Pomegranate juice intake attenuates the increase in oxidative stress induced by intravenous iron during hemodialysis

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Pomegranate juice intake attenuates the increase in oxidative stress induced by intravenous iron during hemodialysis☆,☆☆ Lilach Shema-Didi a , Batya Kristal b, c , Liora Ore d , Galina Shapiro e , Ronit Geron b , Shifra Sela c, f,⁎ a

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

ARTI CLE I NFO

A BS TRACT

Article history:

The hemodialysis (HD) procedure induces oxidative stress (OS), which is further aggravated

Received 16 October 2012

by intravenous (IV) iron administration, aimed at correcting anemia of patients with HD. We

Revised 7 April 2013

have recently shown that 1 year of pomegranate juice (PJ) intake attenuated OS and

Accepted 8 April 2013

inflammation in patients with HD. In the current study, we hypothesized that a single dose of PJ can attenuate the enhanced OS and inflammation induced by both the dialysis

Keywords:

procedure and IV iron administration during HD session. Twenty-seven patients with HD

Oxidative stress

were randomized to receive PJ or placebo during 1 dialysis session with IV iron. Blood

Hemodialysis

samples were drawn before and after the session to asses OS biomarkers such as advanced

Iron

oxidation protein products and myeloperoxidase (MPO), whereas polymorphonuclear

Pomegranate juice

leukocyte (PMNL) counts served as an indirect measure of inflammation. At the end of the dialysis session, an increase in advanced oxidation protein products and MPO levels as well as a decrease in PMNLs counts were observed in the placebo group, whereas no significant changes occurred in the PJ group. The postdialysis increase in MPO levels in the placebo group is a direct result of PMNL degranulation, associated with postdialysis decrease in PMNL counts. Degranulation of PMNLs leads to the release of other cell moieties, such as inflammatory mediators and proteases that enhance inflammation. We conclude that PJ intake attenuated the increase in systemic OS and inflammation induced by IV iron administration during the dialysis session. These beneficial effects illuminate the previously observed attenuation in OS and inflammation in patients with HD on prolonged PJ intake. © 2013 Elsevier Inc. All rights reserved.

Abbreviations: OS, oxidative stress; HD, hemodialysis; IV, intravenous; PJ, pomegranate juice; AOPP, advanced oxidation protein products; MPO, myeloperoxidase; PMNL, polymorphonuclear leukocyte; ESRD, end-stage renal disease; ROS, reactive oxygen species. ☆ Authors' contribution and signatures: We state that this paper and its data have not been previously published. Each author has contributed to the conception and design of the work. We had full access to all of the data in this study, and we take complete responsibility for the integrity of the data and the accuracy of the data analysis. We declare that we have seen and approved the final version. We state that there are no conflicts of interests. We all fulfill the authorship criteria of the Uniform Requirements. ☆☆ Conflict of interest: None to declare. ⁎ Corresponding author. Eliachar Research Laboratory, Western Galilee Hospital, Nahariya, Israel. Tel.: +972 4 9107474, +972 507 887931(mobile); fax: +97249107474. E-mail address: [email protected] (S. Sela). 0271-5317/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nutres.2013.04.004

N U TR IT ION RE S E ARCH 3 3 ( 2 0 13 ) 44 2 –4 4 6

1.

Introduction

Intravenous (IV) iron replacement therapy is routinely used to achieve adequate erythropoiesis in patients with end-stage renal disease. However, the IV iron administration further enhances the oxidative stress (OS) [1–6] associated with hemodialysis (HD) treatment for these patients [7,8]. Upon IV administration of commercial iron formulations, free or labile iron is generated [9] and can be found in the blood of patients with HD [10]. Free iron, by virtue of its ability to flip between reduced and oxidized forms, is a potent inducer and aggravator of the oxidative processes, leading to severe oxidation via the “Fenton reaction.” It is well established that polymorphonuclear leukocytes (PMNLs) from patients with chronic renal failure are primed. Hence, they release faster reactive oxygen species (ROS; inflammatory mediators and chemoattractants), causing increased OS and peripheral counts of PMNLs, with the last reflecting low-grade inflammation [11]. Furthermore, in patients with end-stage renal disease treated with HD, the recurrent blood interaction with the dialyzers triggers increased activation of peripheral PMNLs and monocytes. Activated PMNLs generate and release ROS, proinflammatory cytokines, and intracellular contents such as myeloperoxidase (MPO), which aggravate the already present predialysis OS and inflammation state in patients with HD [11,12]. Such cell activation results in chronic cell recruitment, causing higher peripheral PMNL counts in patients with HD than in healthy subjects [11]. Pomegranate juice (PJ) is an important source of polyphenols, components that can attenuate iron damage by either chelation of free iron and/or scavenging of ROS, hence acting as antioxidants and anti-inflammatory agents [13–17]. We have recently shown that 1 year of PJ intake attenuated OS and inflammation in patients with HD [18]. The beneficial effects of prolonged PJ intake were demonstrated by various biomarkers, decreased PMNL priming, and improvement in clinical outcomes. It was demonstrated that PJ intake attenuated the progression of atherosclerosis and reduced patients' hospitalization rate [18]. The hypothesis of the present study was that intake of PJ, which contains 7 mmol/100mL of polyphenols, will attenuate iron oxidative damage produced during the dialysis session. The objective of the study was to assess the efficiency of a single dose of PJ, taken at the beginning of the dialysis session, to attenuate iron toxicity. We expected that PJ will lower the OS and inflammatory biomarkers observed at the end of the dialysis session that are caused by IV iron administration during the dialysis treatment.

2.

Methods and materials

2.1.

Subjects

Twenty-seven chronic patients with HD, with a mean time on dialysis of 2.9 ± 2.6 years (median, 2.4 years) and a mean age of 65.8 ± 11.8 years, were studied during 1 dialysis session. All patients underwent 4 hours HD sessions on low-flux highperformance cellulose-triacetate (Sureflux-190G; NIPRO,

443

Osaka, Japan) or polysulfone dialyzers (FX 10; Fresenius, Homburg, Germany), 3 times weekly, at the Renal Unit, Western Galilee Hospital, Nahariya, Israel. All patients have been on maintenance IV iron gluconate (62.5 mg 1-3 times/wk; Ferrlecit, U.S. LLC.) for at least 3 months. Exclusion criteria included patients who had undergone HD therapy for less than 3 months and patients with acute infection or inflammatory disease. The study end points were the level of OS and inflammation resulting from the combined effect of the dialysis procedure and IV iron gluconate, with and without PJ intake. The research was conducted in accordance with the Declaration of Helsinki, with all participants signing an informed consent form before study enrollment.

2.2.

Juice and placebo

To choose the commercial PJ with the highest polyphenols levels, several hand-squeezed juices and different commercial brands available in Israel were analyzed by a colorimetric assay for polyphenols, as previously described [19]. Pomegranate juice manufactured in Turkey and purchased from Naturafood (Givat Hen, Israel) was found to contain the highest concentration of polyphenols: 0.7mmol/100 mL of juice. The juice was kept at room temperature (<25°C) until used and consumed immediately after a bottle was opened. The potassium content of the juice was 115 mg/100 mL, less than one-tenth of the recommended daily consumption of potassium by patients with HD (between 1500 and 2000 mg daily). The placebo was prepared especially for this study by food engineers, to resemble the PJ in color and taste, but without polyphenols. During the placebo juice preparation, 2 independent testers performed flavor comparison to verify the similarity between PJ and placebo drinks.

2.3.

Study protocol

After 2 weeks without iron replacement therapy, subjects were randomly assigned in a ratio of 2:1, to receive either 100 mL PJ containing 0.7 mmol of polyphenols (n = 17 patients) or 100 mL placebo juice (n = 10 patients). The juice was given orally to the patients during the first hour of a dialysis session, with IV iron administration. Blood samples were taken from the arterial line, before and after the dialysis, to evaluate OS and inflammatory biomarkers (Scheme 1).

Scheme 1 – Study protocol.

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Patients, nurses, and medical and laboratory teams were blinded to patient group allocation. The smaller randomization ratio of 2:1, with only a modest loss in statistical power [20], was chosen for both ethical and practical reasons because the outcomes of OS and inflammation associated with dialysis, with and without IV iron, were already established by us [2,8].

2.4. Characteristics and assessment of OS and low-grade inflammation related to PMNLs Sera levels of MPO serve as a biomarker of OS and PMNL degranulation [11,21]: briefly, MPO is involved in the production of HOCl, a powerful oxidant and at the same time a marker of PMNL degranulation, because it is released as a result of PMNL priming and activation. Myeloperoxidase was determined by Human MPO Instant ELISA kit (Bender MedSystems GmbH, Vienna, Austria), according to the manufacturer's instructions. Advanced oxidation protein product (AOPP), a marker of protein oxidation, was measured in sera as previously described [22].

2.5. The postdialysis PMNL counts were used to assess inflammation. The differential blood counting was performed using LH 750 Beckman Coulter or Advia 2120 Bayer Health Care instruments (Nyon, Switzerland). To correct for the hemoconcentration resulting from the dialysis treatment, the hematocrit was measured before and after the dialysis session and the values of MPO, AOPP, and PMNLs were measuredafterdialysiswerecorrected,aspreviouslydescribed [2].

2.6.

Statistical analyses

Data analysis was performed using SPSS statistical analysis software (version 11.5; SPSS, Chicago, IL). Continuous data

with normal distribution were reported as means ± SD. t Tests or the Mann-Whitney test, when appropriate, was used to detect differences in continuous variables between the treatment groups. Frequency counts were calculated for categorical data. Differences in these variables were assessed by χ2 tests. In cases when expected values were lower than necessary, the Fisher exact test was used. The effect of IV iron replacement therapy and PJ/placebo intake on OS markers before and after a dialysis session was assessed by the paired t test or the Wilcoxon signed test, when appropriate, in each of the 2 groups (PJ and placebo). Statistical significance was defined as P < .05. The sample size (n = 27) has 80% power (P = .05) to detect a 50% difference in MPO blood levels between the PJ and the placebo groups. It was calculated based on our preliminary results, assuming a mean MPO level of 130 ± 35 ng/mL after a dialysis session.

3.

Results

After randomization, no significant differences were recorded between the PJ and the placebo groups in demographic, dialysis membrane type, comorbidities, and laboratory characteristics (Table 1). High levels of inflammatory markers such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were observed in both groups (Table 1; reference ranges, 0.361.17 pg/mL for IL-6 and 0.55-2.82 pg/mL for TNF-α). The effect of PJ or placebo intake on OS markers is depicted in Table 2. As demonstrated, in the placebo group, OS markers increased during dialysis with IV iron, whereas in the PJ group, no changes were demonstrated. In the PJ group, sera levels of AOPP postdialysis stayed at similar levels, whereas MPO even decreased a little compared with its predialysis levels. In the placebo group, postdialysis MPO and AOPP levels were significantly higher than their predialysis levels.

Table 1 – Baseline characteristics at study initiation (n = 27) Characteristics

Group 1, PJ (n = 17)

Group 2, placebo (n = 10)

Pa

Sex (male %) Age (y), means ± SD Diabetes mellitus (%) CVD (%) b Vascular access (fistula %) Dialysis membrane (cellulose-triacetate %) Vintage (y), means ± SD Systolic blood pressure (mm Hg), means ± SD Total cholesterol (mg/dL), (means ± SD LDL (mg/dL), means ± SD HDL (mg/dL), means ± SD Triglycerides (mg/dL), means ± SD WBC (×103/UL), means ± SD Hematocrit (%), means ± SD Ferritin (ng/mL), means ± SD Transferrin saturation (%) Albumin (g/dL), means ± SD IL-6 (pg/mL), means ± SD TNF-α (pg/mL), means ± SD

58.8 66.5 ± 11.1 58.8 52.9 70.6 76.5 3.1 ± 3.0 146.6 ± 22.8 159.3 ± 22.5 89.5 ± 17.8 30.1 ± 8.1 156.5 ± 49.1 6.8 ± 2.7 34.9 ± 3.1 284.9 ± 205.1 23.7 ± 3.5 4.1 ± 0.3 5.6 ± 4.1 5.5 ± 4.5

60.0 64.7 ± 13.1 60.0 50.0 60.0 80.0 2.5 ± 1.4 135.5 ± 26.2 162.4 ± 50.9 89.8 ± 21.9 36.2 ± 10.2 162.2 ± 58.6 7.7 ± 3.0 35.8 ± 2.8 292.9 ± 144.8 21.6 ± 6.3 4.0 ± 0.2 6.2 ± 3.4 5.7 ± 1.7

.95 .57 .95 .88 .53 .18 .90 .17 .76 .78 .22 .28 .46 .45 .92 .28 .10 .69 .87

a

χ2 Test or t test. Cardiovascular disease—included acute myocardial infarction, congestive heart failure, ischemic stroke, peripheral vascular disease, or unstable angina.

b

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Table 2 – OS marker level before and after dialysis session with IV iron, by marker type and intervention group Marker

Group (n)

Before, means ± SD

After, means ± SD

PV a

PV b

PV c

AOPP (μM)

Placebo (8) PJ (17) Placebo (9) PJ (16) Placebo (9) PJ (17)

146.0 ± 19.3 158.4 ± 39.4 95.7 ± 68.8 82.8 ± 41.9 5.0 ± 2.8 4.18 ± 1.9

217.5 ± 80.8 158.1 ± 55.1 152.9 ± 128.4 69.4 ± 74.4 4.3 ± 2.6 4.4 ± 1.7

.03 .97 .04 .53 .004 .28

.27

.04

.81

.04

.45

.96

MPO (ng/mL) PMNLs (×103/UL)

PV, P value. a Paired t test or Wilcoxon signed ranks test, before vs after a dialysis session. b t Test or Mann-Whitney, before dialysis session, PJ vs placebo. c t Test or Mann-Whitney, after dialysis session, PJ vs placebo.

In the placebo group, postdialysis PMNL counts were significantly lower than predialysis levels, whereas in the PJ group, PMNL counts remained stable. Although it has to be emphasized that after randomization, the placebo group started the study with higher predialysis PMNLs counts than the PJ group; nevertheless, no statistical significance could be shown at this time point. Because the predialysis and postdialysis results are paired for each patient, we can confidently suggest that there is a significant decrease in PMNL counts in the placebo group, whereas no statistically significant change could be seen in the PJ group (Table 2).

4.

Discussion

Various protocols for IV iron administration have been established in order to reduce the iron-induced OS during HD treatment [23]. The results of this study strengthen our hypothesis and suggest a new approach aimed at overcoming the increase in OS caused by the combination of the dialysis process and iron toxicity during one session of HD. We show for the first time that PJ intake during the first hour of dialysis treatment has significant beneficial effects, preventing the aggravation of OS induced by IV iron administration during the dialysis session. These beneficial effects are probably due to the PJ's potent antioxidant contents [13–17], linked to the high concentration of polyphenols in the juice used in this study (70 mM). Our study has demonstrated that a single oral dose of 100 mL PJ significantly reduced OS and abolished the decrease in the peripheral PMNL counts postdialysis. We and others have shown that the interactions between PMNLs and the dialyzer membrane cause PMNL activation, reflected by increased generation of ROS and cell degranulation and resulting in the release of intracellular cell constituents [11]. The findings that patients' postdialysis MPO sera levels increased imply that peripheral PMNLs were involved in this process. In this study, the decreased postdialysis PMNLs counts in the placebo group imply that IV iron administration during HD caused PMNLs degranulation and disintegration, a process that was attenuated by PJ intake. Polymorphonuclear leukocyte disintegration can also result in the release of other inflammatory mediators such as IL-6 and TNF-α [18] and proteases such as elastase and cathepsin G [12], implying an acceleration of the inflammatory cycle. Levels of MPO and AOPP were considerably increased in the placebo group and significantly less so in the PJ group. These

findings are noteworthy because high MPO levels in blood results in increased OS in these patients [21,24]. Our results are supported by other studies that have shown that oral antioxidant therapies such as vitamin E and melatonin have the ability to reduce iron oxidative damage by diminishing the oxidation that occurs during a dialysis session, for example, lipids' peroxidation [6,25]. No decrease in PMNLs count was demonstrated in dialysis session without iron (data not shown). A decrease in PMNL counts at the end of the dialysis session among the placebo group is indicative of cell disintegration because of remarked activation by the dialysis membrane and iron, which was prevented by PJ intake. In patients with HD, the continuous contact between activated PMNLs and the vascular endothelial layer exposes the endothelium to OS and inflammatory mediators, resulting in chronic injury and initiation of the atherosclerotic process [26]. Furthermore, AOPP, beyond serving as a marker of protein oxidation, acts as a proinflammatory mediator, activating leukocytes [27], and is also associated with increased common carotid intima-media thickness [28]. A single dose of PJ was shown to lower postdialysis AOPP levels, emphasizing its beneficial effect on protein oxidation. The limitations of this study lay in the small sample size; we are therefore unable to stratify data by membrane type (cellulose-triacetate or polysulfone membranes). Nonetheless, we do not have any reason to assume that the latter introduced any significant bias because the distribution of the various dialysis membrane types was similar between the 2 groups (PJ and placebo). We did not examine the rate of PJ absorption, and we did not collect data on the concentration of polyphenols in patient's blood; however, because a significant effect is seen only among the PJ group, whereas no other intervention was done, we associated this effect with the PJ intake. Intravenous iron is essential for treating anemia in most patients with HD, but its administration, even in small slow doses, is accompanied by PMNL priming and protein oxidation [2,3,8]. We have found that oral PJ intake, together with IV iron taken during the first hour of the dialysis session, prevented PMNL degranulation and disintegration, reflected by no change in sera MPO levels postdialysis. Preventing the increase in sera MPO level in each dialysis by PJ intake could have accumulating beneficial effects, as was demonstrated in our year-long study of PJ intake [18]. Although predialysis MPO levels increased ~2-fold in patients with HD for 1 year, PJ intake completely abolished this increase. The beneficial effects of a

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single dose of PJ intake during 1 dialysis session can explain the advantageous impact of prolonged PJ intake on OS, inflammation, and clinical outcomes in patients with HD, which were demonstrated in our recently published study [18]. These findings add an important facet to the PJ intervention, which may, in the long run, reduce cardiovascular morbidity, so common in these patients. The beneficial effects of both single-dose and long-term PJ intake [18] should be further validated by multicenter studies to establish a routine antioxidant therapy for patients with HD.

Acknowledgment This study was supported by Grant No. 6186 from the Chief Scientist Office of the Ministry of Health, Israel; Grant No. 2012255 from Jess & Midred Fisher Family Cardiology Research Fund, Office of the Executive Vice President for Research, Technion, Israel; and Iscar Ltd. The authors are indebted to Hadya Sabik (BSc) from the Research Laboratory, Western Galilee Hospital, for the biomarker analysis.

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