The trial was preformed according to a crossover design in spring and summer 2016. Hemodialysis patients were selected from dialysis centers in Namazi and Shahid Faghihi hospitals of Shiraz, Iran. A sample size of 19 was determined based on results provided by previous investigators , a type 1 error of 5%, power of 20%, and considering a 10% drop-out rate.
Inclusion criteria were as follows: ESRD patients on dialysis treatment, dialysis 3 times a week for at least 3 months, an age range of 18 to 65 years, life expectancy more than 1 year, serum potassium level of less than 6 mEq/L, and permission of the physician in charge of the patient. Patients were not included if they were involved in serious diseases such as malignancy, other organs’ failure, taking antioxidant supplements such as vitamin E and vitamin C over the month before the study, iron sucrose (Venofer) usage at least two weeks before the study, malnutrition that required nutritional support, and hospitalization for > 5 days per month. Our primary protocol was to exclude the patients if they lost inclusion criteria, missed PJ consumption for > 3 days a month, changed hypertension and hyperlipidemia medications, did not follow dietary recommendations, and hospitalized during the intervention. However, none of the patients was excluded according to these criteria.
The study was approved by the Ethics Committee of Shiraz University of Medical Sciences (approval number. IR.SUMS.REC.1394.210), and registered at Iranian Registry of Clinical Trials (ID number: IRCT 2016070428797N1). Study protocol was explained and written informed consent was obtained from all participants.
The study was performed in a crossover design with two 8-week sequences separated by a 4-week washout period. At the beginning, the participants were divided into two groups: PJ-treated group (n = 22) who consumed 100 g natural PJ after their dialysis session three times a week and the control group (n = 19) who received the usual care. After the 8-week intervention, there was a 4-week washout course and afterwards the second sequence of the study was started in which the role of the groups was interchanged. The Ethical Committee of our institution did not permit having a control treatment because 1) hemodialysis patients have restrictions for consumption of liquids; 2) it may not be ethically approved to ask hemodialysis patients to drink a placebo beverage when a beneficial effect for which could not be substantiated; 3) making a control drink for PJ required addition of artificial colors and pomegranate flavor which could be harmful for ESRD patients; and 4) in contrast to similar studies on hemodialysis patients, we administered PJ after dialysis session, thus it was crucial to avoid administering unnecessary food items. Because hemodialysis patients spend hours in the dialysis unit, to prevent contamination between intervention and control groups, we recruited the participants from two hospitals and allocated each of the intervention and control groups to a hospital at a time.
Pomegranate was from Rabab variety cultivated in Neyriz, Shiraz. Pomegranates were purchased from Neyriz pomegranate farms in autumn and stored at -20 ⁰C until the time of the intervention. During the intervention, each week some pomegranates were thawed, crushed, and squeezed, and the juice was collected in 500 ml bottles. Thus, the juice was prepared fresh and distributed amongst the patients in PJ condition every week. The patients were asked to keep the juice in a refrigerator until the time of use. One hundred milliliters measuring cups were provided for the participants and they were asked to drink 100 ml PJ three times a week after their dialysis session. In order to ensure consumption of entire amount of PJ, each week 100 ml extra PJ was given to the patients just in case if other family members wanted to taste it.
During the study, each patient was under supervision and care of a nephrologist and medical care team. Blood pressure was measured before and after PJ consumption in order to ensure that PJ would not cause hypotension. For all of the patients, weight maintenance diets were prescribed and the amount of fruit and vegetables that each patient could consume was explained. Diets were set in such a way that people in both groups received equal amounts of fruit and fruit juice. A list of high-potassium fruits was given to the patients and they were asked to abstain from high-potassium fruits on days of PJ consumption. The patients were also recommended to exclude pomegranate and PJ consumption throughout the intervention except for the PJ which was administered.
Total antioxidant capacity and malondialdehyde were considered as the primary and interleukin-6 was measured as the secondary outcome. All parameters were measured at baseline, the end of the first sequence, and before and after the second sequence of the intervention. For weight and body composition, measurements were performed immediately after the dialysis session . Weight was measured by a digital scale (AM-2018) and body composition was determined by bioelectric impedance analyzer (BIA) (InBody, Biospace Co., South Korea). Blood pressure was measured in predialysis state twice with at least 5 min interval by a digital barometer (Microlife BP A200 AFIB(. The mean of two measurements was considered as the subject’s blood pressure.
Blood samples were collected after 12-h fasting. All biochemical variables were measured in serum. Serum lipid profile was analyzed by an auto-analyzer (BT 1500, Biotecnica Instruments, Italy) using specific kits (Pars-Azmun, Iran). Malondialdehyde (MDA) was quantitated by thiobarbituric acid reactive substances (TBARS) method . The method principle is based on the reaction of lipid peroxidation products in serum samples with thiobarbituric acid in the presence of trichloroacetic acid, butylated hydroxyltoluene, hydrochloric acid, and sodium dodecyl sulfate. The reaction mixture was then heated for 30 min at 95 °C and the formed chromogen is read at 532 nm. Interleukin-6 (IL-6) (Diaclone, France) and serum total antioxidant capacity (TAC) (Biocore diagnostics, Hamburg, Germany) were determined by colorimetric assay using commercially available kits.
Three-day dietary recall (two weekdays and one weekend day) was used to assess dietary intake and monitor dietary compliance. Dietary intakes were analyzed by Nutritionist IV software (First Databank, San Bruno, CA, USA) modified for Iranian foods. Mini nutritional assessment (MNA) was used as a tool for detecting malnutrition in hospitalized young and middle-aged adults .
Data were analyzed using SPSS software (version 22; IBM, Armonk, USA). Normality of data distribution was checked by visual examination of the histogram curve of data frequencies. In the case of abnormality, log-transformed data was used for determination of statistical difference between treatment conditions. The intention-to-treat (ITT) approach was used for the analysis. Comparisons between the two conditions were performed by ANCOVA (treatment × time interaction) with age and gender as the covariates. P value < 0.05 was considered statistically significant.