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Effect of Omega-3 Fatty Acids on Serum Lipid Profile and Oxidative Stress in Pediatric Patients on Regular Hemodialysis: A Randomized Placebo-Controlled Study
ORIGINAL RESEARCH
Effect of Omega-3 Fatty Acids on Serum Lipid Profile and Oxidative Stress in Pediatric Patients on Regular Hemodialysis: A Randomized Placebo-Controlled Study Areej Mohamed Ateya, BpharmSci,* Nagwa Ali Sabri, PhD Clinical Pharmacy,* Ihab El Hakim, MD, PhD,† and Sara M. Shaheen, PhD Clinical Pharmacy* Objective: We sought to evaluate the effects of omega-3 fatty acids supplementation on serum lipid profile and oxidative stress markers in pediatric patients with end-stage renal disease on regular hemodialysis (HD). Design: This study was a double-blinded, randomized, placebo-controlled trial conducted on 49 pediatric patients on regular HD for at least 6 months. Intervention: Patients were randomly divided into either omega-3 group (n 5 25) who received 1-g oral omega-3 capsule once daily for 16 weeks or placebo group (n 5 24) who received 1-g matching oral placebo capsule once daily for 16 weeks. Main Outcome Measure: Lipid profile markers including: total cholesterol, triglycerides, high-density lipoprotein cholesterol, lowdensity lipoprotein cholesterol, and oxidative stress markers including the following: malondialdehyde, glutathione peroxidase, and superoxide dismutase were measured at baseline and after 16 weeks of supplementation. Results: By the end of the study, children in omega-3 group showed a highly significant reduction in total cholesterol and a highly significant increase in glutathione peroxidase and superoxide dismutase levels. Conclusion: The administration of omega-3 has a beneficial effect on serum lipid profile and oxidative stress in children undergoing HD. Ó 2016 by the National Kidney Foundation, Inc. All rights reserved.
Introduction
C
ARDIOVASCULAR DISEASE (CVD) is the leading cause of death in children with end-stage renal disease (ESRD), with risk 1,000 times higher when compared to the age-matched non-ESRD population.1 Several risk factors contribute to CVD in these patients.2 Traditional risk factors include hypertension, dyslipidemia, hyperglycemia, and obesity.3 Dyslipidemia is an important cardiovascular risk factor in the general population.4 In patients with ESRD, dyslipidemia is characterized by increased triglycerides (TGs) and low-density lipoprotein cholesterol (LDL-C) levels and reduced levels of high-density lipopro-
*
Department of Clinical Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. † Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Financial Disclosure: The authors declare that they have no relevant financial interests. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Address correspondence to Areej Mohamed Ateya, BpharmSci, Department of Clinical Pharmacy, Faculty of Pharmacy, Ain Shams University, Building 10, Part 1, El Sefarat, Nasr City, Cairo, Egypt. E-mail: areeg.mohamed@
pharma.asu.edu.eg Ó
2016 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2016.11.005
Journal of Renal Nutrition, Vol -, No - (-), 2016: pp 1-6
tein cholesterol (HDL-C).3 These lipid abnormalities persist and are aggravated during hemodialysis (HD)5,6 which may partly explain the high incidence of CVD in these patients.7 However, traditional risk factors alone cannot completely explain the high CVD risk in ESRD population.8 Recently, nontraditional risk factors such as oxidative stress have attracted more interest for the occurrence of CVD events in ESRD patients.9 Oxidative stress is defined as the tissue damage resulting from an imbalance between an excessive generation of oxidant compounds and insufficient antioxidant defense mechanisms.10 Antioxidant mechanisms can be divided into intracellular and extracellular antioxidants. Intracellular antioxidants include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP), which convert substrates (superoxide anion radicals and hydrogen peroxide) into less reactive forms.11 Factors such as exposure of the blood to dialysis membranes, high risk of acute and chronic infection, and dietary limitations in the intake of antioxidant nutrients make patients on HD susceptible to more oxidative stress.12 In chronic kidney disease, oxidative stress is characterized by increased concentrations of malondialdehyde (MDA) as a marker of lipid peroxidation and reduced concentrations of SOD and GP as markers of antioxidants.11 Omega-3 fatty acids are polyunsaturated fatty acids that are mainly obtained from dietary sources especially fatty
1
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fish.13 There is strong evidence from clinical trials that omega-3 fatty acids significantly decrease cardiovascular mortality, improve the lipid status, and have antioxidant effects.14 ESRD patients are at risk for inadequate omega-3 intake because dialysis patients may find foodstuffs less palatable as a result of uremia-associated alterations in taste and renal dietary recommendations that do not encourage fish consumption.15 On the basis of the above data, it was suggested that omega-3 intake may offer a host of benefits to the pediatric patients with ESRD. Thus, the aim of the study was to evaluate the effects of oral omega-3 intake on serum lipid profile, oxidative stress, and antioxidant markers in pediatric patients on regular HD.
Methods Study Design and Population This study was a prospective, randomized, doubleblinded, placebo-controlled study. It was conducted on pediatric patients with ESRD who were undergoing HD from March 2015 to September 2015. Inclusion and Exclusion Criteria Patients between 8 and 18 years old who have been on regular HD for .6 months were included in the study. Patients with malignancy and/or active inflammatory disease and those who received vitamin E during the past 3 months were excluded from the study. The HD regimen of all patients typically consisted of HD sessions of 3-4 hours, 3 days per week. Sample Size Calculation Sample size was calculated according to the oxidative stress marker MDA. Based on a previous study conducted on patients undergoing HD,12 a difference of 0.9 nmol/L was found between groups with a standard deviation (SD) 5 0.9 and 0.7 in the treatment group and the control group, respectively. With this information, we obtained a sample size of 22 patients per group for a 90% statistical power with a 5 0.05. All patients in the dialysis unit who fulfilled the inclusion and exclusion criteria (n 5 53) were included in the study. Only 49 patients completed the study. Three patients were dropped out due to noncompliance, and 1 patient underwent renal transplantation. Randomization The patients were randomly assigned into either omega3 group (n 5 25) or placebo group (n 5 24). Intervention Patients in omega-3 group received 1-g oral omega-3 capsule once daily for 16 weeks. Patients in placebo group received 1-g oral placebo capsule once daily for 16 weeks. Omega-3 capsules were supplied by NOW FOODS company, USA, under the trade name of Ultra Omega-3Ò, containing 500 mg eicosapentaenoic acid and 250 mg docosahexaenoic acid in addition to the standard ingredients of soft gelatin capsules. Placebo capsules contain only the
standard ingredients of soft gelatin capsules (gelatin, water, glycerin, and vitamin E in minute amounts as preservative). Placebo capsules were carefully matched in size, shape, and color with the omega-3 capsules. The dose of omega-3 was given based on the Council on Food and Nutrition of the American Medical Association published in 2002.16 Patients were monitored weekly for any adverse effects of omega-3 fatty acids. Weight was measured after the HD session with subjects bare feet and wearing light clothes. Height was measured using a nonstretchable tape with subjects standing bare feet with heels touching the floor and eyes directed straight ahead. Weight and height z scores were calculated using Centers for Disease Control and Prevention growth charts as recommended by the World Health Organization.
Assays Blood samples were drawn from the patients at baseline and after 16 weeks of supplementation. Blood samples were collected before the dialysis session after 12 hours overnight fasting. Total cholesterol (TC), TG, HDL-C, LDL-C, and GP were assayed by spectrophotometric method using commercial kit manufactured by Biodiagnostic Company, Giza, Egypt. The GP activity was measured indirectly through a coupled reaction with glutathione reductase. Oxidized glutathione produced upon reduction of an organic hydroperoxide by GP was recycled to its reduced state by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). The oxidation of NADPH to oxidized form of NADPH (NADP1) is accompanied by a decrease in absorbance at 340 nm. Enzyme-linked immunosorbent assay method was used to measure MDA and SOD concentrations. A commercial kit OxiSelectÔ MDA Adduct manufactured by Cell Biolabs Inc., USA, was used for MDA assay, while a commercial kit manufactured by Northwest Life Science Specialties, LLC, USA, was used for SOD assay. An informed consent was obtained from either parents or children aged .16 years. The Ethics Committee of the Faculty of Pharmacy, Ain Shams University, approved the study protocol. The study was performed in accordance with the Declaration of Helsinki. Statistical Analysis Data management and analysis were performed using Statistical Package for Social Sciences versus 21. Numerical data were summarized using means and SDs or medians and interquartile ranges, as appropriate. Categorical data were summarized as numbers and percentages. Numerical data were explored for normality using Kolmogrov–Smirnov test and Shapiro–Wilk test. Exploration of data revealed that the collected values were not normally distributed. Comparisons between the 2 groups with respect to numerical variables were done by Mann–Whitney test. Change overtime for each group was tested using Wilcoxon Rank Signed test. These tests were followed by the post hoc
3
EFFECT OF OMEGA-3 ON PEDIATRIC HEMODIALYSIS Table 1. Baseline Demographics and Clinical Characteristics of the 2 Patient Groups Baseline Evaluation Demographics Age, y* Gender, n (%) Male Female Weight z score§ Height z score§ Clinical characteristics etiology of ESRD, n (%) Reflux nephropathy Hypoplastic/dysplastic kidneys Familial nephritis Chronic glomerulonephritis Systemic immunologic diseases Focal segmental glomerulosclerosis Hemolytic uremic syndrome Obstructive uropathy Others Comorbid condition associated with ESRD (%) Hypertension Anemia Congestive heart failure
Omega-3 Group (n 5 25)
Placebo Group (n 5 24)
P Value
14.7 6 2.7
14.6 6 2.7
1.000†
14 (56) 11 (44) 23.8 (27.1, 22.9) 24.3 (26.6, 22.7)
13 (54.2) 11 (45.8) 24.8 (27.6, 23.3) 23.6 (25.3, 23)
1.000‡ .424† .284†
7 (28) 6 (24) 2 (8) 2 (8) 2 (12) 1 (4) 0 (0) 1 (4) 3 (12)
4 (16.7) 4 (16.7) 4 (16.7) 3 (12.5) 1 (4.2) 0 (0) 1 (4.2) 2 (8.3) 5 (20.8)
13 (52) 16 (64) 11 (44)
15 (62.5) 14 (58.3) 11 (45.8)
.458‡ .684‡ .897‡
n, number; SD, standard deviation. *Data expressed as mean 6 SD. †Mann–Whitney test. ‡Data expressed as median with the interquartile range in parenthesis. §Chi-square test.
Bonferroni corrections to adjust the P values. Comparisons between the 2 groups with respect to categorical data were performed by the chi-square test. All P values are 2 sided. P values # .05 were considered significant and ,.001 were considered highly significant.
Results At baseline, there was no significant difference between both patient groups in terms of demographics, clinical characteristics, and laboratory parameters except for TG
and GP levels which were significantly higher in the omega-3 group (Tables 1 and 2). At the end of the 16-week study period, using Wilcoxon test, patients in the omega-3 group showed a highly significant decrease (P ,.001) in serum TC (Fig. 1) and a highly significant increase (P , .001) in GP and SOD when compared to baseline. They also showed a decrease in serum TG and LDL-C, but the decrease was not statistically significant (P 5.104 and P 5.080, respectively). No significant changes were observed in neither HDL-C nor serum MDA when compared to the baseline values (Table 3).
Table 2. Baseline Laboratory Parameters of the 2 Patient Groups Baseline Evaluation Total cholesterol (mg/dL)* Triglycerides (mg/dL)* HDL cholesterol (mg/dL)* LDL cholesterol (mg/dL)* Malondialdehyde (nmol/mL)* Glutathione peroxidase (mu/mL)* Superoxide dismutase (pg/mL)* Hemoglobin (g/dL)‡ Albumin (mg/dL)‡ Systolic BP (mm Hg)‡ Diastolic BP (mm Hg)‡
Omega-3 Group (n 5 25)
Placebo Group (n 5 24)
P Value
172.2 (161.1-190) 137.7 (121.7-147.8) 34.9 (31.6-42.3) 109.6 (84.7-129) 11.5 (10.1-14.8) 92.4 (58.4-121.6) 88.5 (74.9-125.4) 10.2 6 2.00 4.4 6 0.6 130 6 17.6 86 6 18
153.5 (139.2-176.2) 109.6 (101.8-123.3) 35 (29-41) 91.8 (81.6-106.1) 12.9 (11.2-15.2) 58.4 (38.9-68.1) 82.3 (71.6-108.7) 10.6 6 1.7 4.1 6 0.3 121.3 6 19.2 82.5 6 14.5
.475 .001† 1.000 .494 .494 .016† 1.000 1.000 .118 .989 1.000
BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation. Statistical test: Mann–Whitney test. *Data expressed as median with the interquartile range in parenthesis. †Significant at P value # .05. ‡Data expressed as mean 6 SD.
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Figure 1. Median serum total cholesterol in the omega-3 group and the placebo group at baseline and at the end of the study.
In contrast, at the end of the 16 weeks of the study, patients in the placebo group showed a highly significant decrease in HDL-C (P , .001) and a significant increase in serum MDA (P 5.004). They also showed a highly significant increase in SOD, but by calculating the percent change, it was shown that the increase in the omega-3 group was greater than that in the placebo group (P 5.002). No significant changes were observed in neither TC, TG, LDL-C, nor GP when compared to the baseline values (Table 3). The Mann–Whitney test used to compare between the groups revealed that there was no significant difference in the median serum levels of neither TG (115.3 vs. 104.1 mg/dL; P 5 .150), HDL-C (34.6 vs. 33.6 mg/dL; P 5 1.000), nor LDL-C (98.5 vs. 89 mg/dL; P 5 1.000) between the omega-3 and the placebo group at the end of the study. However, there was a significant difference in the median levels of TC (132.9 vs. 149.2 mg/dL; P 5 .005), MDA (11.3 vs. 17.8 nmol/mL; P , .001),
SOD (149.1 vs. 110.5 pg/mL; P 5 .022), and GP (97.7 vs. 50.4 mu/mL; P 5 .003) between both groups at the end of the study. Percent change was also calculated for TG and GP to cancel the significant difference between both groups at baseline. No statistically significant difference was recorded for neither TG nor GP percent change (P 5 .412 and P 5 .818, respectively) Concerning the safety of omega-3 intake, only 2 patients in the omega-3 group and 1 patient in the placebo group suffered from nausea and vomiting during the study. These side effects were mild and patients continued the study.
Discussion Dyslipidemia and oxidative stress play a key role in the development of CVD in ESRD patients.17,18 In the past few years, there has been a growing scientific and public interest in the role of omega-3 fatty acids, mainly obtained from fish and fish oil in CVD, idiopathic Ig A nephropathy,
Table 3. Laboratory Parameters of Both Groups Before and After the Study Omega-3 Group (n 5 25) Laboratory Parameter
Before
After
Placebo Group (n 5 24) P Value
Before
After
P Value
Total cholesterol (mg/dL) 172.2 (161.1-190) 132.9 (118.5-146.1) ,.001* 153.5 (139.2-176.2) 149.2 (140.8-174.7) 1.000 Triglycerides (mg/dL) 137.7 (118.5-146.1) 115.3 (104.9-140.1) .104 109.6 (101.8-123.3) 104.1 (91.8-118.2) 1.000 HDL cholesterol (mg/dL) 34.9 (31.6-42.3) 34.6 (31.5-39.7) 1.000 35 (29-41) 33.6 (28.8-39.3) ,.001* LDL cholesterol (mg/dL) 109.6 (84.7-129) 98.5 (84.1-112.5) .080 91.8 (81.6-106.1) 89 (81.7-105.8) 1.000 Malondialdehyde (nmol/mL) 11.5 (10.1-14.8) 11.3 (10.3-13.8) 1.000 12.9 (11.2-15.2) 17.8 (14.5-24) .004† Glutathione peroxidase (mu/ml) 92.4 (58.4-121.6) 97.7 (66.2-124.6) ,.001* 58.4 (38.9-68.1) 50.4 (40-76.1) 1.000 Superoxide dismutase (pg/mL) 88.5 (74.9-125.4) 149.1 (111.8-215.7) ,.001* 82.3 (71.6-108.7) 110.5 (96.5-124.4) ,.001* HDL, high-density lipoprotein; LDL, low-density lipoprotein. Statistical test: Wilcoxon test. Data are presented as median with the interquartile range in parenthesis. *Highly significant at P value , .001. †Significant at P value # .05.
EFFECT OF OMEGA-3 ON PEDIATRIC HEMODIALYSIS
lupus nephritis, and renal failure.19 Because of its putative cardiovascular benefits, the American Heart Association and various international health organizations now recommend that persons at high cardiovascular risk, such as dialysis patients, consume up to 1-g fish oil daily.20 Several studies have been conducted evaluating the effects of omega-3 fatty acids in patients on regular HD. However, all these studies have focused on the adult population. To the best of our knowledge, our study is the first to assess the effects of omega-3 fatty acids in pediatric patients with ESRD on regular HD. We observed that the administration of oral omega-3 fatty acids to our pediatric patients substantially reduced serum TC from 172.2 6 28.9 mg/dL at baseline to 132.9 6 27.6 mg/dL (P ,.001) at the end of the 16-week study period. These results correlate with those reported by Zhu et al.19 and Bouzidi et al.11 Bouzidi et al. demonstrated that TC values were decreased by 11% after 90 days of supplementation with 2.1 g/day of omega-3 fatty acids when compared to the baseline. On the contrary, Kooshki et al.21 and Naini et al.22 found no significant changes in serum TC levels upon administration of omega-3 fatty acids daily for 10 weeks and 8 weeks, respectively. The discrepancy between our results and those of previous studies may be attributed to the longer duration of our study. In our study, no significant changes were found in neither TG, HDL-C, nor LDL-C levels in the omega-3 group when compared to the placebo group. These findings are in accordance with the results of several previous studies which also reported that omega-3 fatty acids may have no effect on these parameters.15,22,23 Our study reported a highly significant increase in GP and SOD in children of the omega-3 group. These results correlate with those reported by Bouzidi et al.11 and Tayyebi et al.12 who demonstrated that administration of omega-3 fatty acids to patients on regular HD significantly increase SOD and GP levels. Kooshki et al.21,24 and Hassan et al.15 demonstrated that omega-3 fatty acids had no effect on the serum levels of MDA. These results are similar to those of our study where omega-3 fatty acids failed to achieve a significant decrease in the serum levels of MDA. On the other hand, there was a significant increase in the serum levels of MDA in the placebo group as well as a significant difference between groups at the end of the study. This means that although omega-3 fatty acids failed to decrease MDA levels, they succeeded in avoiding its increase. Concerning the safety of omega-3, only nausea and vomiting were reported during the study in both groups. These adverse effects were not resolved after withdrawal of omega-3. This suggests that the reported adverse effects were not provoked by omega-3 fatty acids and may be attributed to any other cause. These results are in accordance with those of Hung et al.25 and Bowden et al.26 who demonstrated in their studies that there were no
5
serious adverse events related to omega-3 intake in HD patients.
Conclusion The administration of 1-g omega-3 oral capsule once daily reduces serum TC and increases both GP and SOD among the pediatric patients with ESRD on regular HD. The study also demonstrated that omega-3 intake is not associated with any serious adverse effects. These findings make omega-3 fatty acids supplementation beneficial for children on regular HD, especially those with dyslipidemia and cardiovascular complications.
Recommendations Future, large-scale, multicenter, randomized, doubleblinded placebo-controlled studies with higher doses and longer durations are recommended to confirm the efficacy and safety profile of omega-3 fatty acids in HD patients especially the pediatric population.
Practical Application Omega-3 fatty acids reduce TC levels and improve the antioxidant defense mechanisms in children with ESRD on regular HD without producing any serious side effects. They can be considered as a safe and effective supplement to reduce the CVD risk, the main cause of death in the pediatric population with ESRD.
References 1. Kaspar C, Bholah R, Bunchman T. A review of pediatric chronic kidney disease. Blood Purif. 2016;41:211-217. 2. Nanayakkara P, Gaillard C. Vascular disease and chronic renal failure: new insights. Neth J Med. 2010;68:5-14. 3. Kumar S, Bogle R, Banerjee D. Why do young people with chronic kidney disease die early. World J Nephrol. 2014;3:143-155. 4. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation. 1998;97:1837-1847. 5. Appel G. Lipid abnormalities in renal disease. Kidney Int. 1991;39:169183. 6. Ma KW, Greene EL, Raij L. Cardiovascular risk factors in chronic renal failure and hemodialysis populations. Am J kidney Dis. 1992;19:505-513. 7. Uhlig K, Levey AS, Sarnak MJ. Traditional cardiac risk factors in individuals with chronic kidney disease. Semin dial. 2003;16:118-127. 8. Tomey MI, Winston JA. Cardiovascular pathophysiology in chronic kidney disease: opportunities to transition from disease to health. Ann Glob Health. 2014;80:69-76. 9. Kao M, Ang D, Pall A, Struthers A. Oxidative stress in renal dysfunction: mechanisms, clinical sequelae and therapeutic options. J Hum Hypertens. 2010;24:1-8. 10. Del Vecchio L, Locatelli F, Carini M. What we know about oxidative stress in patients with chronic kidney disease on dialysis–clinical effects, potential treatment, and prevention. Semin dial. 2011;24:56-64. 11. Bouzidi N, Mekki K, Boukaddoum A, Dida N, Kaddous A, Bouchenak M. Effects of omega-3 polyunsaturated fatty-acid supplementation on redox status in chronic renal failure patients with dyslipidemia. J Ren Nutr. 2010;20:321-328. 12. Tayyebi-Khosroshahi H, Houshyar J, Tabrizi A, Vatankhah A-M, Zonouz NR, Dehghan-Hesari R. Effect of omega-3 fatty acid on oxidative stress in patients on hemodialysis. Iran J kidney Dis. 2010;4:322-326.
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13. Calder PC. Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance. Biochim Biophys Acta. 2015;1851:469-484. 14. Friedman A, Moe S. Review of the effects of omega-3 supplementation in dialysis patients. Clin J Am Soc Nephrol. 2006;1:182-192. 15. 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. 16. Trumbo P, Schlicker S, Yates A, Poos M. Food and Nutrition Board of the Institute of Medicine, The National Academies. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein and amino acids. J Am Diet Assoc. 2002;102:1621-1630. 17. Kuo K-L, Tarng D-C. Oxidative stress in chronic kidney disease. Adaptive Med. 2010;2:87-94. 18. Chen S-C, Hung C-C, Kuo M-C, et al. Association of dyslipidemia with renal outcomes in chronic kidney disease. PLoS One. 2013;8:e55643. 19. Zhu W, Dong C, Du H, et al. Effects of fish oil on serum lipid profile in dialysis patients: a systematic review and meta-analysis of randomized controlled trials. Lipids Health Dis. 2014;13:127. 20. Trumbo P, Schlicker S, Yates AA, Poos M. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein and amino acids. J Am Diet Assoc. 2002;102:1621-1630.
21. Kooshki A, Taleban FA, Tabibi H, Hedayati M. Effects of omega-3 fatty acids on serum lipids, lipoprotein (a), and hematologic factors in hemodialysis patients. Ren Fail. 2011;33:892-898. 22. Naini AE, Keyvandarian N, Mortazavi M, Taheri S, Hosseini SM. Effect of omega-3 fatty acids on blood pressure and serum lipids in continuous ambulatory peritoneal dialysis patients. J Res Pharm Pract. 2015;4:135. 23. Daud Z, 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. 24. Kooshki A, Taleban F, Tabibi H, Hedayati M. Effects of marine omega-3 fatty acids on serum systemic and vascular inflammation markers and oxidative stress in hemodialysis patients. Ann Nutr Metab. 2011;58:197-202. 25. Hung AM, Booker C, Ellis CD, et al. Omega-3 fatty acids inhibit the up-regulation of endothelial chemokines in maintenance hemodialysis patients. Nephrol Dial Transplant. 2015;30:266-274. 26. Bowden RG, Wilson RL, Gentile M, Ounpraseuth S, Moore P, Leutholtz BC. Effects of omega-3 fatty acid supplementation on vascular access thrombosis in polytetrafluorethylene grafts. J Ren Nutr. 2007;17:126-131.
Effect of Omega-3 Fatty Acids on Serum Lipid Profile and Oxidative Stress in Pediatric Patients on Regular Hemodialysis: A Randomized Placebo-Controlled Study Areej Mohamed Ateya, BpharmSci,* Nagwa Ali Sabri, PhD Clinical Pharmacy,* Ihab El Hakim, MD, PhD,† and Sara M. Shaheen, PhD Clinical Pharmacy* Objective: We sought to evaluate the effects of omega-3 fatty acids supplementation on serum lipid profile and oxidative stress markers in pediatric patients with end-stage renal disease on regular hemodialysis (HD). Design: This study was a double-blinded, randomized, placebo-controlled trial conducted on 49 pediatric patients on regular HD for at least 6 months. Intervention: Patients were randomly divided into either omega-3 group (n 5 25) who received 1-g oral omega-3 capsule once daily for 16 weeks or placebo group (n 5 24) who received 1-g matching oral placebo capsule once daily for 16 weeks. Main Outcome Measure: Lipid profile markers including: total cholesterol, triglycerides, high-density lipoprotein cholesterol, lowdensity lipoprotein cholesterol, and oxidative stress markers including the following: malondialdehyde, glutathione peroxidase, and superoxide dismutase were measured at baseline and after 16 weeks of supplementation. Results: By the end of the study, children in omega-3 group showed a highly significant reduction in total cholesterol and a highly significant increase in glutathione peroxidase and superoxide dismutase levels. Conclusion: The administration of omega-3 has a beneficial effect on serum lipid profile and oxidative stress in children undergoing HD. Ó 2016 by the National Kidney Foundation, Inc. All rights reserved.
Introduction
C
ARDIOVASCULAR DISEASE (CVD) is the leading cause of death in children with end-stage renal disease (ESRD), with risk 1,000 times higher when compared to the age-matched non-ESRD population.1 Several risk factors contribute to CVD in these patients.2 Traditional risk factors include hypertension, dyslipidemia, hyperglycemia, and obesity.3 Dyslipidemia is an important cardiovascular risk factor in the general population.4 In patients with ESRD, dyslipidemia is characterized by increased triglycerides (TGs) and low-density lipoprotein cholesterol (LDL-C) levels and reduced levels of high-density lipopro-
*
Department of Clinical Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. † Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Financial Disclosure: The authors declare that they have no relevant financial interests. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Address correspondence to Areej Mohamed Ateya, BpharmSci, Department of Clinical Pharmacy, Faculty of Pharmacy, Ain Shams University, Building 10, Part 1, El Sefarat, Nasr City, Cairo, Egypt. E-mail: areeg.mohamed@
pharma.asu.edu.eg Ó
2016 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2016.11.005
Journal of Renal Nutrition, Vol -, No - (-), 2016: pp 1-6
tein cholesterol (HDL-C).3 These lipid abnormalities persist and are aggravated during hemodialysis (HD)5,6 which may partly explain the high incidence of CVD in these patients.7 However, traditional risk factors alone cannot completely explain the high CVD risk in ESRD population.8 Recently, nontraditional risk factors such as oxidative stress have attracted more interest for the occurrence of CVD events in ESRD patients.9 Oxidative stress is defined as the tissue damage resulting from an imbalance between an excessive generation of oxidant compounds and insufficient antioxidant defense mechanisms.10 Antioxidant mechanisms can be divided into intracellular and extracellular antioxidants. Intracellular antioxidants include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP), which convert substrates (superoxide anion radicals and hydrogen peroxide) into less reactive forms.11 Factors such as exposure of the blood to dialysis membranes, high risk of acute and chronic infection, and dietary limitations in the intake of antioxidant nutrients make patients on HD susceptible to more oxidative stress.12 In chronic kidney disease, oxidative stress is characterized by increased concentrations of malondialdehyde (MDA) as a marker of lipid peroxidation and reduced concentrations of SOD and GP as markers of antioxidants.11 Omega-3 fatty acids are polyunsaturated fatty acids that are mainly obtained from dietary sources especially fatty
1
2
ATEYA ET AL
fish.13 There is strong evidence from clinical trials that omega-3 fatty acids significantly decrease cardiovascular mortality, improve the lipid status, and have antioxidant effects.14 ESRD patients are at risk for inadequate omega-3 intake because dialysis patients may find foodstuffs less palatable as a result of uremia-associated alterations in taste and renal dietary recommendations that do not encourage fish consumption.15 On the basis of the above data, it was suggested that omega-3 intake may offer a host of benefits to the pediatric patients with ESRD. Thus, the aim of the study was to evaluate the effects of oral omega-3 intake on serum lipid profile, oxidative stress, and antioxidant markers in pediatric patients on regular HD.
Methods Study Design and Population This study was a prospective, randomized, doubleblinded, placebo-controlled study. It was conducted on pediatric patients with ESRD who were undergoing HD from March 2015 to September 2015. Inclusion and Exclusion Criteria Patients between 8 and 18 years old who have been on regular HD for .6 months were included in the study. Patients with malignancy and/or active inflammatory disease and those who received vitamin E during the past 3 months were excluded from the study. The HD regimen of all patients typically consisted of HD sessions of 3-4 hours, 3 days per week. Sample Size Calculation Sample size was calculated according to the oxidative stress marker MDA. Based on a previous study conducted on patients undergoing HD,12 a difference of 0.9 nmol/L was found between groups with a standard deviation (SD) 5 0.9 and 0.7 in the treatment group and the control group, respectively. With this information, we obtained a sample size of 22 patients per group for a 90% statistical power with a 5 0.05. All patients in the dialysis unit who fulfilled the inclusion and exclusion criteria (n 5 53) were included in the study. Only 49 patients completed the study. Three patients were dropped out due to noncompliance, and 1 patient underwent renal transplantation. Randomization The patients were randomly assigned into either omega3 group (n 5 25) or placebo group (n 5 24). Intervention Patients in omega-3 group received 1-g oral omega-3 capsule once daily for 16 weeks. Patients in placebo group received 1-g oral placebo capsule once daily for 16 weeks. Omega-3 capsules were supplied by NOW FOODS company, USA, under the trade name of Ultra Omega-3Ò, containing 500 mg eicosapentaenoic acid and 250 mg docosahexaenoic acid in addition to the standard ingredients of soft gelatin capsules. Placebo capsules contain only the
standard ingredients of soft gelatin capsules (gelatin, water, glycerin, and vitamin E in minute amounts as preservative). Placebo capsules were carefully matched in size, shape, and color with the omega-3 capsules. The dose of omega-3 was given based on the Council on Food and Nutrition of the American Medical Association published in 2002.16 Patients were monitored weekly for any adverse effects of omega-3 fatty acids. Weight was measured after the HD session with subjects bare feet and wearing light clothes. Height was measured using a nonstretchable tape with subjects standing bare feet with heels touching the floor and eyes directed straight ahead. Weight and height z scores were calculated using Centers for Disease Control and Prevention growth charts as recommended by the World Health Organization.
Assays Blood samples were drawn from the patients at baseline and after 16 weeks of supplementation. Blood samples were collected before the dialysis session after 12 hours overnight fasting. Total cholesterol (TC), TG, HDL-C, LDL-C, and GP were assayed by spectrophotometric method using commercial kit manufactured by Biodiagnostic Company, Giza, Egypt. The GP activity was measured indirectly through a coupled reaction with glutathione reductase. Oxidized glutathione produced upon reduction of an organic hydroperoxide by GP was recycled to its reduced state by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). The oxidation of NADPH to oxidized form of NADPH (NADP1) is accompanied by a decrease in absorbance at 340 nm. Enzyme-linked immunosorbent assay method was used to measure MDA and SOD concentrations. A commercial kit OxiSelectÔ MDA Adduct manufactured by Cell Biolabs Inc., USA, was used for MDA assay, while a commercial kit manufactured by Northwest Life Science Specialties, LLC, USA, was used for SOD assay. An informed consent was obtained from either parents or children aged .16 years. The Ethics Committee of the Faculty of Pharmacy, Ain Shams University, approved the study protocol. The study was performed in accordance with the Declaration of Helsinki. Statistical Analysis Data management and analysis were performed using Statistical Package for Social Sciences versus 21. Numerical data were summarized using means and SDs or medians and interquartile ranges, as appropriate. Categorical data were summarized as numbers and percentages. Numerical data were explored for normality using Kolmogrov–Smirnov test and Shapiro–Wilk test. Exploration of data revealed that the collected values were not normally distributed. Comparisons between the 2 groups with respect to numerical variables were done by Mann–Whitney test. Change overtime for each group was tested using Wilcoxon Rank Signed test. These tests were followed by the post hoc
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EFFECT OF OMEGA-3 ON PEDIATRIC HEMODIALYSIS Table 1. Baseline Demographics and Clinical Characteristics of the 2 Patient Groups Baseline Evaluation Demographics Age, y* Gender, n (%) Male Female Weight z score§ Height z score§ Clinical characteristics etiology of ESRD, n (%) Reflux nephropathy Hypoplastic/dysplastic kidneys Familial nephritis Chronic glomerulonephritis Systemic immunologic diseases Focal segmental glomerulosclerosis Hemolytic uremic syndrome Obstructive uropathy Others Comorbid condition associated with ESRD (%) Hypertension Anemia Congestive heart failure
Omega-3 Group (n 5 25)
Placebo Group (n 5 24)
P Value
14.7 6 2.7
14.6 6 2.7
1.000†
14 (56) 11 (44) 23.8 (27.1, 22.9) 24.3 (26.6, 22.7)
13 (54.2) 11 (45.8) 24.8 (27.6, 23.3) 23.6 (25.3, 23)
1.000‡ .424† .284†
7 (28) 6 (24) 2 (8) 2 (8) 2 (12) 1 (4) 0 (0) 1 (4) 3 (12)
4 (16.7) 4 (16.7) 4 (16.7) 3 (12.5) 1 (4.2) 0 (0) 1 (4.2) 2 (8.3) 5 (20.8)
13 (52) 16 (64) 11 (44)
15 (62.5) 14 (58.3) 11 (45.8)
.458‡ .684‡ .897‡
n, number; SD, standard deviation. *Data expressed as mean 6 SD. †Mann–Whitney test. ‡Data expressed as median with the interquartile range in parenthesis. §Chi-square test.
Bonferroni corrections to adjust the P values. Comparisons between the 2 groups with respect to categorical data were performed by the chi-square test. All P values are 2 sided. P values # .05 were considered significant and ,.001 were considered highly significant.
Results At baseline, there was no significant difference between both patient groups in terms of demographics, clinical characteristics, and laboratory parameters except for TG
and GP levels which were significantly higher in the omega-3 group (Tables 1 and 2). At the end of the 16-week study period, using Wilcoxon test, patients in the omega-3 group showed a highly significant decrease (P ,.001) in serum TC (Fig. 1) and a highly significant increase (P , .001) in GP and SOD when compared to baseline. They also showed a decrease in serum TG and LDL-C, but the decrease was not statistically significant (P 5.104 and P 5.080, respectively). No significant changes were observed in neither HDL-C nor serum MDA when compared to the baseline values (Table 3).
Table 2. Baseline Laboratory Parameters of the 2 Patient Groups Baseline Evaluation Total cholesterol (mg/dL)* Triglycerides (mg/dL)* HDL cholesterol (mg/dL)* LDL cholesterol (mg/dL)* Malondialdehyde (nmol/mL)* Glutathione peroxidase (mu/mL)* Superoxide dismutase (pg/mL)* Hemoglobin (g/dL)‡ Albumin (mg/dL)‡ Systolic BP (mm Hg)‡ Diastolic BP (mm Hg)‡
Omega-3 Group (n 5 25)
Placebo Group (n 5 24)
P Value
172.2 (161.1-190) 137.7 (121.7-147.8) 34.9 (31.6-42.3) 109.6 (84.7-129) 11.5 (10.1-14.8) 92.4 (58.4-121.6) 88.5 (74.9-125.4) 10.2 6 2.00 4.4 6 0.6 130 6 17.6 86 6 18
153.5 (139.2-176.2) 109.6 (101.8-123.3) 35 (29-41) 91.8 (81.6-106.1) 12.9 (11.2-15.2) 58.4 (38.9-68.1) 82.3 (71.6-108.7) 10.6 6 1.7 4.1 6 0.3 121.3 6 19.2 82.5 6 14.5
.475 .001† 1.000 .494 .494 .016† 1.000 1.000 .118 .989 1.000
BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation. Statistical test: Mann–Whitney test. *Data expressed as median with the interquartile range in parenthesis. †Significant at P value # .05. ‡Data expressed as mean 6 SD.
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Figure 1. Median serum total cholesterol in the omega-3 group and the placebo group at baseline and at the end of the study.
In contrast, at the end of the 16 weeks of the study, patients in the placebo group showed a highly significant decrease in HDL-C (P , .001) and a significant increase in serum MDA (P 5.004). They also showed a highly significant increase in SOD, but by calculating the percent change, it was shown that the increase in the omega-3 group was greater than that in the placebo group (P 5.002). No significant changes were observed in neither TC, TG, LDL-C, nor GP when compared to the baseline values (Table 3). The Mann–Whitney test used to compare between the groups revealed that there was no significant difference in the median serum levels of neither TG (115.3 vs. 104.1 mg/dL; P 5 .150), HDL-C (34.6 vs. 33.6 mg/dL; P 5 1.000), nor LDL-C (98.5 vs. 89 mg/dL; P 5 1.000) between the omega-3 and the placebo group at the end of the study. However, there was a significant difference in the median levels of TC (132.9 vs. 149.2 mg/dL; P 5 .005), MDA (11.3 vs. 17.8 nmol/mL; P , .001),
SOD (149.1 vs. 110.5 pg/mL; P 5 .022), and GP (97.7 vs. 50.4 mu/mL; P 5 .003) between both groups at the end of the study. Percent change was also calculated for TG and GP to cancel the significant difference between both groups at baseline. No statistically significant difference was recorded for neither TG nor GP percent change (P 5 .412 and P 5 .818, respectively) Concerning the safety of omega-3 intake, only 2 patients in the omega-3 group and 1 patient in the placebo group suffered from nausea and vomiting during the study. These side effects were mild and patients continued the study.
Discussion Dyslipidemia and oxidative stress play a key role in the development of CVD in ESRD patients.17,18 In the past few years, there has been a growing scientific and public interest in the role of omega-3 fatty acids, mainly obtained from fish and fish oil in CVD, idiopathic Ig A nephropathy,
Table 3. Laboratory Parameters of Both Groups Before and After the Study Omega-3 Group (n 5 25) Laboratory Parameter
Before
After
Placebo Group (n 5 24) P Value
Before
After
P Value
Total cholesterol (mg/dL) 172.2 (161.1-190) 132.9 (118.5-146.1) ,.001* 153.5 (139.2-176.2) 149.2 (140.8-174.7) 1.000 Triglycerides (mg/dL) 137.7 (118.5-146.1) 115.3 (104.9-140.1) .104 109.6 (101.8-123.3) 104.1 (91.8-118.2) 1.000 HDL cholesterol (mg/dL) 34.9 (31.6-42.3) 34.6 (31.5-39.7) 1.000 35 (29-41) 33.6 (28.8-39.3) ,.001* LDL cholesterol (mg/dL) 109.6 (84.7-129) 98.5 (84.1-112.5) .080 91.8 (81.6-106.1) 89 (81.7-105.8) 1.000 Malondialdehyde (nmol/mL) 11.5 (10.1-14.8) 11.3 (10.3-13.8) 1.000 12.9 (11.2-15.2) 17.8 (14.5-24) .004† Glutathione peroxidase (mu/ml) 92.4 (58.4-121.6) 97.7 (66.2-124.6) ,.001* 58.4 (38.9-68.1) 50.4 (40-76.1) 1.000 Superoxide dismutase (pg/mL) 88.5 (74.9-125.4) 149.1 (111.8-215.7) ,.001* 82.3 (71.6-108.7) 110.5 (96.5-124.4) ,.001* HDL, high-density lipoprotein; LDL, low-density lipoprotein. Statistical test: Wilcoxon test. Data are presented as median with the interquartile range in parenthesis. *Highly significant at P value , .001. †Significant at P value # .05.
EFFECT OF OMEGA-3 ON PEDIATRIC HEMODIALYSIS
lupus nephritis, and renal failure.19 Because of its putative cardiovascular benefits, the American Heart Association and various international health organizations now recommend that persons at high cardiovascular risk, such as dialysis patients, consume up to 1-g fish oil daily.20 Several studies have been conducted evaluating the effects of omega-3 fatty acids in patients on regular HD. However, all these studies have focused on the adult population. To the best of our knowledge, our study is the first to assess the effects of omega-3 fatty acids in pediatric patients with ESRD on regular HD. We observed that the administration of oral omega-3 fatty acids to our pediatric patients substantially reduced serum TC from 172.2 6 28.9 mg/dL at baseline to 132.9 6 27.6 mg/dL (P ,.001) at the end of the 16-week study period. These results correlate with those reported by Zhu et al.19 and Bouzidi et al.11 Bouzidi et al. demonstrated that TC values were decreased by 11% after 90 days of supplementation with 2.1 g/day of omega-3 fatty acids when compared to the baseline. On the contrary, Kooshki et al.21 and Naini et al.22 found no significant changes in serum TC levels upon administration of omega-3 fatty acids daily for 10 weeks and 8 weeks, respectively. The discrepancy between our results and those of previous studies may be attributed to the longer duration of our study. In our study, no significant changes were found in neither TG, HDL-C, nor LDL-C levels in the omega-3 group when compared to the placebo group. These findings are in accordance with the results of several previous studies which also reported that omega-3 fatty acids may have no effect on these parameters.15,22,23 Our study reported a highly significant increase in GP and SOD in children of the omega-3 group. These results correlate with those reported by Bouzidi et al.11 and Tayyebi et al.12 who demonstrated that administration of omega-3 fatty acids to patients on regular HD significantly increase SOD and GP levels. Kooshki et al.21,24 and Hassan et al.15 demonstrated that omega-3 fatty acids had no effect on the serum levels of MDA. These results are similar to those of our study where omega-3 fatty acids failed to achieve a significant decrease in the serum levels of MDA. On the other hand, there was a significant increase in the serum levels of MDA in the placebo group as well as a significant difference between groups at the end of the study. This means that although omega-3 fatty acids failed to decrease MDA levels, they succeeded in avoiding its increase. Concerning the safety of omega-3, only nausea and vomiting were reported during the study in both groups. These adverse effects were not resolved after withdrawal of omega-3. This suggests that the reported adverse effects were not provoked by omega-3 fatty acids and may be attributed to any other cause. These results are in accordance with those of Hung et al.25 and Bowden et al.26 who demonstrated in their studies that there were no
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serious adverse events related to omega-3 intake in HD patients.
Conclusion The administration of 1-g omega-3 oral capsule once daily reduces serum TC and increases both GP and SOD among the pediatric patients with ESRD on regular HD. The study also demonstrated that omega-3 intake is not associated with any serious adverse effects. These findings make omega-3 fatty acids supplementation beneficial for children on regular HD, especially those with dyslipidemia and cardiovascular complications.
Recommendations Future, large-scale, multicenter, randomized, doubleblinded placebo-controlled studies with higher doses and longer durations are recommended to confirm the efficacy and safety profile of omega-3 fatty acids in HD patients especially the pediatric population.
Practical Application Omega-3 fatty acids reduce TC levels and improve the antioxidant defense mechanisms in children with ESRD on regular HD without producing any serious side effects. They can be considered as a safe and effective supplement to reduce the CVD risk, the main cause of death in the pediatric population with ESRD.
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21. Kooshki A, Taleban FA, Tabibi H, Hedayati M. Effects of omega-3 fatty acids on serum lipids, lipoprotein (a), and hematologic factors in hemodialysis patients. Ren Fail. 2011;33:892-898. 22. Naini AE, Keyvandarian N, Mortazavi M, Taheri S, Hosseini SM. Effect of omega-3 fatty acids on blood pressure and serum lipids in continuous ambulatory peritoneal dialysis patients. J Res Pharm Pract. 2015;4:135. 23. Daud Z, 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. 24. Kooshki A, Taleban F, Tabibi H, Hedayati M. Effects of marine omega-3 fatty acids on serum systemic and vascular inflammation markers and oxidative stress in hemodialysis patients. Ann Nutr Metab. 2011;58:197-202. 25. Hung AM, Booker C, Ellis CD, et al. Omega-3 fatty acids inhibit the up-regulation of endothelial chemokines in maintenance hemodialysis patients. Nephrol Dial Transplant. 2015;30:266-274. 26. Bowden RG, Wilson RL, Gentile M, Ounpraseuth S, Moore P, Leutholtz BC. Effects of omega-3 fatty acid supplementation on vascular access thrombosis in polytetrafluorethylene grafts. J Ren Nutr. 2007;17:126-131.