Effects of DASH diet on lipid profiles and biomarkers of oxidative stress in overweight and obese women with polycystic ovary syndrome: A randomized clinical trial

Accepted Manuscript The effects of DASH diet on lipid profiles and biomarkers of oxidative stress in overweight and obese women with polycystic ovary syndrome: a randomised clinical trial Zatollah Asemi, Ph.D Mansooreh Samimi, M.D Zohreh Tabassi, M.D Hossein Shakeri, M.Sc Sima-Sadat Sabihi, B.Sc Ahmad Esmaillzadeh, Ph.D PII:

S0899-9007(14)00142-7

DOI:

10.1016/j.nut.2014.03.008

Reference:

NUT 9255

To appear in:

Nutrition

Received Date: 9 October 2013 Revised Date:

20 November 2013

Accepted Date: 9 March 2014

Please cite this article as: Asemi Z, Samimi M, Tabassi Z, Shakeri H, Sabihi S-S, Esmaillzadeh A, The effects of DASH diet on lipid profiles and biomarkers of oxidative stress in overweight and obese women with polycystic ovary syndrome: a randomised clinical trial, Nutrition (2014), doi: 10.1016/ j.nut.2014.03.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The effects of DASH diet on lipid profiles and biomarkers of oxidative stress in

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overweight and obese women with polycystic ovary syndrome: a randomised

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clinical trial

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Zatollah Asemi Ph.D. a, Mansooreh Samimi M.D. b, Zohreh Tabassi M.D. b, Hossein Shakeri

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M.Sc. a, Sima-Sadat Sabihi B.Sc. a, Ahmad Esmaillzadeh Ph.D. c,d,* a

Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of

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Medical Sciences, Kashan, I.R. Iran

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Department of Gynecology and Obstetrics, School of Medicine, Kashan University of Medical

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Sciences, Kashan, I.R. Iran

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Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran

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Running title: DASH diet for PCOS

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Z.A. conducted the study, carried out the statistical analyses, wrote the manuscript and

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contributed in the interpretation of the findings. M.S,Z.T, H.Sh and S-S. S contributed in data

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collection and assisted in writing the manuscript. A.E. contributed in conception and design and

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also advised on statistical analyses and assisted in interpretation of the findings. None of the

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authors had any personal or financial conflict of interest. All authors approved the final version

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for submission Clinical trial registration number:www.irct.ir:IRCT201304235623N6.

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* Corresponding author. Tel.: 98-311-7922720; fax: 98-311-6682509.

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E-mail address: [email protected] (A. Esmaillzadeh).

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Abstract

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Objective: This study was designed to assess the effects of the Dietary Approaches to Stop

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Hypertension (DASH) diet on lipid profiles and biomarkers of oxidative stress in overweight and

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obese women with polycystic ovary syndrome (PCOS).

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Methods: This randomised controlled clinical trial was done among 48women diagnosed with

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PCOS. Subjects were randomly assigned to consume either the control (n=24) or the DASH

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eating pattern (n=24) for 8 weeks. Both diets were designed to be calorie-restricted. The DASH

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and control diets were consisted of 52% carbohydrates, 18% proteins, 30% total fats. The DASH

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diet was designed to be rich in fruits, vegetables, whole grains, and low-fat dairy products and

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low in saturated fats, cholesterol and refined grains. Fasting blood samples were taken at baseline

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and after 8-wk intervention to measure lipid profiles and biomarkers of oxidative stress including

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plasma total antioxidant capacity (TAC) and total glutathione (GSH).

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Results: Adherence to the DASH diet, compared to the control diet, resulted in a significant

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decrease in weight (-4.4 vs. -1.5 kg; P<0.001) and BMI (-1.7 vs. -0.6 kg/m2; P<0.001), decreased

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serum triglycerides (-10.0 vs. +19.2 mg/dL; P-interaction=0.005) and VLDL-C levels (-2.0 vs.

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+3.9 mg/dL; P-interaction=0.005). Increased concentrations of TAC (+98.6 vs. -174.8 mmol/L;

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P-interaction<0.001) and GSH (+66.4 vs. -155.6 µmol/L; P-interaction=0.005) were also found

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in the DASH group compared with the control group.

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Conclusion: Consumption of DASH diet for 8 weeks led to a significant reduction in serum

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insulin, triglycerides and VLDL-C and a significant increase in TAC and GSH levels.

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KEYWORDS: DASH, polycystic ovary syndrome, lipids, oxidative stress, women

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Introduction

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Polycystic ovary syndrome (PCOS) is the most common endocrinopathy among reproductive-

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aged women [1] affecting6 to 18% of female adults[2].The estimated prevalence of this condition

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in Iran has been reported to be7% based on the National Institute of Health (NIH)criteria, 15.2%

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according to the Rotterdam criteria, and 7.9% according to the Androgen Excess Society

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(AES)criteria [3].Women with PCOS have androgen excess, insulin resistance, variable amounts

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of estrogen exposure, all of which can result in increased lipid profiles and biomarkers of

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oxidative stress [4-5]. In addition, increased visceral fat and the reduction of adiponectin levels in

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PCOS would result in elevated lipid profiles [6-7]. Decreased expression of peroxisome

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proliferators activated receptor (PPAR) in PCOS patients can also result in dyslipidimia and

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increased biomarkers of oxidative stress [8]. PCOS is associated with infertility [9], increased

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risk of endometrial, breast and ovarian cancers [10] and increased incidence of type 2 diabetes

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[11].

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Weight loss and lifestyle modifications including increased physical activity is the first-line

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treatment in the management of PCOS [12]. It has been reported that losing 5% of body weight in

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obese women with PCOS can result in improved hyperandrogenic features[13].Furthermore, the

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use of oral contraceptive pills and metformin may help induce the regular menses [14] and reduce

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hyperinsulinemia and ovarian androgens production in these women[15].Recently, it has been

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suggested that low-carbohydrate, low glycemic load and high-protein diets might beneficially

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influence PCOS than the conventional low-fat, high-carbohydrate diets[16-17].Consumption of a

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low glycemic load diet in combination with medications has been resulted in symptoms relief in

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patients with PCOS [18]. However, adherence to a high-protein and low-glycemicload diet for 12

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weeks did not influence lipid profiles among women with PCOS [19].

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The dietary approaches to stop hypertension (DASH) eating plan is a low-glycemic-index low

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energy-dense diet that has firstly been suggested for lowering blood pressure [20]; however, its

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beneficial effects have also been reported in type 2 diabetes[21],gestational diabetes[22]and

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metabolic syndrome [23]. Although the influence of some dietary components of DASH diet like

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antioxidants[24-25], magnesium[26], dietary fiber, fruit and vegetables[25] in PCOS has been

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assessed in previous studies, we are aware of no study examining the effects of DASH diet on

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metabolic profiles and biomarkers of oxidative stress in patients with PCOS. High contents of

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dietary fiber, antioxidants, phytoestrogens and isoflavones along with its low glycemic index [21,

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23] might help PCOS patients to control their increased levels of lipid profile and oxidative

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stress. Furthermore, adherence to the DASH diet has been associated with lower body fat [21],

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which could in turn result in lower testosterone levels [27], a key component in PCOS. The

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current study was, therefore, performed to investigate the effects of the DASH eating plan on

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lipid profiles and biomarkers of oxidative stress in overweight and obese women with PCOS.

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Materials and Methods

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Participants

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This two-arm parallel randomized controlled clinical trial was carried out in Kashan, Iran, from

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January 2012 to May2012. With the exception of the study dietitian (Z.A.), who provided the

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dietary education, all the study personnel and participants were blinded to dietary assignment.

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Overweight or obese (BMI≥25 kg/m2) women aged 18-40 y diagnosed with PCOS based on

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Rotterdam criteria were recruited in this study. On the basis of sample size formula suggested for

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randomized clinical trials [19], considering the type I error of 5% (α=0.05), type II error of 20%

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(β=0.20; Power=80%) and serum LDL-cholesterol levels as a key variable, we reached the

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sample size of 20 persons for each group. Diagnosis of PCOS was done according to the

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Rotterdam criteria[28]:those with the two of the following criteria were considered as having

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PCOS: oligo- and/or anovulation, excess androgen activity (clinical or biochemical)and

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polycystic ovaries(by gynecologic ultrasound).We did not include women aged<18 or >40 years,

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those with BMI<25 kg/m2, individuals with neoplastic, hepatic, renal or cardiovascular disorders,

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malabsorptive disorders, current or previous (within the last 6 months) use of hormonal,

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antidiabetic, or anti-obesity medications and intention to adopt a diet and/or a specific physical

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activity program. A total of 150 women attended gynecology clinics affiliated to Kashan

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University of Medical Sciences, Kashan, Iran, were screened for PCOS. Females who reported

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menstrual irregularity and/or had a modified Ferriman Gallwey (mF-G) score of 8 [29] were

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invited for a clinical examination. Those who did not have these criteria were not further

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evaluated and were deemed not to have PCOS. Taking a medical history and focusing on

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symptoms of PCOS, specifically menstrual irregularities, clinical hyperandrogenism and

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medication use including hormone therapy was done by trained midwifes. Menstrual irregularity

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was assessed as the presence of chronic amenorrhea or a menstrual cycle length of less than 21

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days or more than 35 days, or more than four days variation between cycles. Clinical

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hyperandrogenism was assessed as the self-reported degree of hirsutism using the modified

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Ferriman Gallwey (mF-G) scoring method based on a chart displaying degree of hair growth in

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nine regions. Polycystic ovaries were diagnosed by Ultrasonography (US) in participants with

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menstrual dysfunction and/or hirsutism (12 or more small follicles observed in an ovary on

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ultrasound examination).Furthermore, hormone profiles including serum prolactin, T3, T4 and

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TSH levels were assessed for all individuals at study baseline and having abnormal levels of

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these hormones was considered as a criteria for PCOS rejection based on the Rotterdam criteria.

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Finally, 54 women met the inclusion criteria and were enrolled in the study (Fig. 1).Subjects

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were stratified according to BMI (<30 and ≥30 kg/m2) and age (<30 and ≥30 y) and then were

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randomly assigned to the control (n=27) or the DASH diet (n=27) for 8 weeks. Random

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assignment was done by the use of computer-generated random numbers [30].The study was

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conducted according to the guidelines laid down in the Declaration of Helsinki. The ethical

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committee of Kashan University of Medical Sciences approved the study (no. P/29/5/1/9213) and

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informed written consent was obtained from all participants.

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Study design

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Participants were randomly assigned to consume the control or the DASH diet for 8 weeks. They

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were asked not to alter their routine physical activity, and not to receive any lipid-lowering

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medications as well as medications that might affect their reproductive physiology during the 8-

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wk intervention. Compliance with the consumption of diets was monitored once a week through

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phone interviews. The compliance was also double-checked by the use of three-day dietary

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records completed throughout the study. Dietary intakes of participants were assessed by means

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of three-day dietary records (two week days at weeks 3 and 6 and one weekend day at week 8 of

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intervention) completed throughout the study. The dietary records were based on estimated

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values in household measurements.To obtain nutrient intakes of participants based on these three-

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day food diaries, we used Nutritionist IV software (First Databank, San Bruno, CA) modified for

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Iranian foods.

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Diets

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Calorie requirements of each subject were estimated based on resting energy expenditure (by the

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use of Harris-Benedict equation) and physical activity level [31]. As all study participants were

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overweight or obese, both diets were designed to be calorie-restricted (350-700 kcal less than the

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computed energy requirement for each participant; 350 kcal for subjects with the BMI in the

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range of 25-27.5 kg/m2; 500 kcal for those with the BMI in the range of 27.5-31 kg/m2; and 700

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kcal for those with the BMI >31 kg/m2) to avoid ethical problems. We used two dietary plans: 1)

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a DASH diet that was consisted of 52% carbohydrates, 18% proteins, 30% total fats. The DASH

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diet was designed to be rich in fruits, vegetables, whole grains, and low-fat dairy products and

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low in saturated fats, cholesterol, refined grains, and sweets. Prescribed sodium in the DASH diet

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was less than 2,400 mg/day.2) The control diet was also designed to contain52% carbohydrates,

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18% protein and 30% total fat [23]; however, the two diets were different in terms of food groups

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contained (Table 1). It must be kept in mind that this study was not a feeding trial; therefore, we

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did not prepare foods for participants. In this study we just provided 7-day menu cycles to

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participants. The diets were individually planned using a ‘calorie count’ system. To facilitate the

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compliance to the diets, subjects were given and instructed an exchange list. To control for

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dietary intakes of participants throughout the study, the dietitian was calling the participants to

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resolve their probable problems. Furthermore, to examine the compliance to the diets, we asked

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participants to record their dietary intakes every two weeks. All participants spent about 45

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minutes with a dietitian learning the basics of their diets.

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Assessment of anthropometric measures

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Weight was assessed at baseline and after 8 weeks of intervention in gynecology clinics by

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trained midwifes. Body weight was measured in an overnight fasting status without shoes in a

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minimal clothing state by the use of a digital scale (Seca, Hamburg, Germany) to the nearest 0.1

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kg. Height was measured using a non-stretched tape measure (Seca, Hamburg, Germany) to the

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nearest 0.1 cm. BMI was calculates as weight in kg divided by height in meters squared.

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Biochemical assessment

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Fasting blood samples (10 mL) were taken at baseline and after 8-wk intervention at Kashan

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reference laboratory in an early morning after an overnight fasting. Serum total cholesterol and

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triglycerides concentrations were assayed using commercial kits (Parsazmun, Tehran, Iran) by

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enzymatic colorimetric tests with cholesterol oxidase p-aminophenazone and glycerol phosphate

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oxidase, respectively. Serum HDL-cholesterol was measured after precipitation of the

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apolipoprotein B containing lipoproteins with phosphotungistic acid. Serum VLDL- and LDL-

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cholesterol levels were also measured using available kits. Plasma total antioxidant capacity

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(TAC) was assessed by means of the FRAP method developed by Benzie and Strain [32]. Plasma

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GSH levels were measured using the method of Beutler et al [33].

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Statistical analysis

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We used Kolmogrov-Smirnov test to examine the normal distribution of variables. Log

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transformation was applied for non-normally distributed variables. The analyses were done based

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on intention-to-treat analysis. Missing values were treated based on Last-Observation-Carried-

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Forward method. Independent samples Student’s t test was used to detect differences in general

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characteristics and dietary intakes between the two groups. Energy-adjusted dietary intakes of

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nutrients were computed using residual method and compared using analysis of covariance. To

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determine the effects of DASH diet on lipid profiles and biomarkers of oxidative stress, we used

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one-way repeated measures analysis of variance. In this analysis, the treatment (DASH vs.

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control diet) was regarded as between-subject factor and time with two time-points (baseline and

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week 8 of intervention) was considered as within-subject factor. An additional model was

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constructed to control for the effect of age. We further adjusted for weight changes to determine

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of if the effect of DASH diet is independent of weight change. P<0.05 was considered as

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statistically significant. All statistical analyses were done using the Statistical Package for Social

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Science version 17 (SPSS Inc., Chicago, Illinois, USA).

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Results

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Among individuals in the control diet, 3 women [IVF treatment (n=1), became pregnant (n=1) or

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the use of medications (n=1)] were excluded. The exclusions in the DASH diet were 3 persons

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[IVF treatment (n=1), health problems (n=1) and the use of medications (n=1)]. Finally, 48

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participants [control diet (n=24) and the DASH diet (n=24) completed the trial (Fig. 1).

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Mean age of study participants was not statistically different between the DASH and control

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groups. Comparison of weight and BMI both at baseline and end of trial did not reveal a

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significant difference between the two groups (Table 2). When we compared the changes in

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weight and BMI between the two groups, we found greater reductions in both weight (-4.4±2.7

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vs. -1.5±2.6 kg, P<0.001) and BMI (-1.7±1.1 vs. -0.6±0.9 kg/m2, P<0.001) in DASH group than

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those in the control group.

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Based on the three-day dietary records that participants provided throughout the study, no

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statistically significant difference was seen between the two groups in terms of dietary intakes of

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energy, carbohydrates, protein and fat; however, significant differences were found in dietary

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intakes of saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA), cholesterol, dietary

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fiber, simple sugar, sodium, potassium, magnesium and calcium between the two groups (Table

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3).

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Compared to the control diet, consumption of DASH eating pattern led to decreased serum

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triglycerides (-10.0 vs. +19.2 mg/dL; P-interaction=0.005) and VLDL-C levels (-2.0 vs. +3.9

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mg/dL; P-interaction=0.005) (Table 4). Further control for age did not alter the findings. After

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additional adjustments for weight change, we found that those in the DASH group tended to have

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reduced levels of serum triglycerides (-5.9±7.5 vs. +15.2±7.5 mg/dL, P-interaction=0.07) and

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VLDL-C levels (-1.2±1.5 vs. +3.0±1.5 mg/dL, P-interaction=0.07) compared with individuals in

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control group. Increased concentrations of plasma TAC (+98.6 vs. -174.8 mmol/L; P-

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interaction<0.001) and total GSH (+66.4 vs. -155.6 µmol/L; P-interaction=0.005) were also

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found in the DASH group compared with the control group. When age and weight changes were

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taken into account, we observed that individuals in the DASH diet tended to have lower levels of

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plasma GSH levels than those in the control group (+34.1±57.6 vs. -123.3±57.6µmol/L, P-

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interaction=0.07).Adherence to the DASH eating pattern, compared to the control diet, resulted in

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a significant reduction in serum insulin levels (-1.88 vs. +2.89 µIU/mL, P-interaction=0.03), but

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did not significantly affect fasting plasma glucose levels (-0.5 vs. +5.7 mg/dL; P-

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interaction=0.20).We failed to find significant differences in mean changes of serum total

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cholesterol, HDL- and LDL-C levels between the two diets. Within-group changes revealed a

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significant decrease of serum triglycerides levels (P=0.04) and a significant increase of plasma

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TAC levels (P= 0.03) in the DASH diet, but a significant elevation of serum triglyceride levels

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(P=0.03) and a significant reduction of plasma TAC levels (P=0.001) was seen in the control diet.

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Furthermore, a significant within-group reduction in plasma total GSH levels was also seen in the

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control group (P=0.005).

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Discussion

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We found that adherence of the DASH eating pattern for 8 weeks among overweight and obese

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women with PCOS had beneficial effects on weight, BMI, serum triglycerides, VLDL-C, insulin,

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plasma TAC and total GSH levels compared with the control diet. However, the effects of DASH

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eating pattern on serum total cholesterol, HDL- and LDL-C were not significantly different

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compared with the control diet.

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Few studies evaluated the beneficial effects of diet therapy on metabolic status in women PCOS.

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According to our knowledge, no information is available indicating the effect of DASH diet on

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metabolic profiles of PCOS patients. Although the efficacy of DASH diet has been examined in

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metabolic syndrome [23], childhood obesity [34] and patients with stroke [35], we are aware of

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no study examining the effect of DASH diet on metabolic profiles of PCOS patients.

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Furthermore, there is still no definite dietary recommendation for these patients. In a previous

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study, Mehrabani et al [19] reported the beneficial effects of a high-protein, low-glycemic-load

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hypocaloric diet (30% of daily energy from protein plus low-glycemic-load foods) in overweight

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and obese women with PCOS after 12 weeks. Mehrabani et al have examined the effect of a high

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protein diet, while in the current study we assessed the effect of DASH diet. The high protein

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diets are totally different from the DASH diet. Furthermore while both diets in our study were

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designed to be calorie-restricted (350-700 kcal less than the computed energy requirement for

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each participant), Mehrabani et al [19] prescribed a fixed range of dietary energy (1200-1700

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kcal/d) for all participants. Finally, Mehrabani et al did not examine the effect of their dietary

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intervention on biomarkers of oxidative stress, while we assessed the effect of DASH diet on

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these biomarkers as well.

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The current study showed that consumption of the DASH diet compared with the control diet led

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to a significant reduction in weight, BMI and serum insulin levels. In line with our study, weight

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loss and improved insulin sensitivity was also seen following energy restriction for 12 weeks in

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overweight women with PCOS [17].Even short-term energy restrictions for 4 weeks in PCOS

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patients resulted in a lower fasting insulin [36-37]. It is possible that the family of insulin-like

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growth factors (IGFs) and their binding proteins get involved, as they have been shown to be

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differentially influenced by energy restriction [37] and weight loss [36]. Specifically, decreased

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insulin levels would result in increased IGF-binding protein-1 during short-term energy

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restriction [17].

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It has been shown that PCOS is associated with decreased levels of adiponectine concentrations,

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which in turn is associated with disturbances of the mitogen-activated protein kinases (MAPK)

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and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway.

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These conditions would lead to increased inflammatory markers and biomarkers of oxidative

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stress [7]. Increased lipid profiles and biomarkers of oxidative stress would result in several

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aberrations [38-39].Our study demonstrated that consumption of the DASH eating plan

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significantly reduced serum triglycerides and VLDL-C levels, but could not affect serum total-,

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HDL- and LDL-C levels among women with PCOS. Previous studies have shown the beneficial

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effects of DASH diet on serum lipid profiles of patients with hypertension, cardiovascular

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disease, metabolic syndrome and type 2 diabetes [21, 23, 40-41]. Our previous study among

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pregnant women with gestational diabetes mellitus (GDM) has also indicated a significant

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reduction of serum triglycerides, total cholesterol and LDL-C concentrations following

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consumption of the DASH eating patternfor4 weeks [22]. Azadbakht et al [21] have shown a

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significant decrease of serum LDL-C and an increase of HDL-C levels after intervention with the

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DASH diet intype 2 diabetic patients.Similar findings were also seen with the administration of

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DASH dietfor8 weeks in patients with the metabolic syndrome [23], for 30 days in subjects with

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elevated blood pressure [42]and for 8 weeks in hypercholestrolemic patients[43].Several

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mechanisms can explain the beneficial effects of DASH diet on lipid profiles in PCOS women. In

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this study, dietary intake of simple sugar in the DASH diet was about half that of the control diet.

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Furthermore, its fiber content was 1.5-2.0 times higher than that of the control diet. Earlier

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studies have shown that high-sugar diets might induce mitochondrial dysfunction in adipose

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tissue, which can in turn lead to metabolic impairment and elevated serum triglycerides levels

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[44].The DASH diet contain high amounts of arginine-rich foods including fish, soy, beans,

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lentils, whole grains, and nuts, parsley and fresh basil. The high arginine content of DASH diet

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might also explain its beneficial effects on insulin resistance and serum triglycerides levels.

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Decreased serum triglycerides levels following arginine intake have been attributed to shifting

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nutrient partitioning to promote muscle over fat gain and may in turn provide a useful treatment

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for improving the metabolic profile [45]. The DASH diet is also a rich source of dietary calcium

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and magnesium, through which it can affect triglyceride levels. Increased intracellular calcium in

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liver may stimulate microsomal triglyceride transfer protein (MTP) which is implicated in the

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formation and secretion of VLDL, and then result in decreased serum triglycerides levels

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[46].Furthermore, magnesium intake has been associated with lower levels of serum lipids due to

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suppressing endothelial injury, reducing the peroxidation of lipids and increasing the antioxidant

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capacity in serum and tissues [47].

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The current study revealed that consumption of the DASH diet resulted in a significant increase

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in plasma TAC and total GSH levels among overweight and obese women with PCOS. Our

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previous study has indicated that consumption of DASH diet increased plasma TAC and total

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GSH levels in pregnant women with GDM after 4 weeks[48].Similar findings has also been

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reported following consumption of the DASH eating pattern in free-living obese hypertensive’s

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after 4 weeks [49].Adherence to the low-sodium DASH diet in salt-sensitive (SS) subjects has

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also been resulted in decreased urine F2-isoprostanes, a biomarker of oxidative stress, than a

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standardized usual low fruit and vegetables diet (ULFV) [50].However, consumption of the

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DASH eating plan after 3 months led to a non-significant increase in plasma TAC in healthy

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individuals [51]. Overall, as PCOS is associated with increased oxidative stress [52],

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consumption of the DASH diet in patients with PCOS could help them control the complications

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these conditions might led to. The favorable effects of DASH diet on biomarkers of oxidative

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stress could be attributed to its high content of antioxidant-rich fruit and vegetables [49, 53-54].

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Individuals in the DASH group received vitamin C nearly 2 times greater than that in the control

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group. Vitamin C, a major component of TAC, can decrease the NADPH oxidase activity, which

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is a major superoxide-generating enzyme and increased oxidative stress [55]. High intakes of

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calcium and magnesium in the DASH diet might also help explain it effects on decreased

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oxidative stress. Calcium can act as a DNA damage reducing agent [56], which in turn results in

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the free radical scavenging and increased plasma TAC and total GSH levels. In addition,

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restoring the activity of anti-oxidative enzymes [57] and scavenging oxygen radicals [58] that

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results from magnesium intake can provide some reasons for our findings. Finally, the arginine

308

content of DASH diet might also explain its beneficial effects on decreased oxidative stress.

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Arginine intake has been shown to reduce serum levels of angiotensin II and measures of

310

oxidative stress [59].

311

Our findings must be interpreted in the context of some limitations. The first limitation is the

312

relatively short duration of intervention. Long-term interventions might result in greater changes

313

in lipid profiles. Second, we could not assess the effects of DASH eating plan on other

314

biochemical indicators of oxidative stress and factors of peroxidation. Further studies are required

315

to examine the effects of DASH diet on other measures of oxidative stress.

316

In conclusion, consumption of DASH eating pattern for 8 weeks among overweight and obese

317

women with PCOS resulted in a significant decrease in weight, BMI, serum insulin, triglycerides

318

and VLDL-C levels as well as a significant increase in plasma TAC and total GSH levels. Given

319

the high prevalence of PCOS and based on the findings of the current study, we believe that the

320

DASH diet could be recommended as a suitable diet for these patients to control abnormal

321

metabolic profiles.

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Acknowledgment

324

The present study was supported by a grant (no. 9213) from the Vice-chancellor for Research,

325

KUMS, Kashan, Iran. The authors would like to thank the staff of Naghavi and Shaheed Beheshti

326

gynecology Clinics (Kashan, Iran) for their assistance in this project.

327

Clinical trial registration number: www.irct.ir:IRCT201304235623N6.

328

Name of trial registry: Effects of the hypocaloric DASH diet on metabolic parameters,

329

inflammatory factor and biomarkers of oxidative stress in overweight and obese women with

330

polycystic ovary syndrome.

331

Accessed protocol: www.irct.ir:IRCT201304235623N6.

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Table 1

555

Constituents of the DASH and control diets used in the study‫٭‬ DASH diet

Grains †

9

6

Simple sugar

5

Vegetables

2

Fruits

2

Dairy ††

2

Meats, poultry and fish†††

4

Nuts, seeds and legumes

1

Fats and oils

3

556

DASH, Dietary Approaches to Stop Hypertension

557

‫٭‬

558

†

559

††

560

†††

SC

3

4 2

3

4 servings from lean meat in the DASH diet and 2 servings in the control diet

TE D EP AC C

567

5

Low-fat (<2%) in the DASH diet

563

566

4

At least 3 servings from whole grains in the DASH diet

562

565

2

Data are presented for a calorie intake of 1700 kcal/day

561

564

RI PT

Control diet

M AN U

Food group

568 569 570

24

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571

Table 2

572

General characteristics of the study participants‫٭‬ DASH diet (n=24) 22.1±3.2

P value†

Height (cm)

161.3±6.0

160.5±4.9

0.60

Weight at study baseline (kg)

74.6±16.2

78.0±12.0

0.41

Weight at end-of-trial (kg)

73.1±15.5

73.6±12.1

0.90

-4.4±2.7

<0.001

30.3±4.5

0.27

BMI at study baseline (kg/m2)

28.6±5.8

BMI at end-of-trial (kg/m2)

28.0±5.7

28.6±4.4

0.74

BMI change (kg/m2)

-0.6±0.9

-1.7±1.1

<0.001

* Data are means ± SD.

575

† Obtained from independent t test.

TE D

574

576

582 583

AC C

581

EP

577

580

M AN U

-1.5±2.6

BMI, Body mass index

579

0.07

Weight change (kg)

573

578

SC

Age (y)

RI PT

Control diet (n=24) 24.7±6.0

584 585 586

25

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587

Table 3

588

Dietary intakes of study participants throughout the study‫٭‬ P value†

DASH diet

(n=24)

(n=24)

Energy (kcal/d)

1887±138

1839±122

0.20

Fat (g/d)

62.9±4.6

61.3±4.0

0.20

Protein (g/d)

84.9±6.2

82.8±5.5

0.20

245.3±17.9

239.1±15.8

0.20

Crude

13.8±1.0

<0.001

Model 1

13.6±0.1

8.8±0.1

<0.001

Crude

11.3±0.8

18.6±1.2

<0.001

Model 1

11.1±0.1

18.8±0.1

<0.001

Crude

185.5±14.6

141.6±20.7

0.001

Model 1

182.3±1.0

144.8±1.0

0.001

Crude

12.1±1.5

17.2±1.3

<0.001

Model 1

11.9±0.2

17.4±0.2

<0.001

Crude

3.5±0.4

4.8±0.4

<0.001

Model 1

3.5±0.1

4.9±0.1

<0.001

Crude

2815.5 ±460.4

1729.8±103.5

<0.001

Model 1

2785.5 ±41.2

1699.1±15.4

<0.001

Crude

3190.4±278.3

4864.4±329.3

<0.001

Model 1

3140.3±29.0

4914.3±29.0

<0.001

PUFA (g/d)

TE D

Cholesterol (mg/d)

Dietary fiber (g/d)

AC C

Insoluble fiber (g/d)

M AN U

8.7±0.6

EP

SFA (g/d)

SC

Carbohydrate (g/d)

RI PT

Control diet

Sodium (mg/d)

Potassium (mg/d)

Magnesium (mg/d)

26

ACCEPTED MANUSCRIPT

Crude

233.1±17.9

411.5±15.2

<0.001

Model 1

230.9±2.3

413.7±2.3

<0.001

Crude

1038.6±42.2

1717.2±36.2

<0.001

Model 1

1032.3±1.7

1724.3±1.7

<0.001

Fruit (servings/day)

2.8±0.4

5.2±0.4

<0.001

Vegetables (servings/day)

2.6±0.5

4.3±0.4

<0.001

Nuts (servings/day)

1.0±0.2

2.5±0.4

<0.001

Fats and oils (servings/day)

3.5±0.5

3.6±0.5

0.40

SC

RI PT

Calcium (mg/d)

DASH, Dietary Approaches to Stop Hypertension; PUFA, Polyunsaturated fats; SFA, Saturated fatty

590

acids

591

* Data are means ± SD.

592

† Obtained from independent t test.

593

Model 1: Adjusted for energy intake (data are means± standard error)

594 595

M AN U

589

.

AC C

EP

TE D

596

27

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Table 4

P value†

DASH diet (n=24) Wk8

Change

Time

Group

Time x Group

162.8±33.6

-4.2±24.7

0.95

0.33

0.29

166.4±6.2

-3.2±6.2

0.34

0.10

0.45

165.9±6.7

1.2±6.5

0.72

0.33

0.84

109.2±49.7

99.2±45.2‫٭‬

-10.0±22.3

0.35

0.93

0.005

112.5±9.9

104.6±11.3

-7.9±7.0

0.18

0.48

0.01

113.5±10.7

107.6±12.1

-5.9±7.5

0.30

0.39

0.07

21.8±9.9

19.8±9.0‫٭‬

-2.0±4.5

0.35

0.93

0.005

22.5±2.0

20.9±2.3

-1.6±1.4

0.18

0.48

0.01

3.0±1.5

22.7±2.1

21.5±2.4

-1.2±1.5

0.30

0.39

0.07

46.7±7.9

-0.2±9.9

48.4±9.9

48.2±12.8

-0.2±9.3

0.89

0.59

0.95

46.5±2.3

46.9±2.2

0.4±2.0

48.8±2.3

48.0±2.2

-0.8±2.0

0.20

0.68

0.54

Model 2

46.6±2.5

46.6±2.4

EP

Control diet (n=24)

RI PT

Means (±standard deviation) of serum lipid profiles and biomarkers of oxidative stress at baseline and after the intervention

Wk0

Wk8

Change

Wk0

Crude

153.5±42.0

158.3±31.5

4.8±34.4

167.0±33.7

Model 1

151.0±7.8

154.6±6.2

3.6±6.2

169.6±7.8

Model 2

155.8±8.1

155.1±6.7

-0.7±6.5

164.7±8.1

Crude

93.3±47.9

112.5±67.3‫٭‬

19.2±42.8

Model 1

89.9±9.9

107.1±11.3

17.2±7.0

Model 2

88.9±10.7

104.1±12.1

15.2±7.5

Crude

18.6±9.6

22.5±13.5‫٭‬

3.9±8.6

Model 1

18.0±2.0

21.4±2.3

3.4±1.4

Model 2

17.8±2.1

20.8±2.4

Crude

46.9±11.8

Model 1

0.0±2.1

48.7±2.5

48.3±2.4

-0.4±2.1

0.18

0.57

0.89

Crude

88.0±30.0

89.0±23.2

1.0±25.9

96.8±30.6

94.8±29.5

-2.0±22.7

0.89

0.65

0.65

Model 1

86.5±6.3

86.3±5.2

-0.2±5.0

98.3±6.3

97.5±5.2

-0.8±5.0

0.19

0.13

0.93

Model 2

91.4±6.4

87.7±5.5

-3.7±5.2

93.3±6.4

96.1±5.5

2.8±5.2

0.48

0.53

0.40

3.4±0.8

3.4±0.7

0.0±0.48

3.6±1.0

3.6±1.1

0.0±0.7

0.67

0.43

0.54

TE D

VLDL-C(mg/dL)

AC C

HDL-C (mg/dL)

LDL-C(mg/dL)

M AN U

Triglycerides (mg/dL)

SC

Total cholesterol(mg/dL)

Total: HDL-C ratio Crude

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ACCEPTED MANUSCRIPT

Model 1

3.3±0.2

3.4±0.2

0.1±0.1

3.6±0.2

3.7±0.2

0.1±0.1

0.03

0.24

0.99

Model 2

3.4±0.2

3.4±0.2

0.0±0.1

3.6±0.2

3.7±0.2

0.1±0.1

0.14

0.50

0.27

Crude

968.5±233.1

793.7±103.4‫٭‬

-174.8±218.0

921.7±226.7

1020.3±301.9‫٭‬

98.6±213.7

0.22

0.12

<0.001

Model 1

978.0±47.7

795.7±47.4

-182.3±45.0

912.2±47.7

1018.0±47.4

105.8±45.0

0.27

0.20

<0.001

Model 2

983.7±51.1

795.8±51.2

-187.9±48.5

906.5±51.5

1018.0±51.2

111.5±48.5

0.34

0.29

<0.001

Crude

601.8±267.9

446.2±131.9‫٭‬

-155.6±242.8

478.3±195.9

544.7±220.2

66.4±279.0

0.24

0.79

0.005

Model 1

586.8±47.9

445.0±38.1

-141.8±53.9

493.4±47.8

545.9±38.1

52.5±53.9

0.27

0.93

0.01

Model 2

566.0±50.9

442.7±41.2

-123.3±57.6

514.1±50.9

548.2±41.2

34.1±57.6

0.44

0.63

0.07

RI PT

TAC (mmol/L)

M AN U

SC

GSH (µmol/L)

DASH, Dietary Approaches to Stop Hypertension; FPG, Fasting plasma glucose; GSH, Total glutathione; HOMA-IR, Homeostasis Model of Assessment-Insulin Resistance; HDL-C, High density lipoprotein-cholesterol; hs-CRP, High-sensitivity C-reactive protein; LDL-C, Low density lipoprotein-cholesterol; TAC, total antioxidant capacity; VLDL-C, Very low density lipoprotein-cholesterol ‫٭‬Different from wk 0, P < 0.05with paired samples t test. Model 1: Adjusted for age (data are means± standard error)

TE D

†Obtained from repeated measures ANOVA test.

AC C

EP

Model 2: Adjusted for Model 1+ weight change (data are means± standard error)

29

ACCEPTED MANUSCRIPT

Assessed for screening (n=150)

Lost to follow-up (n=3)

M AN U

Allocated to control (n=27)

Allocated to intervention (n=27)

Lost to follow-up (n=3)

♦ IVF treatment (n=1) ♦ Health problems (n=1) ♦ The use of medications (n=1)

Analyzed (n=24)

Analyzed (n=24)

EP

TE D

♦ IVF treatment (n=1) ♦ Became pregnant (n=1) ♦ The use of medications (n=1)

AC C

Analysis

Follow-up

Allocation

Randomized (n=54)

SC

RI PT

Enrollment

Excluded (n=96) ♦ Not living in Kashan (n=24) ♦ Taking excluded medications (n=17) ♦ Within normal weight range (n=15) ♦ Outside the age range required (n=14) ♦ Unable to commit to study (n=12) ♦ Excluded health condition (n=6) ♦ Not diagnosed with PCOS (n=4) ♦ Recent weight loss (n=4)

Fig. 1.Summary of patient flow

IVF, In vitro fertilisation; PCOS, Polycystic ovary syndrome

30