ORIGINAL RESEARCH

Effects of Oral Supplementation With Omega-3 Fatty Acids on Nutritional State and Inflammatory Markers in Maintenance Hemodialysis Patients Afshin Gharekhani, PharmD,* Mohammad-Reza Khatami, MD,† Simin Dashti-Khavidaki, PharmD,*,† Effat Razeghi, MD,† Alireza Abdollahi, MD,‡ Seyed-Saeed Hashemi-Nazari, MD, MPH, PhD,§ and Mohammad-Ali Mansournia, MD, MPH, PhD{ Objective: The objective was to determine the effects of omega-3 supplementation on nutritional state and inflammatory markers of hemodialysis patients. Design and Methods: This was a randomized, placebo-controlled trial. Adult patients undergoing maintenance hemodialysis were included. Patients with malignancy, pregnancy, concurrent inflammatory or infectious diseases, or concomitant use of any medication affecting inflammation status were excluded. The omega-3 group received 6 soft-gel capsules of fish oil (180 mg eicosapentaenoic acid and 120 mg docosahexaenoic acid in each) daily for 4 months, and the placebo group received corresponding paraffin oil capsules.Nutrition indices including body mass index; mid-arm muscle circumference; serum concentrations of albumin, prealbumin, and transferrin; and serum levels of inflammatory/anti-inflammatory markers including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-a, C-reactive protein, ferritin, parathyroid hormone, and ratios of IL-10 to TNF-a and IL-10 to IL-6 were measured before and after 4 months of intervention. Results: Twenty patients in the placebo and 25 patients in the omega-3 group completed the study. There were no significant changes in nutritional markers between the omega-3 and placebo groups after 4 months of intervention. Regression analysis adjusting post-treatment values of nutrition markers for baseline values, omega-3 treatment, and patients’ baseline demographic and clinical data revealed that omega-3 treatment was a significant independent predictor of increased serum prealbumin level (182.53; 95% confidence interval 21.14, 511.18; P 5 .11). Although slight reduction of inflammatory state was observed in the omega-3 group, no significant differences were evident in the mean changes of inflammatory and anti-inflammatory markers between the 2 groups with the exception of serum ferritin level and the IL-10 to IL-6 ratio, which significantly changed in favor of omega-3 supplementation (P , .001 and P 5 .003, respectively). Conclusions: Omega-3 supplementation in hemodialysis patients produced a slight attenuation in systemic inflammation without any remarkable effects on nutritional markers. Ó 2014 by the National Kidney Foundation, Inc. All rights reserved.

Introduction

P

ROTEIN-ENERGY WASTING (PEW) or malnutrition exists in approximately 20% to 50% of chronic hemodialysis (HD) patients and sharply increases the risk of * Department of Clinical Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. † Nephrology Research Center, Tehran University of Medical Sciences, Tehran, Iran. ‡ Laboratory Department, Vali-e-Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran. § Department of Epidemiology, School of Public Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran. { Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Support: See Acknowledgments on page 184. Address correspondence to Simin Dashti-Khavidaki, Pharm D, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, 1417614411, PO Box 14155/6451. E-mail: [email protected] Ó 2014 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2014.01.014

Journal of Renal Nutrition, Vol 24, No 3 (May), 2014: pp 177-185

morbidity and mortality in this population.1-3 It frequently coexists with inflammation and atherosclerosis, which together form a complex syndrome termed malnutritioninflammation-atherosclerosis syndrome.4 Cardiovascular diseases represent major causes of mortality in HD patients.5 Malnutrition-inflammation-atherosclerosis syndrome may play a pivotal role in the etiology of premature cardiovascular disease in HD patients.6 One or more defective cycles can explain concurrent occurrence of malnutrition, inflammation, and atherosclerosis. Malnutrition induces some degree of immune paresis, predisposing individuals to infection and inflammation. In the meantime, inflammatory and infectious processes promote the production of catabolic and anorectic cytokines, leading to increased risk of malnutrition and progressive atherosclerosis.1,4 Despite recent advancements in improving the nutritional and inflammatory state of chronic HD patients, no single medication has thus far been able to correct malnutrition-inflammation complex syndrome.5,7 Several published studies have recently demonstrated the 177

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beneficial effects of omega-3 fatty acids in HD patients, with most focusing on markers of inflammation and nutrition.8-12 Perunicic-Pekovic and colleagues reported a significantly less amount of omega-3 fatty acids in the composition of erythrocyte membrane phospholipids in HD patients than healthy controls, leading to a worsened underlying malnutrition-inflammation condition.9 However, clinical trials examining the ability of omega-3 fatty acids to alleviate malnutrition-inflammation in HD patients have thus far been inconclusive. Given the controversial reports about the effects of omega-3 fatty acids on inflammatory and nutritional state, this trial was intended to test that administration of omega3 fatty acids (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) could correct inflammation and improve the nutritional state of maintenance HD patients.

Methods Study Population The study presented here was conducted as a randomized, single-blind, placebo-controlled trial. Adult patients who were undergoing regular HD treatment for at least 3 months at the HD units of 2 teaching hospitals (Imam Khomeini Hospital Complex and Sina Hospital) affiliated with the Tehran University of Medical Sciences, Tehran, Iran, were screened. Patients were not qualified for the study if they had a malignancy, malabsorption syndrome, inflammatory or infectious diseases, medical or surgical illness in the previous 3 months, hypothyroidism, asthma, chronic obstructive pulmonary disease, hemoglobinopathies, coagulopathies, or known psychiatric disorders; if they were participants in a concurrent or recent research study; if they did not comply with HD or medication regimens; or if they were pregnant. Subjects taking nonsteroidal anti-inflammatory drugs, corticosteroids, anticoagulants, omega-3 fatty acid supplementation during the previous 3 months, immunosuppressants or immunomodulators, or those with hypersensitivity to fish or fish-derived products were also excluded. The local Ethics Committee of Tehran University of Medical Sciences approved the study presented here. Informed written consent was obtained from all participants. The trial was registered in the Iranian Registry of Clinical Trials (registry number IRCT201202203043N5). Study Protocol Eligible patients were given 2 soft-gel capsules of either omega-3 or placebo 3 times daily, provided by Zahravi Pharmaceutical Company, Tabriz, Iran. Each omega-3 capsule contained 180 mg EPA and 120 mg DHA (a total of 1,800 mg omega-3 fatty acids per day) whereas placebo capsules were composed of paraffin oil. The study lasted for 4 months. The size, color, shape, and packaging pattern of placebo and omega-3 capsules were the same. All participants were asked to maintain their drug regimen, dietary

habits, and level of physical activity unchanged during the study period. Compliance was ascertained by providing participants with the weekly dose of capsules and having them give the unused capsules back from the prior week as well as by interviewing each participant individually thrice weekly to encourage adherence. Moreover, a nephrologist met each patient monthly during a routine HD session to increase adherence to the recommended supplement regimens and to monitor the tolerability and adverse effects of supplementation. Any adverse effects or medication changes were recorded over the course of the study. At baseline and month 4, a 10-mL fasting blood sample was drawn from each patient immediately before HD commencement. Serum samples were separated from blood by centrifugation at 3,000 rpm for 10 minutes and then stored at 270 C for later measurement of serum prealbumin, albumin, transferrin, ferritin, intact parathyroid hormone (iPTH), and proinflammatory (interleukin [IL]-6, tumor necrosis factor [TNF]-a, C-reactive protein [CRP]) and anti-inflammatory cytokine (IL-10) concentrations.

Measurements Nutritional status of the patients was assessed using measurement of serum albumin by the bromocresol green colorimetric method (Autoanalyzer BT3000, Biotechnica, Italy), transferrin by the turbidimetry method (Selectra E, Elitec, France), and prealbumin by an enzyme-linked immunosorbent assay kit (Bioassay Technology Laboratory, Shanghai Crystal Day Biotech Co., Ltd., Shanghai, China). Mid-arm muscle circumference (MAC), dry body weight (weight after HD treatment), and body mass index (BMI) were also measured. Serum CRP level was measured by a turbidimetric method (Autoanalyzer BT3000, Biotechnica, Italy). Serum IL-6, TNF-a, and IL-10 were measured using enzymelinked immunosorbent assay kits (Bioassay Technology Laboratory). Dialysis dose (termed as single-pool Kt/V urea) was calculated for each patient at the beginning and after 4 months of intervention using existing software that is based on pre- and postdialysis serum urea nitrogen concentrations, dialysis time, ultrafiltration volume, and postdialysis weight. Statistical Methods All statistical tests were performed using Stata software (StataCorp, 2011, Stata Statistical Software: Release 12. College Station, TX, StataCorp LP). The normality of distributions of continuous variables was tested by quantile normal plot, normal probability plot, and the KolmogorovSmirnov test. Categorical variables were compared between groups using the c2 test. Two independent-sample t tests and the Wilcoxon-Mann-Whitney test were used for a crude comparison of means for variables with normal and non-normal distributions, respectively. Furthermore,

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Figure 1. Study flowchart of patient screening and randomization procedure.

we estimated the effect of omega-3 treatment on nutrition indices using the linear regression model with omega-3 treatment, the baseline value of nutrition marker, the preand postintervention values of cytokines, and patients’ demographic and clinical data as explanatory variables. Because of the non-normality and heterogeneity of variances of the residual distributions, we used a nonparametric bootstrap method with 2,000 replications to obtain empirical standard errors and bias-corrected and accelerated confidence intervals. Categorical data were reported as number and percentage whereas continuous data were expressed as mean 6 standard deviation (SD). P values less than .05 or a confidence interval not including a null value were considered statistically significant.

Results Figure 1 displays the screening and randomization procedure of the study. Sixty-four of 155 patients that were

screened for eligibility fulfilled the entrance criteria (41%). Of those, 54 patients consented to consume the supplements and were included in the trial. There were 9 dropouts (2 in the omega-3 and 7 in the placebo group) during the study; thus, 45 subjects (20 in the placebo and 25 in the omega-3 group) completed the intervention period of the study, and their data were included in the final analysis. A summary of baseline characteristics of patients who completed the study is presented in Table 1. Both groups were comparable at baseline with respect to the demographic (gender and age) and clinical (HD duration and underlying causes of end-stage renal disease) data. Dialysis dose was significantly lower in the placebo group than the omega-3 group at baseline, but this difference did not persist during the study so that 2 groups were comparable for dialysis dose at the end of the study (data not shown). In search to clarify the effect of omega-3 fatty acids on systemic inflammation in HD subjects, we observed that

Table 1. Patients’ Demographic and Clinical Data at Baseline in the Placebo and Omega-3 Group Characteristics Age (y) (6SD) Sex, n (%) Females Males Hemodialysis duration (mo) (6SD) Dialysis adequacy (Kt/V) Cause of end-stage renal disease, n (%) Diabetes Hypertension Other causes

Placebo (n 5 20)

Omega-3 Fatty Acids (n 5 25)

P

57.2 (615.19)

56.8 (613.09)

.925 .592

8 (40) 12 (60) 72.05 (660.51) 1.25 6 0.17

12 (48) 13 (52) 59.88 (645.69) 1.43 6 0.24*

7 (35) 8 (40) 5 (25)

Age and hemodialysis duration are expressed as mean 6 SD. *P , .05 was considered as statistically significant.

12 (48) 9 (36) 4 (16)

.450 .010* .624

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omega-3 supplementation decreased serum concentrations of TNF-a, IL-6, and CRP, and it increased serum levels of IL-10 and ratios of IL-10 to TNF-a and IL-10 to IL-6. In addition, there was an increase in serum iPTH and ferritin levels, as surrogates of inflammatory state, in both groups throughout the study period, with much more of an increase in the placebo group. However, nonparametric comparison of mean change (post-treatment minus baseline) between the 2 groups demonstrated a significant difference exclusively in the IL-10 to IL-6 ratio and the serum ferritin level (Table 2). Moreover, a regression analysis with bootstrap method showed that omega-3 treatment was a significant independent predictor of reduced serum ferritin and iPTH levels and an increased IL-10 to IL-6 ratio when their post-treatment values were adjusted for their baseline values and patients’ demographic and clinical data (Table 3). In our study, in comparison with the placebo group, supplemental use of omega-3 fatty acids did not produce significant changes in nutrition indices, including BMI, dry body weight, MAC, and serum transferrin concentration (Table 2). On the other hand, serum prealbumin concentration increased in omega-3-treated patients whereas its concentration decreased in the placebo group during the study period. Nonetheless, no significant difference was observed in the mean change of serum prealbumin concentration between the 2 groups at the end of the study. In addition, despite a lack of significant difference in serum albumin concentration between the 2 groups at baseline,

the mean change in the omega-3 group was significantly lower than that in the placebo group (P 5.018). Table 4 depicts statistically significant determinants of postintervention values of nutrition indices in a linear regression model. HD vintage had negative associations with postintervention BMI and dry body weight (P ,.001 for both). In addition, HD vintage was the sole significant and positive predictor of postintervention serum transferrin concentration (P 5 .005). A significant negative association was also found between serum albumin concentration and age of patients (P , .05). Among the nutrition indices measured in our study, omega-3 treatment was only a significant positive predictor of serum prealbumin concentration. To know whether the beneficial effects of omega-3 fatty acids on serum prealbumin are mediated through their ameliorative effects on the inflammatory process, serum prealbumin concentration was adjusted for inflammation markers in different regression models. Regression analysis revealed that the ameliorative effect of omega-3 treatment on serum prealbumin level was closely related to its suppressive effects on serum iPTH level because omega-3 treatment was no longer a significant predictor of postintervention serum prealbumin level in the presence of iPTH as a covariate in linear regression. Over the course of the study, omega-3 and placebo capsules were well tolerated without any reports of serious adverse events. Mild transient gastrointestinal complaints including nausea, burping, and loose stool were frequently

Table 2. Indices of Inflammation and Nutrition in the Omega-3 and Placebo Groups Inflammation/ Nutrition Indices IL-6 (ng/L) TNF-a (ng/L) IL-10 (pg/mL) IL-10/IL-6 IL-10/TNF-a CRP (mg/L) Ferritin (ng/mL) iPTH (pg/mL) Prealbumin (mg/mL) Albumin (g/dL) Transferrin (mg/dL) Dry body weight (kg) BMI (kg/m2) MAC (cm)

Preintervention

Postintervention

Omega-3

Placebo

Omega-3

144.38 6 167.66 122.09 6 171.69 123.94 6 71.91 1.32 6 0.55 1.35 6 0.29 9.24 6 8.79 893.36 6 743.54

97.75 6 138.48 77.16 6 62.47 108.55 6 64.59 1.59 6 0.41 1.85 6 1.13 7.74 6 4.68 356.52 6 163.08

136.85 6 183.56 116.57 6 172.32 132.06 6 94.81 1.96 6 1.19 1.44 6 0.28 8.65 6 7.83 997.22 6 1,105.47

330.72 6 553.69 272.26 6 233.51 495.31 6 928.55 380.55 6 509.69 258.68 6 428.56 414.28 6 559.04 4.41 6 0.82

3.98 6 0.37

4.01 6 0.36

222.10 6 51.72 246.65 6 86.86 175.50 6 53.55

Placebo 106.48 6 136.51 77.43 6 59.82 107.24 6 63.01 1.39 6 0.35 1.69 6 0.95 12.69 6 14.02 1,236 6 885.85 624.93 6 1,057.51 151.25 6 94.71

Change Omega-3 27.53 6 126.01 25.52 6 46.92 8.11 6 57.63 0.64 6 1.14* 0.09 6 0.25 21.25 6 5.68 165.45 6 886.59*

Placebo 2.94 6 206.17 23.5 6 49.21 24.51 6 90.79 20.18 6 0.53 20.17 6 1.17 4.96 6 12.59 890.05 6 862.71

P .11 .97 .44 .003* .399 .143 .000*

14.65 6 235.88 383.39 6 927.49 .17 33.73 6 556.27 2122.40 6 398.78 .196

4.28 6 0.48

20.33 6 0.79*

185.12 6 48.39

251.06 6 55.08

0.34 6 0.64

.018*

261.21 6 104.14 .713

62.16 6 9.70

64.20 6 10.11

62.74 6 9.99

64.80 6 12.01

0.58 6 1.99

0.74 6 3.83

.891

23.84 6 3.82 26.32 6 2.88

23.29 6 3.24 24.75 6 3.68

24.07 6 3.91 27.82 6 6.14

23.88 6 4.46 25.83 6 3.63

0.23 6 0.81 1.5 6 4.16

0.48 6 1.51 1.11 6 2.08

.892 .653

BMI, body mass index; CRP, C-reactive protein; IL, interleukin; iPTH, intact parathyroid hormone; MAC, mid-arm muscle circumference; TNF-a, tumor necrosis factor-a. All data are expressed as mean 6 SD. P values, pertaining to comparisons of changes between groups, were derived from Mann-Whitney U test with the exception of transferrin and IL-10/TNF-a, which were derived from unpaired Student’s t test. *P , .05 was considered as statistically significant.

CI, bias-corrected and accelerated confidence interval; CRP, C-reactive protein; IL, interleukin; iPTH, intact parathyroid hormone; pre-, preintervention; post-, postintervention; TNF, tumor necrosis factor. *All analyses were adjusted for age, sex, hemodialysis duration, and baseline values of Kt/V and outcome variables. †P , .05 or 95% CI (with bootstrap method) not including null value was considered as statistically significant.

.356 .001* .053† (29.260, 2.417) (21,533.274, 2412.422) (21,150.008, 2129.641) .633 (20.672, 0.226)

2414.247 213.935 2922.484 282.992 22.789 3.022 20.103 0.216

Omega-3 treatment Coefficient (b) 225.877 26.566 25.389 0.894 46.403 11.973 20.366 0.338 Bootstrap standard error P .577 .583 .791 .008* 95% CI (2147.422, 42.715) (232.778, 14.654) (263.172, 22.849) (0.319, 1.603)

Post-iPTH Post-ferritin Post-CRP Post-IL-10/IL-6 Post-IL-10/TNF-a Post-IL-6

Post-TNF-a

Post-IL-10

Dependent Variable*

Independent variable

Table 3. Linear Regression Analysis Demonstrating the Effect of Omega-3 Treatment on Postintervention Values of CRP, Ferritin, iPTH, and Cytokines After Adjustment for Their Baseline Values and Patients’ Demographic and Clinical Data

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the reported side effects and mostly occurred in omega-3treated patients.

Discussion It has been well established that inflammation is a prominent feature of chronic kidney disease in predialysis and dialysis patients.13 Proinflammatory comorbid conditions (e.g., infections, diabetes mellitus, and atherosclerosis), uremic toxins, modality of dialysis, retained inflammatory mediators, oxidative stress, insulin resistance, and diet are several different factors that may contribute to the high prevalence of inflammation in this patient population.1,13 Given the established role of chronic inflammation in developing PEW,14 interventions with anti-inflammatory properties may improve the nutritional state of chronic HD patients. Although HD patients consume inadequate omega-3 fatty acids15 and have lower plasma levels of these fatty acids,16 no well-established recommendation has been made on omega-3 supplementation in this population. Our study examined the potential of omega-3 fatty acid supplementation to improve the nutritional and inflammatory state of HD patients. Although a significant difference, in favor of omega-3 treatment, was exclusively observed in the mean change of serum ferritin and the ratio of IL-10 to IL-6 between the 2 groups, administration of omega-3 fatty acids also produced slight desirable changes in serum inflammatory markers, as evident in the serum concentrations of IL-6, TNF-a, CRP, IL-10, iPTH, and the IL-10 to TNF-a ratio. Failure to obtain significant anti-inflammatory effects by omega-3 supplementation in our study may partly be explained by the relatively small dosage of omega-3 fatty acids or the short follow-up period. It is important to note that most of the studies investigating the effects of omega-3 fatty acids on the inflammatory markers of HD patients are not comparable because of a wide variation in study design, supplement dosage, study duration, and the EPA to DHA ratio. Saifullah and colleagues demonstrated that oral supplementation of 1.3 g of EPA and DHA daily for 3 months significantly lowered serum CRP levels.10 Himmelfarb and colleagues examined the effects of a gamma-tocopherolenriched mixture of tocopherols and DHA (providing 924 mg gamma-tocopherol and 1,200 mg DHA per day) on the inflammation markers of HD patients.17 After 6 months of supplementation, the serum IL-6 level showed a significant reduction compared with the baseline value whereas no significant change was observed in serum CRP level. In addition, Perunicic-Pekovic and colleagues found a significant decrease in the serum IL-6 and TNFa levels of HD subjects treated with 2.4 g of omega-3 fatty acids daily for 2 months.9 Bowden and colleagues showed that administration of 2 soft-gel capsules of omega-3 supplement with each meal for 6 months (providing a total of 960 mg EPA and 600 mg DHA per day) produced a significant reduction in serum CRP level.18 On the other

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Table 4. Regression Analysis Reflecting the Effect of Omega-3 Treatment on Nutrition Indices After Adjustment for Their Baseline Values, Age, Sex, Baseline Kt/V, and HD Vintage Dependent Variable Post-albumin

Post-transferrin

Post-MAC

Post-BMI

Post-dry weight

Baseline Value

Age

Sex

Baseline Kt/V

HD Vintage

b 5 20.18 95% CI (20.53, 0.06) P 5 .233 b 5 182.53 95% CI (21.14, 511.18)* P 5 .110 b 5 21.31 95% CI (236.04, 29.52) P 5 .936 b 5 0.88 95% CI (21.27, 4.73) P 5 .542 b 5 20.57 95% CI (21.34, 0.08) P 5 .117 b 5 20.89 95% CI (22.53, 0.83) P 5 .295

b 5 0.05 95% CI (20.22, 0.23) P 5 .645 b 5 0.54 95% CI (0.13, 0.92)* P 5 .055 b 5 0.17 95% CI (20.10, 0.52) P 5 .274 b 5 1.14 95% CI (0.61, 2.12) P 5 .001* b 5 1.07 95% CI (0.94, 1.17) P 5 .000* b 5 1.09 95% CI (0.99, 1.18) P 5 .000*

b 5 20.01 95% CI (20.02, 20.001) P 5 .048* b 5 22.67 95% CI (212.29, 5.06) P 5 .519 b 5 1.11 95% CI (20.34, 2.44) P 5 .123 b 5 20.03 95% CI (20.15, 0.05) P 5 .537 b 5 20.02 95% CI (20.05, 0.01) P 5 .141 b 5 20.06 95% CI (20.13, 0.03) P 5 .151

b 5 20.01 95% CI (20.26, 0.32) P 5 .950 b 5 78.08 95% CI (2194.62, 318.25) P 5 .554 b 5 25.35 95% CI (246.72, 21.80) P 5 .743 b 5 1.33 95% CI (20.60, 4.83) P 5 .308 b 5 20.09 95% CI (20.80, 0.71) P 5 .817 b 5 0.93 95% CI (20.97, 3.06) P 5 .370

b 5 20.23 95% CI (20.70, 0.27) P 5 .350 b 5 572.07 95% CI (232.79, 1,159) P 5 .050 b 5 234.26 95% CI (296.89, 60.35) P 5 .374 b 5 23.75 95% CI (213.67, 0.85) P 5 .274 b 5 0.71 95% CI (21.01, 2.44) P 5 .391 b 5 2.73 95% CI (22.06, 8.28) P 5 .253

b 5 20.0005 95% CI (20.004, 0.002) P 5 .742 b 5 0.89 95% CI (20.88, 3.32) P 5 .402 b 5 0.48 95% CI (0.14, 0.81) P 5 .005* b 5 0.01 95% CI (20.01, 0.05) P 5 .596 b 5 20.01 95% CI (20.02, 20.01) P 5 .000* b 5 20.03 95% CI (20.05, 20.02) P 5 .000*

CI, bias-corrected and accelerated confidence interval; HD, hemodialysis; post-albumin, postintervention albumin; post-BMI, postintervention body mass index; post-dry weight, postintervention dry weight; post-MAC, postintervention mid-arm circumference; post-prealbumin, postintervention prealbumin; post-transferrin, postintervention transferrin. *P , .05 or 95% CI (with bootstrap method) not including null value was considered as statistically significant.

GHAREKHANI ET AL

Post-prealbumin

Independent Variables Omega-3

OMEGA-3, NUTRITION, INFLAMMATION IN HD PATIENTS

hand, a recent study on HD patients using 2.08 g of omega3 fatty acids for 10 weeks failed to exert any effects on serum CRP, IL-6, and TNF-a concentration.19 Consistent with our findings, Hassan and colleagues reported that omega3 supplementation (2.4 g DHA and 1 g EPA per day orally) to peritoneal dialysis patients for 8 weeks produced an insignificant decrease in the serum inflammatory markers, including CRP, IL-6, and TNF-a.20 Our results were also in agreement with those obtained by Poulia and colleagues, in which supplemental use of omega-3 fatty acids (1,680 mg per day) for 4 weeks caused an insignificant reduction in serum CRP levels in chronic HD patients.21 Furthermore, omega-3 treatment in our study was a significant and independent predictor of reduced serum ferritin and iPTH level. With implementing a careful monitoring during the study period and having free access to intravenous iron sucrose and erythropoietin, it seems that a large amount of the increase or decrease in serum ferritin level is related to a systemic inflammatory state in HD patients. In addition, none of the patients in our study received cinacalcet to control hyperparathyroidism, and the number of patients taking calcitriol was comparable between the 2 groups. Taken together, significant association of omega3 treatment with reduced serum ferritin and iPTH levels might reasonably be translated into a relative attenuation of systemic inflammation after 4 months of omega-3 supplementation. PEW is involved in a complex syndrome induced by nutritional and non-nutritional factors such as poor food intake secondary to uremia, restrictive diets, a persistent inflammatory state, enhanced catabolism due to treatment modalities, loss of nutrients into the dialysate, and metabolic acidosis.5 Given the importance of the problem, as well as the complexity of the relationship between chronic inflammation and PEW, it is obviously conceivable that preventive and/or therapeutic options would be complex and critical. To date, no single treatment approach has been able to successfully alleviate such a complex syndrome of uremia. We thought that omega-3 fatty acids with a suppressive effect on the inflammatory response might improve the nutritional state of maintenance HD patients. We found that the serum albumin level significantly decreased in the omega-3 group compared with the placebo group during the study. This finding was in contrast to what was reported by Perunicic-Pecovic and colleagues, in which administration of 2.4 g of omega-3 fatty acids daily for 8 weeks significantly increased the serum albumin level in chronic HD patients.9 Kalantar-Zadeh and colleagues used a novel nutritional oral supplement (containing 1,680 mg omega-3 fatty acids) during each HD session for 4 weeks to improve the nutritional status of HD patients.7 In contrast to our finding, a significant increase appeared in the serum albumin concentration and total iron binding capacity, an important surrogate of serum transferrin, after 4 weeks of intervention. Although our

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study used a relatively higher dose of omega-3 supplement (1,800 mg per day) for a longer period (4 months), we could not compare our results with those of KalantarZadeh and colleagues because the different composition of the supplement given in their pilot study made it impossible to distinguish the potential benefits of omega-3 fatty acids from other constituents. Vernaglione and colleagues observed no change in the serum albumin level of chronic HD patients after 4 months of supplemental use of 2,100 mg of omega-3 fatty acids.22 In addition, and in agreement with our finding, Mat Daud and colleagues failed to indicate a significant improvement in the nutrition indices of maintenance HD patients, including BMI, malnutrition-inflammation score, normalized protein equivalence of nitrogen appearance, and serum albumin level, when subjects were given 30 mL of liquid protein supplement (providing 18 g protein) combined with 4 omega-3 capsules (providing a total of 1,800 mg EPA and 600 mg DHA) during regular dialysis sessions for 6 months.5 In our study, the observed discordance of reduced serum albumin level with slight attenuation of inflammatory state may be explained by the results of some previous studies. Kaysen and colleagues showed that serum albumin, as a surrogate of protein intake, is not an exclusive correlate of inflammation.23 Friedman and Fadem proposed that serum albumin should be considered as a marker of illness rather than nutrition.24 On the other hand, in this study, regression analysis adjusting post-treatment serum albumin for demographic factors revealed a marginally significant and negative association between serum albumin and patients’ age. This finding may be explained by the poor food and energy intake of HD patients with aging as illustrated by other researchers.25 Altogether, difficulty in interpreting our finding might partly be because serum albumin is influenced by other nutritional and non-nutritional factors that were not controlled in our study. Thus, future studies should consider other potential confounding factors. In the study presented here, we found no significant changes in dry body weight, BMI, MAC, and serum concentrations of transferrin and prealbumin in the omega-3 group compared with the placebo group. These results are consistent with those obtained by Szklarek-Kubicka in that they also found no significant effects of intradialytic intravenous administration of omega-3 emulsion (providing 2 g EPA and 2 g DHA) on BMI and serum concentrations of albumin and transferrin after 11 consecutive HD sessions.26 On the other hand, Ewers and colleagues showed a significant increase in the body weight of HD patients who were given a dietary supplement providing 3 g of omega-3 fatty acids per day for 6 weeks.4 In agreement with our results, no significant changes were observed in the nutritional parameters of HD patients, including BMI, when Moreira and colleagues treated them with a sardine supplement (containing 3.87 g omega-3 fatty acids) during

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each HD session, thrice weekly, for 8 weeks.12 In our study, although the increased serum prealbumin level in omega-3 group did not reach statistical significance versus the placebo group, regression analysis revealed that omega-3 treatment was a significant independent predictor of increased serum prealbumin level. According to the fact that serum prealbumin is a sensitive nutrition marker with the shortest serum half-life among serum nutrition markers measured in this trial, we might likely find significant improvement in serum prealbumin with a larger dose of omega-3 and/or a somewhat longer intervention period. In our study, adjusted regression analysis illustrated that among patients’ demographic and clinical data, HD duration was a significant independent predictor of decreased dry body weight and BMI. In line with our finding, a 9year longitudinal study on HD patients by Mekki and colleagues showed a negative significant correlation between BMI and HD duration.27 It is a wise thought that decreased energy and food intake in prolonged HD treatment may contribute to reduced BMI and body weight in this population. On the contrary, HD duration showed a positive significant association with serum transferrin concentration in this study. Although positive correlation between serum albumin and HD duration has been reported in previous studies,9 we failed to find such relationship between transferrin and the duration of HD treatment in the literature. However, a recent study by Formanowicz and colleagues noted that transferrin variant 3, likely a crucial variant in iron transport, was positively associated with the duration of HD treatment in the presence of a worsened inflammatory state.28 We were unfortunately not able to know which of the transferrin variants comprised the dominant variant of measured serum transferrin. As evident in Table 4, among the nutrition indices measured in this trial, serum prealbumin was the exclusive marker correlated significantly and positively with the omega-3 treatment. Serum iPTH level was the sole inflammation marker that largely contributed to improvement of serum prealbumin level by omega-3 treatment. Adding to the difficulty in interpreting this finding may be the relatively less clear role of iPTH in the inflammatory processes and the paucity of studies on the relationship between iPTH and inflammation. Although the usefulness of omega-3 supplementation to improve the nutritional and inflammatory state of HD patients was not remarkable in our study, there were several strengths to our trial, including the placebo-controlled design, the relatively longer follow-up period (4 months), the relatively comprehensive evaluation of nutrition and inflammation state of HD patients, no serious adverse events leading to patient withdrawal, and more frequent interviews with patients to enhance their compliance. However, we admit some limitations, which should be considered in the interpretation of the results. There was low statistical power to detect meaningful differences be-

tween groups because of the small population size. As examples, to detect difference between 2 groups regarding mean changes of serum CRP, IL-10, and prealbumin at an a level of 0.05 and a power of 80%, approximately 39, 570, and 150 patients in each group were respectively required. The length of our study seemed to be shorter compared with some previous studies. We used the conventional practice of ‘‘pill counting’’ to monitor patient compliance rather than measurement of erythrocyte membrane fatty acid composition as the standard compliance measurement. Our study lacked an interim analysis to follow a potential trend over time and lower variance of estimates of treatment effects. The ‘‘fishy’’ smells of omega-3 capsules, although not mentioned as a complaint by any patients, did not appear in placebo capsules, which might have affected the blindness of subjects in the placebo arm of the study. Finally, the assessment of the nutritional state of patients did not include their dietary intake as a potential confounder to our intervention. In conclusion, our study showed that a 4-month administration of omega-3 fatty acids in chronic HD patients was well tolerated and useful in regard to the relative attenuation of systemic inflammation. Although omega-3 supplementation failed to produce pronounced alleviation in systemic inflammation, its positive, although slight, effects on inflammatory state were encouraging by considering systemic inflammation as a correlate of increased cardiovascular mortality in maintenance HD patients. Given the lack of any remarkable effects of omega-3 fatty acids on nutritional parameters, it can be speculated that additional nutritional support, along with omega-3 supplementation, might prevent/treat PEW in maintenance HD patients. However, we propose further well-designed investigations to definitely determine the effectiveness of omega-3 fatty acids in improving the nutrition and inflammatory state of HD patients.

Practical Application Considering the cardiovascular benefits of continuous dietary ingestion of omega-3 fatty acids as a minimum, their continuous consumption coupled with nutritional support seems to be beneficial in improving the malnutritioninflammation complex in maintenance HD patients.

Acknowledgments This study was supported by Tehran University of Medical Sciences (grant no. 17020). The authors thank Zahravi Pharmaceutical, Co., Tehran, Iran, for providing placebo free of charge and Dr. Amir Hekmat for his kind coordinating between the researchers’ team and the pharmaceutical company. The authors also appreciate the valuable help of the HD nursery teams of the Imam-Khomeini Hospital complex and Sina Hospital, especially Mrs. Abdpoor.

References 1. Ikizler TA. Nutrition, inflammation and chronic kidney disease. Curr Opin Nephrol Hypertens. 2008;17:162-167.

OMEGA-3, NUTRITION, INFLAMMATION IN HD PATIENTS 2. Arslan Y, Kiziltan G. Nutrition-related cardiovascular risk factors in hemodialysis patients. J Ren Nutr. 2010;20:185-192. 3. Cano NJ, Heng AE, Pison C. Multimodal approach to malnutrition in malnourished maintenance hemodialysis patients. J Ren Nutr. 2011;21: 23-26. 4. Ewers B, Riserus U, Marckmann P. Effects of unsaturated fat dietary supplements on blood lipids, and on markers of malnutrition and inflammation in hemodialysis patients. J Ren Nutr. 2009;19:401-411. 5. Mat Daud ZA, Tubie B, Adams J, et al. Effects of protein and omega-3 supplementation, provided during regular dialysis sessions, on nutritional and inflammatory indices in hemodialysis patients. Vasc Health Risk Manag. 2012;8:187-195. 6. Stenvinkel P, Heimburger O, Paultre F, et al. Strong association between malnutrition, inflammation, and atherosclerosis in chronic renal failure. Kidney Int. 1999;55:1899-1911. 7. Kalantar-Zadeh K, Braglia A, Chow J, et al. An anti-inflammatory and antioxidant nutritional supplement for hypoalbuminemic hemodialysis patients: a pilot/feasibility study. J Ren Nutr. 2005;15:318-331. 8. Zabel R, Ash S, King N, Naslund E, Bauer J. Gender differences in the effect of fish oil on appetite, inflammation and nutritional status in haemodialysis patients. J Hum Nutr Diet. 2010;23:416-425. 9. Perunicic-Pekovic GB, Rasic ZR, Pljesa SI, et al. Effect of n-3 fatty acids on nutritional status and inflammatory markers in hemodialysis patients. Nephrology. 2007;12:331-336. 10. Saifullah A, Watkins BA, Saha C, Li Y, Moe SM, Friedman AN. Oral fish oil supplementation raises blood omega-3 levels and lowers C-reactive protein in haemodialysis patients-a pilot study. Nephrol Dial Transpl. 2007;22:3561-3567. 11. Rasic-Milutinovic Z, Perunicic G, Pljesa S, et al. Effects of N-3 PUFAs supplementation on insulin resistance and inflammatory biomarkers in hemodialysis patients. Ren Fail. 2007;29:321-329. 12. Moreira AC, Gaspar A, Serra MA, Simoes J, Lopes da Cruz J, Amaral TFD. Effect of a sardine supplement on C-reactive protein in patients receiving hemodialysis. J Ren Nutr. 2007;17:205-213. 13. Neade T, Uribarri J. Diet, inflammation, and chronic kidney disease: getting to the heart of the matter. Semin Dial. 2008;21:331-337. 14. Kalantar-Zadeh K, Ikizler TA, Block G, Avram MM, Kopple JD. Malnutrition-inflammation complex syndrome in dialysis patients: causes and consequences. Am J Kidney Dis. 2003;42:864-881. 15. Noori N, Dukkipati R, Kovesdy CP, et al. Dietary omega-3 fatty acid, ratio of omega-6 to omega-3 intake, inflammation, and survival in long-term hemodialysis patients. Am J Kidney Dis. 2011;58:248-256.

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16. Madsen T, Christensen JH, Svensson M, Witt PM, Toft E, Schmidt EB. Marine n-3 polyunsaturated fatty acids in patients with endstage renal failure and in subjects without kidney disease: a comparative study. J Ren Nutr. 2011;21:169-175. 17. Himmelfarb J, Phinney S, Ikizler TA, Kane J, McMonagle E, Miller G. Gamma-tocopherol and docosahexaenoic acid decrease inflammation in dialysis patients. J Ren Nutr. 2007;17:296-304. 18. Bowden RG, Wilson RL, Deike E, Gentile M. Fish oil supplementation lowers c-reactive protein levels independent of triglyceride reduction in patients with end-stage renal disease. Nutr Clin Pract. 2009;24: 508-512. 19. Kooshki A, Taleban FA, Tabibi H, Hedayati M. Effects of marine omega3 fatty acids on serum systemic and vascular inflammation markers and oxidative stress in hemodialysis patients. Ann Nutr Metab. 2011;58:197-202. 20. Hassan KS, Hassan SK, Hijazi EG, Khazim KO. Effects of omega-3 on lipid profile and inflammation markers in peritoneal dialysis patients. Ren Fail. 2010;32:1031-1035. 21. Poulia KA, Panagiotakos DB, Tourlede E, et al. Omega-3 fatty acids supplementation does not affect serum lipids in chronic hemodialysis patients. J Ren Nutr. 2011;21:479-484. 22. Vernaglione L, Cristofano C, Chimienti S. Omega-3 polyunsaturated fatty acids and proxies of cardiovascular disease in hemodialysis: a prospective cohort study. J Nephrol. 2008;21:99-105. 23. Kaysen GA, Chertow GM, Adhikarla R, Young B, Ronco C, Levin NW. Inflammation and dietary protein intake exert competing effects on serum albumin and creatinine in hemodialysis patients. Kidney Int. 2001;60:333-340. 24. Friedman AN, Fadem SZ. Reassessment of albumin as a nutritional marker in kidney disease. J Am Soc Nephrol. 2010;21:223-230. 25. Oliveira CMC, Costa SP, Costa LC, Pinheiro SM, Lacerda GA, Kubrusly M. Depression in dialysis patients and its association with nutritional markers and quality of life. J Nephrol. 2012;25:954-961. 26. Szklarek-Kubicka M, Fijalkowska-Morawska J, ZarembaDrobnic D, Ucinski A, Czekalski S, Nowicki M. Effect of intradialytic intravenous administration of u-3 fatty acids on nutritional status and inflammatory response in hemodialysis patients: a pilot study. J Ren Nutr. 2009;19:487-493. 27. Mekki K, Remaoun M, Belleville J, Bouchenak M. Hemodialysis duration impairs food intake and nutritional parameters in chronic kidney disease patients. Int Urol Nephrol. 2012;44:237-244. 28. Formanowicz D, Formanowicz P. Transferrin changes in haemodialysis patients. Int Urol Nephrol. 2012;44:907-919.