipids profile19

, blood pressure20

and risk of type 2 diabetes21

. In vitro and

animal experiments also suggest that soy intake has selective effects on up-regulation of genes

involved in glucose and lipid metabolism, enhances insulin sensitivity, and promotes a select loss

of visceral adipose tissue7. However, in clinical trials, soy products are often provided as dietary

supplements, and the benefits have typically been small and inconsistent 22 23

. Soy protein isolate

and isoflavones are the mostly studied soy components in human trials. The discrepancies

between the inconsistent results of clinical trials that using individual soy components and the

generally positive results of habitual soy foods in observational studies suggest that nutrients in

supplements may not reduce risk to the same extent as the nutrients in foods. Most of traditional

Asian soy foods such as tofu, soy flour soy milk, soy nuts and dried bean curd etc. are minimally

processed and belong to whole soy foods/diet. The more noted health effect of whole soy than a

selected soy component(s) could be due to the alteration of amino acid or phytochemical

compositions as a result of complex methods for extraction of soy components thus influence the

nutritional value22

. Application of single nutrients rather than the whole food may ignore

interactions between dietary components. Recent researches on diet and chronic disease risks

have also been focused on whole foods or complete diets rather than on individual dietary

components 22

.

The evidence gap of researches on whole soy replacement diet

Whole soy diet are not only healthy food choice, but can also act indirectly on health

through the displacement of foods of lower nutritional density and quality24

. The displacement

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value are unique to soy since other foods or food components are added to the diet rather than

exchanging for suboptimal foods, which makes soy a particularly valuable tool in the dietary

armamentarium to reduce cardiovascular risks22

. A recent systemic review24

using a predictive

model in NHANES Ⅲ survey data suggested that the soy consumption (13~58 g/d) had an

additional 3.6 to 6.6% LDL-c reduction due to displacement of saturated fat and cholesterol from

animal foods. Thus, the combined intrinsic (4.3%) and extrinsic effects of soy foods on LDL-c

ranged from 7.9 to 10.3%. The American Heart Association (AHA) review also suggested that

although soy protein with isoflavones has minor effects on LDL-C (3~5%), the whole soy foods

may be beneficial to cardiovascular and overall health if used to replace fatty foods25

. Our recent

6-m randomized controlled trial among 270 pre-hypertensive equol-producing postmenopausal

women also indicated that whole soy, but not purified daidzein, had a beneficial effect on

reduction of LDL-C and inflammatory marker26

. However, the replacement effect of soy has not

been specifically examined24

. In literature, several trials27-30

used one kind of whole soy food

(tofu, soy milk or soy nuts) in substitution of one animal food (cheese, cow milk or red meat) as

treatment and all reported beneficial effects of soy on lipids. However, all the studies had

relatively small sample size (10~50) and short duration (3~8 weeks). In addition, most of the

findings are from laboratory studies, not in free-living conditions. Positive findings from a

laboratory conditions, however, provide only a theoretical maximum effect that may or may not

be achievable under free-living status. Thus, clinical trials exploring the potential displacement

value of whole soy diet and the longer-term effect are warranted 24

.

Soy foods, red or processed meat and dairy products consumption in Hong Kong

Historically, soy was deemed as ‘the meat without bones’ and ‘the meat of the field’

among the less affluent people of Asia. As Hong Kong (South China) increases its rate of

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modernization and westernization, more people are likely to consume energy-dense western-

style diets (inadequate plant food and increase in red-meat and high fat dairy product etc.) while

the traditional soy foods consumption is notably reduced. A health behavior survey

commissioned by Hong Kong Health Department in 2007 indicated more than 94% Hong Kong

women consume less than 1 serving of soy foods (tofu or soy milk etc) per day and 34.8% had

no intake of any soy foods in recent week before interview31

. The average daily intakes of soy

protein are 30g in Japan, 20g in Korea, but only 7g in Hong Kong32

. Soy consumption is even

less in elderly women33

. Our previous population-based study indicated women aged 50y above

had significant lower soy intake (29.2g/wk soy protein) than man (36.5 g/wk) in the same age or

women less than 50y (46.4g/wk)33

. A survey in 200934

reported that the average amount of red

meat intake in Hong Kong was 130g/d, 57.8% consume processed meat at least once per week,

and 65% choose full-fat milk rather than low fat or skim milk. The figures fall short of the

recommendation of the Chinese Dietary Guideline35

, suggesting room for improvement.

Study aim

Thus, we propose a 12-month randomized controlled trial (RCT) among Hong Kong

postmenopausal women at risk of MetS or early MetS to examine the effect of whole soy foods

in place of red or processed meat and high fat dairy products on metabolic risk factors (central

obesity, serum lipids profile, glucose and BP etc.). We hypothesize that whole soy replacement

diet will significantly improve metabolic syndrome features in postmenopausal women at risk of

MetS or early MetS.

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Participants

Women aged 45~70 y will be recruited by advertisements in newspaper, health talk or

referrals. They will be initially screened via telephone or in person using a prescreening

questionnaire, in which a detailed medical history and medications will be reviewed and the risk

of MetS will be evaluated based on established risk factors of MetS (obesity, elevated BP,

fasting glucose and lipids, family history of hypertension, diabetes or hyperlipidemia, as well as

physical inactivity etc.). Subjects who meet the initial criteria will be invited for a clinic visit to

determine their eligibility by assessment of central obesity, blood pressure, glucose and lipids etc.

Inclusion and exclusion criteria

Participants will be recruited if they are aged 45~70y within 15 years after menopause;

women at risk of MetS or early MetS will be identified based on a modified National Cholesterol

Education Program Adult Treatment Panel (NCEP ATP III) criteria36

, which have considered the

ethnic difference in the definition of central obesity and the modified American Diabetes

Association criteria for impaired fasting glucose. Participants who meet 2 or more of the

following items will be enrolled (i) waist circumference (WC) ≥80 cm; (ii) triglyceride

concentration ≥1.7 mmol/l; (iii) HDL-c <50 mg/dl (1.29 mmol/l); (iv) SBP/DBP ≥130/85 mm

Hg; (v) fasting glucose ≥5.6 mmol/l.

Women will be excluded if they are on use of medications known to affect body weight,

lipids and glucose within past 3-month such as hypoglycemic or hypocholestrolemic or weigh

reduction agents or hormone therapy; medical history or presence of severe systemic or

endocrine diseases such as thyroid disease, stroke, cardiac infarction, severe liver and renal

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dysfunction, gout; present or history of breast, endometrial or ovarian cancer, abnormal uterine

bleeding after menopause; on prescribed or vegetarian diet and known soy allergy.

Study design (the study flow chart please see Figure 1)

This will be a 12-month, randomized, single-blind, controlled, parallel trial. Before

randomization, a 4-week run-in exercise will be performed to make subjects familiar with the

study requirements. Subjects will take training on estimation of foods amount and fulfillment of

a 7-day dietary record, as well as 24h urine collection for the study adherence assessment.

Subjects who are qualified in dietary record and urine collection and willing to continue after

run-in will be randomly assign to either one of two intervention groups, whole soy replacement

diet group (Whole soy group) and usual diet group (Control group), each for 12 months.

Measurements will be obtained at baseline, 6 and 12 months. Participants are free-living

during the study and prepare their own meals. The participants will be asked to maintain their

habitual physical activity and record their dietary intake for consecutive 3-day in each month

during the study. The completed dietary records will be mailed back to research center in the pre-

paid envelopes. All the participants will be in touch with the nutritionist or research staff every

month via telephone. The preceding dietary record will be assessed by the nutritionist and

discussed with the participant during the interview. Participants, who completed the study

according to the protocol, will be given a one year membership in health center as incentive.

Intervention

Participants who are assigned to the usual diet group will receive a 5~10 min

conventional lifestyle education on MetS by a research staff in which the general

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recommendation for macronutrient composition of the control diet will be 50–60% of energy as

carbohydrate, 15–20% of energy as protein, and <30% of energy as total fat.

Participants who are allocated to whole soy group will receive a 30~40 min counseling

session by an experienced nutritionist on: 1) conventional lifestyle education on MetS; 2) The

benefits of whole soy diet; 2) Practical techniques to incorporate of 4 servings of whole soy

foods (equivalent to 25g soy protein) into their daily diet and reduce/replace high saturated fat

and cholesterol rich animal foods (including red/processed meat and full-fat dairy products)

based on their prior 7-day dietary record during run-in; (3) Participants will also receive a

pamphlet and an 30 min DVD which will provide practical cooking recipes with both illustration

and demonstration on how to prepare whole soy foods/diet in an easy and fun way to replace

fatty animal meat and dairy products; the food composition table with major nutrients in

common soy and animal foods/products; the updated knowledge on soy intake and women’s

health; the tips on how to estimate food amounts, utensil size and record of dietary intake; as

well as a detailed and practical soy food exchange/replacement list applied in daily meal

schedules.

Rationale of whole soy dosage

Soy intake recommendation is based on three considerations: Asian soy intake, clinical

and epidemiologic studies assessing the health consequences of soy consumption as well as

general principles of dietary practice37

. Existing epidemiologic studies and clinical trials on soy

and health indicate the optimal adult soy intake is two to four servings per day38

. The proposed

25g soy protein exceeds the soy protein intake of at least 90% of the Japanese and Shanghai

(China) populations but it is still within the dietary range 39 40

. Higher dosage may not be

practical and may affect the participants’ compliance. Participants are required to consume 4

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servings of whole soy foods (tofu, soy milk, soy flour, bean curb or soy nuts etc) per day. One

serving of soy foods contains 6.25 g soy protein. Twenty-five gram soy protein is equivalent to

300g tofu, or 600ml soy milk, or 120g dried bean curd or 65g dried soy nuts, which will iso-

calorically substitute for high saturated fat/cholesterol animal sources foods such as red meat

(pork, beef and mutton), processed meat (bacon, sausage, roast etc.), full-fat dairy products (cow

milk, cheese and ice-cream etc.). Each 30 g soy nut would be exchanged as one serving of red

meat41

. Dietary records will then be coded according to the prescribed protocol and analyzed for

content of energy and the other nutrients based on Chinese Food Composition Table 2007.

Sample size planning

Based on our previous RCTs on soy, we assume whole soy replacement diet will result

in a 5 mmHg (SD of change, 10 mmHg) reduction in SBP42

, 5mg/dl (SD of 12mg/dl) reduction

in fasting glucose23

, and 2% (SD of 4%) in body weight 43

and 5% (SD of 12%) in LDL-c 44

.

Based on the change and SD of change of fasting glucose or body weight (the largest ratio of

SD/change among above outcomes), 90 subjects per group will yield at least 80% power at 5%

level of significance (2-side) to detect a difference in above four metabolic components. Our

previous RCTs on soy reported a 7.8% (RGC-CUHK 4450/06M)42

and 6.3% (RGC-GRF

465810)45

drop-out rates and more than 90% good compliance (>80% required amount) with soy

supplementation in Chinese postmenopausal women. Thus, assuming 15% non-compliance

including drop-outs, 104 subjects per group and total 208 participants will be appropriate for the

project.

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Randomization and blindness

A block randomization procedure will be used for subject allocation in a block size of 8

using the SPSS (21.0, SPSS Inc., Chicago, USA) procedure. In brief, 208 continuous serial

numbers (1-208) will be divided into 26 sub-blocks. Two treatments will be randomly allocated

to the two groups. A total 208 numbers will be assigned to eligible participants according to the

sequence of their visits after run-in. The allocation code will be placed in numbered envelopes to

be opened by the nutritionist or research staff in the presence of the participants. The statistician,

investigators, and laboratory staff who analyze the samples or conduct data collection and

analyses will be unaware of participant allocation.

Adherence assessment

Adherence will be estimated mainly based on the monthly 3-day dietary records and

24h urinary isoflavones concentration at basal and final term visits. The good compliance of

whole soy diet is defined as daily average soy protein intake more than 20 g (80% of target

amount of 25g) and urinary total isoflavones levels notably exceeding baseline level in whole

soy group.

Code breaking and conditions for withdrawal

The principal investigator will be responsible for breaking the randomization code after

the completion of data analyses or in emergency situations (if subjects have adverse reaction

/side effects to the treatments). Conditions for withdrawal include any situation where, in the

opinion of the investigator, continuation of the study would not be in the best interest of the

subject, including but not limited to reaction or discomfort from the treatments; subjects

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developed conditions or on medications as specified under exclusion criteria; or by subject

request.

Data collection/outcome measures

The primary outcome of the study is to examine the effect of whole soy replacement

diet on the metabolic components of MetS. Data collection will be performed at baseline, 6 and

12-month after treatment. Twenty-four hours urine and overnight fasting (10-12h) blood sample

will be collected at baseline and at 12-month after intervention. Plasma/serum will be isolated

within 2h after collection. Specimens will be stored at -80°C freezer until analyses. All samples

from each subject will be run in the same batch to avoid inter-assay variability. Structured

questionnaire interview and anthropometric measurements will be performed at baseline, 6 and

12-month.

1) Anthropometric measures:

Body weight, height, waist and hip circumferences will be measured according to

standard procedures. Body mass index (BMI) and waist to hip ratio (WHR) will be calculated.

Body fat percentage (BF%), fat mass (FM) and free fat mass (FFM) will be measured by a bio-

electrical impedance analyzer (TBF-410-GS Tanita Body Composition Analyzer, Japan).

2) Blood pressure (BP):

BP will be measured twice on a standardized procedure after the participants sit for 15

min using cycling Dinamaps (GE Medical System Information Technologies, Inc, Milwaukee,

Wis) at the baseline, 6 and 12-month. Two readings will be obtained at least 1 min apart. If there

is more than 5 mm Hg difference in systolic BP between the 2 readings, a third reading will be

obtained.

3) Serum lipids and glucose levels:

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A fasting blood samples (10~12h) will be collected into plain tubes and centrifuged at 4

°C and 3000 × g for 10 min to separate the serum. Fasting serum glucose, total cholesterol and

triglycerides will be measured by standardized enzymatic colorimetric methods. Serum HDL-C

and LDL-C will be measured by enzymatic clearance assay. All analyses will be performed on

automated analyzer at a certified clinical laboratory.

4) Number of metabolic syndrome characteristics: The number of metabolic

characteristics (a maximum 5 for WC, BP, glucose, TG and HDL-C) will be counted

at baseline and final of the trial.

5) Estimation of 10-year risk for ischemic cardiovascular disease (ICVD):

The 10-year ICVD risk score will be estimated based on an established equation model

recalibrated in Chinese population46

.

Covariates and biomarkers for compliance and safety

1) Socio-demographic data: collected by face-to-face interview based on structured and

previously validated questionnaire;

2) Habitual physical activities: collected by a modified Baecke questionnaire validated in Hong

Kong population;

3) Urinary isoflavones: will be determined by high performance liquid chromatography

(HPLC).

4) Serum thyroid stimulating hormone (TSH): Given that soy may increase iodine requirements,

serum TSH level will be measured at the baseline and end of the trial. Serum TSH will be

measured by a standardized immunoassay.

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Statistical analysis

The primary analysis will be an intention to treat analysis that included all subjects

who are randomized. A secondary per protocol analysis will be performed including subjects

with good compliance (defined as subjects who consumed 80% of required amounts and

completed all assessments and sample collections). The non-compliant subjects will be described

and compared to the compliant subjects. Skewed variables or variables with significant

heterogeneity will be log-transformed first. Relevant parametric and non-parametric tests will be

used for test of differences in the baseline characteristics of the two study groups. Comparisons

of means of outcome measures (MetS components) at 6 and 12 months between groups will be

made using both repeated-measures analysis of variance and analysis of covariance (ANCOVA)

with baseline data as covariate. All results will be considered significant if the two-tailed P

value is less than 0.05. Statistical analysis will be performed using SPSS 21.0 software.

Discussion

The study is specifically designed among postmenopausal women at risk of MetS and

explores the effect of whole soy diet in place of high saturated fat and cholesterol rich animal

foods on features of metabolic syndrome. Application of whole soy replacement diet model

would be a safe, practical, and economical diet strategy to improve metabolic diseases and

cardiovascular health. The modality may obtain more effective compliance than other dietary

restrictions. If proven effective, this diet strategy will offer an additional or alternative nutritional

approach to the prevention and management of MetS. The study will have important public

health implications when the findings are disseminated in communities. With the increasing

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prevalence of MetS and its complications in postmenopausal women, this study will explore an

area with important public health implications both locally and internationally.

Ethics and dissemination:

Written informed consent will be obtained from all participants before the intervention.

Ethics approval has been obtained from the Ethics Committee of the Chinese University of Hong

Kong (CRE2013.121). The results will be disseminated via conference presentations and papers

in academic peer reviewed journals. The protocol will be performed in accordance with the

Declaration of Helsinki. A report will be submitted to the ethics committee yearly. The scientific

committee does not require auditing for this study. Data files will be deposited in an accessible

repository.

Conflicts of Interest:

All the authors declared no conflict of interest.

Abbreviations

The following abbreviations have been used in this manuscript:

MetS: Metabolic syndromes

IDF: International Diabetes Federation

AHA: American Heart Association

BMI: Body mass index

WHR: Waist to hip ratio

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BF%: Body fat percentage

FFM: Free fat mass

BP: Blood pressure

WC: Waist circumference

NCEP ATP: National Cholesterol Education Program Adult Treatment Panel

RCT: Randomized controlled trial

ICVD: Ischemic cardiovascular disease

Authors contribution

ZML conceived and developed the idea for the study protocol. All authors critically

commented and revised the protocol.

Competing interests:

None declared.

Acknowledgement

The cost of manuscript publication in open access is supported by the Direct Grant of

the Chinese University of Hong Kong (No. 4054150).

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18. Messina M, Messina V. The role of soy in vegetarian diets. Nutrients. 2010;2(8):855-88.

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concentrations: a cross-sectional study of 1033 pre- and postmenopausal women in the

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21. Villegas R, Gao YT, Yang G, Li HL, Elasy TA, Zheng W, et al. Legume and soy food intake

and the incidence of type 2 diabetes in the Shanghai Women's Health Study. The

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for healthcare professionals from the Nutrition Committee of the AHA. Circulation.

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23. Liu ZM, Chen YM, Ho SC. Effects of soy intake on glycemic control: a meta-analysis of

randomized controlled trials. The American journal of clinical nutrition 2011;93(5):1092-

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24. Wakai K, Egami I, Kato K, Kawamura T, Tamakoshi A, Lin Y, et al. Dietary intake and

sources of isoflavones among Japanese. Nutrition and cancer 1999;33(2):139-45.

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32. [cited]; MM. Guidelines for Healthy Soy Intake.

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45. Liu ZM, Ho SC, Chen YM, Woo J. A six-month randomized controlled trial of whole soy

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Study flow of chart based on CONSORT guideline.

187x200mm (300 x 300 DPI)

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1

SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*

Section/item Item No

Description Addressed on page number

Administrative information

Title 1 Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym __1___________

Trial registration 2a Trial identifier and registry name. If not yet registered, name of intended registry ___3__________

2b All items from the World Health Organization Trial Registration Data Set ___3__________

Protocol version 3 Date and version identifier In documents for

ethical approval_

Funding 4 Sources and types of financial, material, and other support __4___________

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors __4__________

5b Name and contact information for the trial sponsor __4__________

5c Role of study sponsor and funders, if any, in study design; collection, management, analysis, and

interpretation of data; writing of the report; and the decision to submit the report for publication, including

whether they will have ultimate authority over any of these activities

NA

_____________

5d Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint

adjudication committee, data management team, and other individuals or groups overseeing the trial, if

applicable (see Item 21a for data monitoring committee)

_15-6_______

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2

Introduction

Background and

rationale

6a Description of research question and justification for undertaking the trial, including summary of relevant

studies (published and unpublished) examining benefits and harms for each intervention

___5-8______

6b Explanation for choice of comparators ___5-8______

Objectives 7 Specific objectives or hypotheses ____8_______

Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group),

allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory)

___9-10______

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will

be collected. Reference to where list of study sites can be obtained

_8-9_______

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and

individuals who will perform the interventions (eg, surgeons, psychotherapists)

_9________

Interventions 11a Interventions for each group with sufficient detail to allow replication, including how and when they will be

administered

_9-11________

11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose

change in response to harms, participant request, or improving/worsening disease)

_NA_________

11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence

(eg, drug tablet return, laboratory tests)

__10________

11d Relevant concomitant care and interventions that are permitted or prohibited during the trial __NA_______

Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood

pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation

(eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen

efficacy and harm outcomes is strongly recommended

__13-15______

Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits

for participants. A schematic diagram is highly recommended (see Figure)

___NA_________

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Sample size 14 Estimated number of participants needed to achieve study objectives and how it was determined,

including clinical and statistical assumptions supporting any sample size calculations

___11-2_____

Recruitment 15 Strategies for achieving adequate participant enrolment to reach target sample size ___8-10______

Methods: Assignment of interventions (for controlled trials)

Allocation:

Sequence

generation

16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any

factors for stratification. To reduce predictability of a random sequence, details of any planned restriction

(eg, blocking) should be provided in a separate document that is unavailable to those who enrol

participants or assign interventions

__12______

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered,

opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned

__12________

Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to

interventions

_12________

Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome

assessors, data analysts), and how

__12________

17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s

allocated intervention during the trial

_13_______

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related

processes to promote data quality (eg, duplicate measurements, training of assessors) and a description

of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known.

Reference to where data collection forms can be found, if not in the protocol

__13-14________

18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be

collected for participants who discontinue or deviate from intervention protocols

___12______

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Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality

(eg, double data entry; range checks for data values). Reference to where details of data management

procedures can be found, if not in the protocol

____15___

Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of

the statistical analysis plan can be found, if not in the protocol

____15_____

20b Methods for any additional analyses (eg, subgroup and adjusted analyses) ____15______

20c Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any

statistical methods to handle missing data (eg, multiple imputation)

____15______

Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement

of whether it is independent from the sponsor and competing interests; and reference to where further

details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is

not needed

_____16_____

21b Description of any interim analyses and stopping guidelines, including who will have access to these

interim results and make the final decision to terminate the trial

_16__________

Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse

events and other unintended effects of trial interventions or trial conduct

___13__________

Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent

from investigators and the sponsor

__16______


Research ethics

approval

24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval ___16___

Protocol

amendments

25 Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes,

analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals,

regulators)

___16_______

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Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and

how (see Item 32)

___16____

26b Additional consent provisions for collection and use of participant data and biological specimens in

ancillary studies, if applicable

In documents for

ethical approval_

Confidentiality 27 How personal information about potential and enrolled participants will be collected, shared, and

maintained in order to protect confidentiality before, during, and after the trial

____16_______

Declaration of

interests

28 Financial and other competing interests for principal investigators for the overall trial and each study site ___16_______

Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that

limit such access for investigators

In documents for

ethical approval_

Ancillary and post-

trial care

30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from

trial participation

In documents for

ethical approval_

Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals,

the public, and other relevant groups (eg, via publication, reporting in results databases, or other data

sharing arrangements), including any publication restrictions

____16______

31b Authorship eligibility guidelines and any intended use of professional writers ___4_______

31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code In documents for

ethical approval_

Appendices

Informed consent

materials

32 Model consent form and other related documentation given to participants and authorised surrogates In documents for

ethical approval_

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular

analysis in the current trial and for future use in ancillary studies, if applicable

_In documents for

ethical approval__

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items.

Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons

“Attribution-NonCommercial-NoDerivs 3.0 Unported” license.

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