One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: A randomized placebo-controlled trial

Free Radical Biology and Medicine 53 (2012) 297–304

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Original Contribution

One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: A randomized placebo-controlled trial Lilach Shema-Didi a, Shifra Sela b,c,d,n, Liora Ore e, Galina Shapiro c, Ronit Geron f, Goldfeld Moshe g, Batya Kristal d,f a

Quality Assurance Department, Western Galilee Hospital, Nahariya, Israel Clinical Microbiology Lab, Western Galilee Hospital, Nahariya, Israel Eliachar Research Lab, Western Galilee Hospital, Nahariya, Israel d Faculty of Medicine, Bar-Ilan University, Zefat, Israel e School of Public Health, Faculty of Social Welfare & Health Sciences, University of Haifa, Israel f Nephrology Department, Western Galilee Hospital, Nahariya, Israel g Radiology Department, Western Galilee Hospital, Nahariya, Israel b c

a r t i c l e i n f o

abstract

Article history: Received 17 October 2011 Received in revised form 7 May 2012 Accepted 9 May 2012 Available online 17 May 2012

Increased systemic inflammation and oxidative stress are well established as nontraditional key players in the pathogenesis of atherosclerosis and are also involved in the innate immunity dysregulation in hemodialysis (HD) patients. The study aim was to investigate the effect of 1-year intake of pomegranate juice, an antioxidant source, on oxidative stress, inflammation, and long-term clinical outcomes. A randomized placebo controlled double-blind trial was designed, enrolling 101 chronic HD patients to receive during each dialysis 100 cc of pomegranate juice, or matching placebo, three times a week for 1 year. The primary endpoints were levels of oxidative stress and inflammation biomarkers. Secondary endpoints were hospitalization due to infections and the progression of atherosclerotic process based on a composite of variables of the carotid arteries: intima media thickness (IMT), number, and structure of plaques. Pomegranate juice intake yielded a significant time response reduction in polymorphonuclear leukocyte priming, protein oxidation, lipid oxidation, and inflammation biomarkers levels. These beneficial effects were abolished 3 months postintervention. Pomegranate juice intake resulted in a significantly lower incidence rate of the second hospitalization due to infections. Furthermore, 25% of the patients in the pomegranate juice group had improvement and only 5% progression in the atherosclerotic process, while more than 50% of patients in the placebo group showed progression and none showed any improvement. Prolonged pomegranate juice intake improves nontraditional CV risk factors, attenuates the progression of the atherosclerotic process, strengthens the innate immunity, and thus reduces morbidity among HD patients. & 2012 Elsevier Inc. All rights reserved.

Keywords: Inflammation Oxidative stress Pomegranate juice Hemodialysis

Introduction Atherosclerosis, cardiovascular disease (CVD), and infections are the main causes of morbidity, hospitalization, and mortality among chronic hemodialysis (HD) patients [1]. While traditional risk factors dominate the scene in the general population, in chronic kidney disease (CKD), nontraditional risk factors play an

n Corresponding author at: Clinical Microbiology and Eliachar Research Laboratory, Laboratory of Clinical Microbiology and Eliachar Research Laboratory, Western Galilee Hospital, Nahariya, ISRAEL 22100, Nahariya, Israel. Tel.: þ 972 507887931; fax: þ 972 4 9107474. E-mail address: [email protected] (S. Sela).

0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.013

increasingly important role being even more dominant in ESRD patients. Oxidative stress and inflammation are well-established key players in the pathogenesis of atherosclerosis and vascular disease among CKD patients [2]. Moreover, oxidative stress has also been related to the immune system dysregulation in patients with uremia [3,4], as reflected by elevated oxidative biomarkers and the activation of circulating leukocytes [5]. The recurrent blood interactions with the dialyzers trigger peripheral polymorphonuclear leukocytes (PMNLs) and monocytes to generate and release proinflammatory cytokines and reactive oxygen species (ROS), aggravating the already existing oxidative stress and inflammation state in HD patients [6,7]. Even though antioxidative treatment is a reasonable therapeutic approach in HD patients, controversial results were reported [8–10].

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Two randomized-controlled trials have yielded promising results, showing that antioxidant therapies with vitamin E and N-acetylcysteine (NAC) are a worthwhile strategy for ameliorating cardiovascular morbidity risk in dialysis patients [11,12], but not adequate enough to warrant widespread use of antioxidants as a sole base in dialysis patients [DOQI 2005[. Many fruits and vegetables, with their natural flavonoid/ polyphenol contents, have antioxidative and anti-inflammatory properties that are known to scavenge diverse ROS or inhibit their formation [13,14]. Indeed, few small short-term clinical studies demonstrated the beneficial effects of polyphenolic-rich natural juice intake on oxidative stress in HD patients [15–17]. The antioxidant activity of pomegranate juice in vitro was found to be linked to diverse groups of polyphenols, including ellagitanins, gallotannins, and ellegic acid, as well as flavonoids, such as anthocyanins. The antioxidant activity of pomegranate juice in vitro was found to be three times higher than that of red wines and green teas, and two- to six-fold stronger than other natural beverages [15]. Evidence for the clinical benefits of pomegranate juice was reported in several studies: reduction of systolic blood pressure in hypertensive patients, decreasing common carotid artery intimamedia thickness (IMT) [18], attenuating myocardial ischemia, and improving lipid profile in diabetic patients [19,20]. Although patients on chronic HD have a high prevalence of all the above comorbidities and are exposed to severe systemic oxidative stress and inflammation, the effect of pomegranate juice intake among these patients has never been studied. As such, the present randomized clinical trial was designed to explore the effect of a 1-year intake of pomegranate juice by HD patients, on oxidative stress, inflammation, and clinical outcomes.

Materials and methods Study population A single dialysis center at the Western Galilee Hospital, Nahariya, with 149 HD patients, participated in the study. Inclusion criteria were chronic HD patients, aged 418 years who underwent 4 h dialysis sessions thrice weekly using low-flux high-performance cellulose-triacetate [SureFlux 190 G, NIPRO], or polysulfone membranes [FX 10, Fresenius]. Dialysis was performed with dialysate calcium concentration of 2.5 meq/L and sodium concentrations of 140 meq/L. Exclusion criteria were HD treatment for less than 3 month, participation in other clinical trials, refusing or unable to give informed consent due to mental or physical state, and patients who were pregnant or planning to become pregnant during the duration of the study. All individuals gave informed, written consent to participate in the study which was approved by the Ethics Committee at the study center (Helsinki Committee approval No. 2008-06-06). The study was registered in ClinicalTrials.gov registration, Identifier number: NCT00727519. Study design The study was a double-blind, placebo-controlled, randomized, clinical trial. The recruited patients where randomly assigned to two groups: one group (n¼66) received 100 cc of pomegranate juice, and the other (n¼35) received a matching 100 cc placebo juice, three times a week during the first dialysis hour, for 12 months. Patients, as well as the medical and laboratory staff, were blinded to patient’s group allocation. The smaller randomization ratio of 2:1with only a modest loss in statistical power [21] was chosen due to ethical/practical issues. The study sample size (n¼101), with a ratio of 2:1, pomegranate juice:placebo, had 80%

power (P¼0.05) to detect 25% decrease in CD11b (PMNL priming), assuming a mean level of 29.3711.9 MFI according to preliminary results. During the study period patients were instructed to drink the pomegranate juice during the first hour of their dialysis treatment, and considered it a single fruit serving in their daily recommended diet. The follow-up after the juice intake was carried out and documented by the study coordinator. Noncompliant patients were excluded from the study after refusing to drink the juice more than 3 consecutive times. Ultrasound of the carotid artery was performed for mobile patients twice: first at the study initiation, and then after 12 months of intervention. Blood was always drawn from the arterial line before the dialysis initiation, for measurements of oxidative stress and inflammation biomarkers at 0, 3, 6, and 12 months following study initiation, and 3 months after cessation. Clinical updates regarding patients’ hospitalization due to infections were carried out daily. Pomegranate juice In order to choose the commercial pomegranate juice with the highest polyphenol levels, several hand-squeezed and various commercially available juices in Israel were analyzed using the colorimetric assay, as previously described [22,23]. Briefly, after 1:10 dilution of the juices in water containing 20% of Na2CO3, the phenols were determined by the Folin-Ciocalteu reagent (using Quercetin as a standard. The absorbance was measured at 760 nm. Pomegranate juice manufactured in Turkey and purchased from Naturafood [Israel] was used in this study as it showed the highest concentration of polyphenols (0.7 mmol/100 cc juice). The juice was used as recommended by the manufacturer and kept at room temperature (o25 1C) until opened. Placebo juice The placebo was engineered to resemble the pomegranate juice in color and taste and was prepared especially for this study by food engineers. The placebo contained artificial pomegranate extract of Frutarom Ltd, corrosive acid, caramel as color material, and aspartame and acesulfame as sugar substitutes. Analyzing the placebo juice using the colorimetric assay verified that the juice has no polyphenols. During placebo juice preparation, comparisons tests of flavor between placebo juice and pomegranate juice were performed by two independent testers to verify that placebo and pomegranate juice had a similar taste. Outcomes Primary endpoints The levels of oxidative stress and inflammation biomarkers included: PMNL priming changes followed by the expression of CD11b; the higher the priming, the easier to generate ROS by a second stimulant, resulting in oxidative stress in these patients. Myeloperoxidase (MPO) is involved in the production of HOCl, a powerful oxidant. MPO is a product of PMNL degranulation, as a result of priming and activation; hence its sera levels serve as a biomarker of oxidative stress. Oxidative stress was evaluated by sera levels of advanced oxidation protein products (AOPP), oxidized fibrinogen, and malondialdehyde (MDA); inflammation was evaluated by sera levels of IL-6, TNF-a, albumin, and fibrinogen. CD11b on PMNLs in whole blood was determined using a FC500 flow cytometer (Beckman-Coulter). Fifty microliters of

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blood was incubated for 10 min with anti-CD11b-R phycoerythrin (PE)-conjugated monoclonal antibody (IOTest, Immunotech, a Beckman Coulter Company, Marseille, France) followed by RBC lysis (Q-prep method; Coulter Corporation, Hialeah, FL, USA). The PMNL population was gated after staining with anti-human-CD16 R-phycoerythrin-cyanin 5.1 (PC5)-conjugated antibody (IOTest, Immunotech, a Beckman Coulter Company, Marseille, France). CD11b on PMNLs is expressed as mean fluorescence intensity (MFI) after subtracting nonspecific background. Human MPO instant ELISA kit (Bender MedSystems GmbH, Vienna, Austria) was used according to the manufacturer instructions. Sera AOPP was measured as previously described [24] and carbonylated fibrinogen, reflecting fibrinogen oxidation, was measured in the 340-kDa band on the SDS-PAGE as was previously published by us [25]. MDA, a marker of lipid peroxidation, was measured as described by Morel et al. [26]. IL-6 and TNF-a were measured by Quantikine ELISAs (R&D Systems) according to their kits instructions. Albumin levels were determined using a bromocresol green (BCG)-based assay (using AEROSET chemical analyzer, ABBOTT Laboratories, USA). Due to a technical issue (change in albumin analyzer) the 1-year results of albumin were not included. Fibrinogen was measured by an immunoassay kit manufactured by Kamiya Biomedical Company (Seattle, WA, USA) analyzed on a Cobas Mira analyzer (Roche Diagnostics).

used. In order to compare changes over time in oxidative stress and the inflammatory biomarkers, in the two groups, with an attempt to minimize the effects of informative missingness as much as possible, we used a linear mixed model with unstructured covariance structure, using treatment by time interaction as a fixed effect, while patients as well as intercepts, as random effects. This model enables all possible data on all randomized subjects to be used. Changes over time in each of the groups were analyzed by linear mixed models with an unstructured covariance structure, using group as a fixed effect and patients as well as intercept as a random effect. In addition, we examined the difference between the two groups in oxidative stress and inflammatory biomarkers at the end of the patient’s follow-up, using the t test or Mann Whitney test when appropriate, using last observation carried forward method for missing data to retain the intention to treat analysis. Survival curves comparing the effect of treatment on the secondary endpoints were calculated by the Kaplan-Meier method using the intention-to-treat (ITT) principle. All statistical tests were two-sided, and statistical significance was defined as Po0.05.

Clinical secondary outcome The atherosclerotic process at the carotid artery was evaluated twice: at study initiation and after 12 months of intervention using Logic 9 ultrasonography, linear probe 10 mhz (GE Healthcare, Milwaukee, WI, USA). The common carotid artery (CCA) and the proximal internal carotid artery (ICA) far wall were interrogated for IMT and plaques, and measured with electronic calipers. In order to minimize technical differences between the two examinations, the sonographer made a maximum effort to obtain the same plaque profile in both examinations. The examinations were read by an experienced radiologist who was unaware of the patient’s group allocation. The difference between the two measurements regarding the number of plaques, structure of plaque, and IMT (when IMT was measurable) were used to define a new composite variable indicating whether the atherosclerotic process in the carotid arteries improved, worsened or did not change during the study. It must be noted that due to practical issues, patients who were not mobile (on wheelchair or bed bound) were unable to undergo the US examination. This limitation restricts our capability to generalize the results regarding the relation between pomegranate juice consumption and the improvement in the atherosclerotic process.

One hundred and one HD patients from 149 eligible HD patients, 101 were recruited and randomly assigned to two groups to receive either pomegranate juice or placebo (Scheme 1). The recruited patients did not differ from the eligible patients in demographic, dialysis, and comorbidities variables (data not shown). As described in Table 1, the two groups were similar at study initiation in patients’ demographics, comorbidities, treatment, and biochemical characteristics and the average interdialytic weight gain was between 2 and 4 kg/48–72 h, reflecting the ultrafiltration target loss. Median follow-up time for the two groups was 15 months (range 0.25–15.6 months). The dropout rate was 33.7%, higher among the pomegranate juice group (37.8%) compared to placebo (25.7%). This difference did not reach significance (P¼0.22). The main reasons for dropping out were mortality rates of 15.2 and 20%, as well as consent withdrawal rates of 15.2 and 5.7% among the pomegranate juice group and the placebo group, respectively. The latter included patients who disliked the flavor of the juice, more among the pomegranate juice group, and those who did not believe in the juice’s effects. Adverse events such as stomach upset or other GI-related effects were not demonstrated. Patients

Results Study population

149 Eligible patients

Infections events Infection events were defined as any hospitalizations due to fever. Events of fever among patients who were not admitted were not taken into consideration. Statistics Data analysis was done using SPSS statistical analysis software. Continuous data were reported as means and SD or median and IQR (interquartile range) when appropriate. The t test for independent samples or nonparametric tests such as Mann Whitney tests when appropriate were used to detect differences in continuous variables between the groups. Categorical data were described as frequency, and differences were assessed by the chi square test. In cases where expected values were lower than necessary, the Fisher exact test (nonparametric test) was

101 Patients randomized

Not included: N= 30- other clinical studies N= 18- refused to participate

Placebo N=35

Study initiation

Pomegranate juice N=66

N= 32 (91.4%)

3M of intervention

N= 63 (95.5%)

N= 27 (77.1%)

6M of intervention

N= 54 (81.8%)

N= 26 (74%)

12M of intervention

N= 41 (62%)

Scheme 1. Study flowchart.

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Table 1 Baseline characteristics of the study cohort at study initiation, pomegranate juice vs placebo. Characteristic Gender (%) Diabetes mellitus (%) CVD (%) Basic renal disease (%)

Dialysis access (%)

Dialysis membrane (%) Vasodilators (%) Beta blockers (%) Ca blockers (%) ACE inhibitors (%) EPO (%) Age (years) BMI (kg/m2) Vintage (years), Kt/V Systolic blood pressureb (mm Hg) Diastolic blood pressureb (mm Hg) Hemoglobin (g%) White blood cells (  103/mL) PMNLs (  103/mL) Total cholesterol (mg/dl) LDL (mg/dl) HDL (mg/dl) Triglycerides (mg/dl), MPO (ng/ml) CD11b (MFI) MDA (mM) AOPP (mM) Oxidized fibrinogen (nmol carbonyls/mg fibrinogen) IL-6 (pg/ml) TNF-a (pg/ml), Fibrinogen (mg/dl) Albumin (g/dl) CRP (mg/L),

Male Yes Yes Diabetic nephropathy Unknown Polycystic renal disease Glomerular nephritis others Fistula Catheter Graft Cellulose-triacetate Polysulfone Yes Yes Yes Yes Yes Mean7 SD Mean7 SD Median (IQR) Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean7 SD Median (IQR) Mean7 SD Mean7 SD Mean7 SD Mean7 SD Mean 7 SD Mean7 SD Median (IQR) Mean7 SD Mean7 SD Median (IQR)

Pomegranate Juice (N ¼ 66)

Placebo (N ¼35)

P value

56.1 62.1 63.6 47 19.7 15.2 6.1 12.1 56.1 37.9 6.1 71.2 28.7 16.7 39.4 43.9 7.6 80.3 65.97 11.2 27.97 7.8 2.3 (3.9) 1.37 0.3 145.07 22.1 70.17 13.5 11.27 1.1 7.77 2.4 5.27 2.1 164.8 737.1 94.67 31.8 33.17 9.5 150.5 (98.2) 97.17 767.4 27.16 712.2 4.47 2.0 184.6 772.6 1.377 0.75 6.167 4.3 4.8 (2.0) 515.3 7187.1 3.997 0.3 7.6 (13.6)

51.4 65.7 57.1 51.4 22.9 8.6 8.6 8.6 57.1 37.1 5.7 85.7 14.2 17.1 34.3 34.3 14.3 74.3 67.5 7 13.0 26.3 7 5.2 2.1 (3.4) 1.3 70.1 139.0 7 28.2 66.1 7 16.9 11.2 7 1.2 7.5 72.7 5.1 72.1 163.6 7 33.3 95.8 7 28.9 36.6 7 10.7 158.0 (103.0) 94.38 7 78.6 24.7 7 11.2 3.88 7 1.78 162.3 7 58.5 1.08 70.39 6.1 74.8 5.0 (2.5) 507.0 7114.1 3.92 7 0.3 5.5 (8.3)

0.65 0.72 0.54 0.84

0.99

0.39 0.95 0.61 0.35 0.28 0.49 0.53 0.28 0.99a 0.56 0.20 0.11 0.68 0.78 0.85 0.86 0.85 0.10 0.98a 0.85 0.31 0.20 0.12 0.07 0.98 0.34a 0.83 0.26 0.28a

MPO, myeloperoxidase; AOPP, advanced oxidation protein product; MDA, malondialdehyde. a b

Mann-Whitney test. Mean of three measurements taken before three dialysis sessions.

who dropped out from the study did not differ in demographic, comorbidities, treatment, and biochemical characteristics (data not shown). Furthermore, despite the 33.7% dropout rate, it should be noted that the baseline characteristics of patients who participated until the end of the study were similar between the two groups (data not shown), indicating retained randomization success.

initiation. No significant changes were demonstrated in the placebo group postintervention (Table 3). In the interest of simplification, fewer representative biomarkers are depicted in Fig. 1. In these, CD11b represents PMNL priming, oxidative stress is represented by protein (AOPP) and lipid (MDA) oxidation, and IL-6 represents inflammation. These biomarkers were also followed up in the 3-month post-1-year intervention, as shown in Table 3.

Primary outcome—Levels of biomarkers Secondary clinical outcomes All biomarkers measured showed amelioration during 1 year of pomegranate juice intake compared to placebo, using last observation carried forward method for missing data. A significant lower level of all oxidative stress and inflammatory biomarkers is demonstrated among the pomegranate juice group compared to the placebo group (Table 2). Oxidative stress and inflammatory biomarkers were also measured 3 months postintervention, among a randomly selected group of patients from the pomegranate juice and the placebo groups (n¼31 and 18, respectively, Table 3). The beneficial effects of pomegranate juice on oxidative stress and inflammatory biomarkers, demonstrated at the end of 1-year follow-up, were abolished, and were similar to those observed at the study

Ultrasound of the carotid arteries (Table 4) Ultrasound of the carotid arteries was performed only for the mobile patients (18 and 13 patients in the pomegranate juice and placebo groups, respectively). Improvements in the composite of IMT, as well as number and structure of plaques of carotid arteries, were observed in more than a quarter of the patients in the pomegranate juice group (27.8%), compared to no improvement in the placebo group. While, worsening in the composite measurements of the carotid arteries was observed in 53.8% of the placebo group, compared to only 5.6% of the pomegranate juice group. These differences were statistically significant (P¼ 0.004).

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Table 2 Oxidative stress and inflammatory biomarkers at end of follow-up (1 year), pomegranate juice vs placebo. Biomarkers

CD11b MFI MPO ng/ml AOPP mM Oxidized fibrinogen (nmol carbonyls/mg fibrinogen) MDA mM Albumin g/dl (half yeara) Fibrinogen mg/dl IL-6 pg/ml TNF-a pg/ml, median (IQR)

Pomegranate juice

Placebo

P.V

N

Mean7 SD

N

Mean7 SD

66 66 66 57 66 66 57 66 66

20.3 77.4 88.9 7 62.0 151.2 7 39.9 0.86 70.26 3.8 71.3 3.9 70.3 526.9 7 144.2 4.1 72.8 3.9 (1.6)

35 35 35 26 35 35 26 35 35

27.1 7 11.2 181.2 7 152.9 177.2 7 49.7 1.03 70.34 6.9 7 2.6 3.6 7 0.3 611.7 7 194.9 7.5 7 4.5 6.7 (6.8)

0.002 0.002 0.005 0.03 o0.001 o0.001 0.05 o0.001 0.03

Statistical analysis was performed by t test or Mann Whitney test. Last observation carried forward was used for missing data. a

See Materials and methods.

Table 3 Oxidative stress and inflammation 3 months post 1-year intervention. Biomarkers

Time

Pomegranate juice (n¼ 31)

Placebo (n¼ 18)

P.V1

P.V2

P.V3

AOPP (mM)

12 m of intervention 3 m postintervention 12 m of intervention 3 m postintervention 12 m of intervention 3 m postintervention 12 m of intervention 3 m postintervention

148.4 7 39.6 187.9 7 46.3 20.8 7 6.8 33.0 7 11.9 3.9 7 1.3 6.3 7 1.9 4.1 7 1.9 8.5 7 5.8

186.7 7 57.9 193.2 7 63.2 28.6 7 9.1 31.5 7 12.1 6.6 7 1.9 5.9 7 1.4 6.7 7 3.3 8.2 7 5.7

0.01 0.74 0.004 0.67 o0.001 0.50 0.001 0.88

o0.001

0.68

o0.001

0.29

o0.001

0.15

o0.001

0.06

CD11b (MFI) MDA (mM) IL-6 (pg/ml)

P.V1, pomegranate juice vs placebo, t test. P.V2, 12 m of intervention vs 3 m postintervention, among pomegranate juice group, paired t test. P.V3, 12 m of intervention vs 3 m postintervention, among placebo group, paired t test.

Hospitalization due to infections (events) Treatment with pomegranate juice was associated with lower incidence rates of the first (1.8 times lower) and second (5.3 times lower) infection events, compared to the placebo. In addition, the reduction in the incidence rate of the first event compared to the second event in each group was more pronounced in the pomegranate juice group (7.8 times reduction in pomegranate juice, compared to 2.6 reductions in placebo). Due to a low number of events, the difference in the first infection event between the two groups, as expressed by the Kaplan-Meier survival analysis, did not reach significance (P¼0.07, Fig. 2). However, Kaplan-Meier survival analysis regarding the second event exhibited a significantly higher proportion free of second infection event among pomegranate juice compared to placebo (P¼0.01, Fig. 2).

Discussion The present study provides an answer to the need for a wellconducted, prolonged clinical trial, in the field of natural antioxidant therapy among HD patients. This is the first study to show that 1 year of pomegranate juice intake reduced PMNL priming, oxidative damage, and inflammation; significantly lowered the incidence of hospitalization due to infections; and attenuated the atherosclerotic process in HD patients. Pomegranate juice intake had an accumulating effect on the biochemical parameters as expressed by a time–response relation between pomegranate juice intake and levels of the biomarkers of oxidative stress and inflammation. Interestingly, these beneficial effects were abolished 3 months following pomegranate juice discontinuation, validating our results.

Serum albumin, a well-established independent cardiovascular risk factor, has been reported by Kalantar-Zadeh et al. to decrease during the time a patient is treated by dialysis and is related to a significant increase in cardiovascular mortality [27]. In our study, not only did pomegranate juice intake prevent such a decrease, it also yielded a significant rise in serum albumin already after 6 months of intervention. The prevention of hypoalbuminemia concomitantly with the decrease in AOPP, and the improvement in the atherosclerotic process, which was also demonstrated by others [18], may forecast an improvement in cardiovascular morbidity and survival among these patients [27,28]. Decreased fibrinogen levels may also curb the exceedingly high cardiovascular risk of HD patients [29]. In our study, pomegranate juice intake for 12 months prevented the significant increase in fibrinogen which was demonstrated among the placebo group and yields a significant decrease in its oxidized form. The significant reduction in the rate of the second hospitalization due to infections demonstrates the beneficial accumulating effect of pomegranate juice on the innate immunity system. The significant reduction in the incidence of infections in our study is fascinating, suggesting that the attenuation in systemic inflammation due to pomegranate juice intake, as expressed by lowering the levels of interleukins, cytokines, and oxidative stress, in fact strengthened the innate immunity, thereby preventing infections. This link between the improvement in systemic inflammation followed by reduction in infections is supported by a previous report [5] The effect of antioxidant therapy on CVD reduction was demonstrated by others using antioxidant drugs such as NAC and Vit E [11,12]. Similar to these studies, we also demonstrated that antioxidants decreased morbidity but not mortality. We assume that a longer intervention period, as well as bigger sample

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Fig. 1. Mean (SE) of oxidative stress and inflammation biomarkers during the study period. P.V from linear mixed model with unstructured covariance structure.

Table 4 US of the carotid artery, pomegranate juice vs placebo. Group

Pomegranate juice Placebo a

Improvement

Worsening

No change

N (%)

P.Vna

N (%)

P.Va

N (%)

P.Va

5 (27.8) 0

0.05

1 (5.6) 7 (53.8)

0.004

12 (66.7) 6 (46.2)

0.25

Fisher exact test.

size, is required in order to demonstrate the antioxidant effect on mortality. Although the above studies suggest that the use of antioxidants is beneficial, they are not adequate enough to warrant widespread use of antioxidants as a sole treatment in these patients. Moreover, the effect of such intervention on the infection rates was not measured in these studies. In the field of natural antioxidants, the beneficial effect of polyphenolic rich fruit juices on HD patients was examined on a few, small, short-term clinical studies which focused only on oxidative stress-related parameters and not on long-term clinical outcomes [15–17]. Spormann et al. demonstrated a significant decrease of DNA oxidation, nuclear factor kB-binding activity, and an increase in glutathione level after uptake of 200 ml/day of red fruit juice for 4 weeks [17]. Also, red grape juice consumption was found to be associated with reduced plasma concentration of total cholesterol, oxidative LDL, and ex vivo

neutrophil NADPH oxidase activity as well as an increase in the concentration of antioxidant capacity of plasma [15,16]. What could be the common denominator of reduced infection rates and decreased atherosclerosis? The mechanism by which pomegranate juice intake reduced oxidative stress and inflammation is unclear. There are several assumptions, including scavenging free radicals [30], gene regulation [31], fructose-mediated uric acid production due to an hepatic metabolism that leads to a transient decrease in hepatic ATP and inorganic phosphate, which are important inhibitors of 50 -nucleotidase and AMP deaminase, respectively, and thus increased degradation of AMP to uric acid [32], and inhibition of enzymes related to inflammation, such as peroxisome proliferators active receptors (PPARs), nuclear transcription factor kB (NF-kB), and NSAID activated gene-1 (NAG-1) [33]. In addition, other studies have demonstrated that pomegranate juice has the ability to protect nitric oxide against oxidative destruction [34], to enhance the nitric oxide synthase bioactivity, and to up-regulate paraoxonase 1 and 2 which have an atheroprotective role in CVD and improves the innate immunity [35]. We have shown that pomegranate juice intake yields a prolonged reduction in PMNL priming. Primed PMNLs, expressed here by increased CD11b levels, are recently reported as contributors to oxidative stress and inflammation in chronic diseases associated with atherosclerosis and cardiovascular diseases [4]. Hence the correction of PMNL priming state, expressed herein also by the

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and innate immunity dysregulation [5], originating from PMNL priming, can affect the atherosclerotic process and the infection rates, defining PMNL priming as a nontraditional risk factor for both clinical states. In interpreting the above results, it should be asked whether the relations found are causal. In addressing this issue, one can see that all Hill’s criteria were met [37]: The clinical trial methodology enabled us to assure temporal relationship issues; our findings regarding the association between polyphenol-rich juice and oxidative stress reduction were shown to be consistent across other studies of divergent types and/or in divergent populations [15–17]; a time–response relationship was demonstrated between duration of intervention and oxidative stress/inflammation improvement; hence, there is a possible explanation for the relationship between pomegranate juice intake and the demonstrated results as previously described. Some methodological limitations should be noted: Although a single-center small study, the sample size was adequate to examine most of our assumptions. AOPP, a measure of protein oxidation, was determined as originally described by Witko-Sarsat [24], and not by the improved version [38]. Nevertheless, all oxidative modifications measured were attenuated in a similar manner. Last, the improvement in the accelerated atherosclerotic process was based on a semiquantitative evaluation by a single blinded radiologist and fits the other results of this study.

Conclusion One year of pomegranate juice intake ameliorates nontraditional CV and innate immunity risk factors in HD patients and improves clinical outcomes. Since these are novel findings, it is of crucial importance to replicate and validate them by multicenter studies, in order to formulate pomegranate juice intake as an integral part of the dialysis replacement therapy.

Acknowledgments This study was supported by Grant 6186 from the Chief Scientist Office of the Ministry of Health, Israel; Grant 2012255 from Jess & Midred Fisher Family Cardiology Research Fund, Office of the Executive Vice President for Research, Technion, Israel; and by Iscar Ltd. The authors are indebted to Hadya Sabik (B.Sc) from the Research Laboratory, Western Galilee Hospital, for the biomarkers analysis. The authors declare no financial conflict of interest. References Fig. 2. Kaplan-Meier survival curves from first (a) and second (b) infection event.

decreased degranulation of MPO, explains why pomegranate juice is beneficial in these patients. Since the immune system dysfunction and increased oxidative stress and inflammation processes are prevalent among CKD and hemodialysis (HD) patients [3], we suggest that primed PMNLs are the culprit of these clinical manifestations. PMNLs, which are already primed in CKD, are further activated by the interactions of blood cells with the dialyzer membrane, increasing generation of ROS and inflammatory mediators, undergoing increased apoptosis, ending in endothelial dysfunction and reduced phagocytic function [4,36]. Hence, the triad of oxidative stress, inflammation,

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