Effect of omega-3 fatty acid supplementation on serum lipids and vascular inflammation in patients with end-stage renal disease: a meta-analysis

Omega-3 fatty acids (O3FAs) are associated with lower cardiovascular disease (CVD) risk in adults. However, this association in patients with end-stage renal disease (ESRD) remains controversial prompting the need for investigation into the role of O3FAs on serum lipids and vascular inflammation markers. The present meta-analysis summarized the effects of O3FA supplementation on serum lipids and vascular inflammatory markers in patients with ESRD. PubMed, EmBase, and the Cochrane Library were searched to identify randomized controlled trials (RCTs) focused on serum lipids and vascular inflammation markers in patients with ESRD. Standard mean differences (SMD) were used to measure the effect of O3FA supplementation on serum lipids and vascular inflammatory markers. The final pooled analysis included 20 RCTs involving 1,461 patients with ESRD. The results indicated that O3FA supplementation reduced TG by 0.61, LDL by 0.35 and CRP by 0.56. However, O3FA had no significant effect on TC, HDL, albumin, hemoglobin, homocysteine, DBP, glucose, lipoprotein(a), and ferritin. O3FA supplementation is associated with lower several serum lipids and vascular inflammation markers in patients with ESRD.

Data relating the effect of O3FA supplementation on TG were collected from 16 RCTs. Overall TG level was significantly reduced (fixed model: SMD, − 0.35, 95%CI: − 0.49 to − 0.20, P < 0.001; random model: SMD, − 0.61, 95% CI: − 1.01 to − 0.22, P = 0.002; Fig. 2A) in patients treated with O3FA. The extent of significant effect of O3FA was noted across all RCTs regardless of substantial heterogeneity (P < 0.001), and the conclusion was not affected after sequential exclusion of each study from pooled analyses (Table S1).
Data relating the effect of O3FA supplementation on TC were collected from 17 RCTs. No significant difference in TC level was observed in patients treated with O3FA supplementation compared to control (fixed model: SMD, −0.10, 95%CI: −0.23 to 0.03, P = 0.132; random model: SMD, −0.30, 95%CI: −0.68 to 0.08, P = 0.118; Fig. 2B). Furthermore, substantial heterogeneity was noted across the trials (P < 0.001). The Taziki's study 24 was excluded from meta-analysis as younger non-diabetic patients on maintenance hemodialysis alone were enrolled in this trial. It was found that O3FA supplementation was significantly associated with reduced TC in patients with ESRD (fixed model: SMD: −0. 15 Fig. 3A). Furthermore, the pooled analysis using the fixed model revealed that O3FA supplementation was associated with a higher level of HDL (SMD: 0.20; 95%CI: 0.07 to 0.34; P = 0.003), whereas no significant difference was observed by random model (SMD: 0.46; 95%CI: − 0.02 to 0.94; P = 0.062; Fig. 3B). Substantial heterogeneity was observed in the magnitude of the effect across the studies (P < 0.001),  the Khosroshahi's study was excluded from pooled analysis and we noted O3FA supplementation has little or no significant effect on LDL; furthermore, when excluding Khosroshahi or Lee's study, O3FA supplementation significant increased the level of HDL (Table S1).
Data relating the effect of O3FA supplementation on CRP, albumin, and hemoglobin were collected from 9, 7 and 5 trials, respectively. We noted that O3FA supplementation significantly reduced the level of CRP (fixed model: SMD, − 0.55, 95%CI: − 0.75 to − 0.35, P < 0.001; random model: SMD, − 0.56, 95%CI: − 1.01 to − 0.11, P = 0.016; Fig. 4A). Although the summary results using fixed model indicated that O3FA supplementation was significantly associated with a reduction of albumin levels (SMD: − 0.27; 95%CI: − 0.52 to − 0.02; P = 0.037), no significant differences were seen between O3FA and control using the random model (SMD: − 0.33; 95%CI:     In the subgroup analysis, we stratified studies into groups to evaluate the sources of heterogeneity and explore the effect of O3FA in specific subpopulations (Table 2). First, O3FA supplementation was associated with lower TG level in multiple subsets except that the duration of follow-up was greater than 3 months. Second, O3FA supplementation significantly reduced the levels of TC in patients with BMI greater than 25.0. Third, O3FA supplementation was not associated with altered levels of LDL if the study was published before 2010, and the patients were residents of other countries, with a mean age greater than 60 years, BMI less than 25.0, and the trial showed a high quality. Forth, O3FA supplementation was associated with increased level of HDL if patients' BMI  was greater than 25.0. In addition, publication year, country, and dose of O3FA contributed significant heterogeneity between subgroups to LDL and HDL; Age and follow-up duration acted as the source of heterogeneity between subgroup for the effects of HDL; Significant heterogeneity between subgroups for TG was based on BMI; Study quality produced significant heterogeneity between subgroups for TC ( Table 2). Review of the funnel plots could not rule out the potential for publication bias for TG, TC, LDL, and HDL (Fig. 6). The Egger and Begg tests revealed no publication bias for TC (P values: 0.067 and 0.096, respectively), LDL (P values: 0.086 and 0.685, respectively), and HDL (P values: 0.141 and 0.711, respectively). Although the Begg test showed no evidence of publication bias for TG (P value: 0.053), the Egger test showed potential evidence of publication bias for TG (P value: 0.005). The conclusions were not changed after adjustment for publication bias by using the trim and fill method.

Discussion
In the present meta-analysis, the effects of O3FA supplementation on serum lipids and vascular inflammation markers such as TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin in patients with ESRD were investigated. In this comprehensive systemic review, 1,461 patients with ESRD in 20 RCTs were included. In the absence of statistical heterogeneity (I 2 < 50%), the fixed-effect model was used, otherwise the random effects model was applied. Therefore, the meta-analysis indicated that O3FA supplementation significantly reduced TG, LDL and CRP levels, whereas no significant effect was found in TC, HDL, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a), and ferritin levels using random models. According to sensitivity analysis, O3FA supplementation was beneficial in regulating TC. Subgroup analyses revealed that O3FA supplementation significantly reduced the TG and LDL levels in several subpopulations. Furthermore, O3FA supplementation was associated with a reduced TC, LDL and increased HDL levels in patients with BMI greater than 25.0. The methodological evaluation of each included study was limited by randomization, blinding, allocation concealment, withdrawals and dropouts, and use of intention-to-treat analysis. Our meta-analysis of RCTs provides clear randomization, blinding, and allocation concealment. Although most trials reported withdrawals and dropouts, and use of intention-to-treat analysis, the other forms of bias contributed to heterogeneity in every study. Ultimately, considering the unsatisfactory quality of included studies, we critically analyzed our recommendations for the treatment of patients with ESRD.
A previous meta-analysis 10 suggested that O3FA significantly lowered the serum TG levels. However, there was no significant effect on LDL, TC, and HDL. The inherent limitations of the previous review were as follows several important factors such as CRP, homocysteine, SBP, DBP, glucose, albumin, hemoglobin, lipoprotein(a), and ferritin were not summarized, and the study failed to reveal the effect of O3FA supplementation on serum lipids and vascular inflammation markers in several specific subpopulations. Furthermore, a previous meta-analysis 35,36 evaluated the effect of O3FA supplementation on the risk of major cardiovascular events. The effects of treatment on serum lipids and vascular inflammation markers remain unclear. Hence, a comprehensive systematic review and meta-analysis was undertaken to assess the effect of O3FA supplementation on serum lipids and vascular inflammation markers in patients with ESRD. Further subgroup analyses were performed to evaluate the effect of O3FA in subpopulations. In the present meta-analysis, the pooled SMD was < 0 for TG, TC, LDL, CRP, albumin, homocysteine, SBP, DBP, lipoprotein(a) and ferritin whereas it was > 0 for HDL hemoglobin, and glucose, which reflected a potential protective effect of O3FA. However, these trends were not obvious and require further validation. Several RCTs included in this systemic review have reported inconsistent results. Many RCTs have reported that there is no significant difference between O3FAs and control on major CVD risk factors. Kooshki et al. 12 conducted a RCT including 34 hemodialysis patients and found that marine O3FA supplementation could reduce serum TG, whereas it could not affect other serum lipids, lipoprotein, and hematologic factors among hemodialysis patients. Furthermore, Bouzidi et al. 13 study indicated that O3FA supplementation could improve hypertriglyceridemia and oxidative stress in patients with chronic renal failure, which might lead to a decreased rate of cardiovascular complications. Khalatbari Soltani et al. 14 study indicated that flax seed consumption could improve lipid abnormalities and reduce systemic inflammation in hemodialysis patients. Ando et al. 15 concluded that eicosapentanoic acid administration was an effective and safe treatment to decrease plasma remnant lipoproteins. Khosroshahi et al. 16 study indicated that O3FA supplementation could significantly reduce the serum homocysteine level; however, in contrast, high serum TC levels also reported. Similarly, Taziki et al. 24 pointed out that O3FA was associated with higher TC levels. Khosroshahi et al. 16 and Bowden et al. 18 suggested that O3FA significantly reduced the LDL, whereas other trials reported no significant difference 13,14 . The reason could be that the sample population size was smaller than expected, and these trials were designed to evaluate other lipid factors as primary end point. Hence, clinically significant differences in LDL were not found. Similarly, Khajehdehi et al. 25 and Lee et al. 27 reported inconsistent results relevant to HDL when compared to other studies 14,16,18 . Some RCTs suggested that O3FA supplementation was associated with elevated HDL level 14,18,25 . On the contrary, other two trials reported that the O3FA supplementation was related with reduced HDL level 16,27 . The reason might be that the enrolled patients had significant heterogeneity in disease status. Moreover, two trials reported that O3FA supplementation resulted in significant reduction of homocysteine levels 16,21 . Lok et al. 19 study suggested that O3FA was correlated with lower DBP and SBP. It was also found that O3FA supplementation could reduce the risk of intravascular clots, indicating its potential on reducing the risk of CVD.
The subgroup analysis indicated that O3FA supplementation had a significant relationship in reducing TG among multiple subsets. Notably, O3FA had no significant effect on LDL if the study published before 2010, the patients were residents of other countries, with a mean age greater than 60 years, BMI less than 25.0, and the study was a high-quality trial probably because of fewer number of trials included in these subsets. In addition, it was found that O3FA played a different role on serum lipids in different follow-up subsets. The possible reason could be that patients had different platelet count, alkaline phosphatase, serum sodium and potassium, and total iron binding capacity affected the treatment effects 37 . Another important reason could be that long-term O3FA supplementation might result in reduction of platelet activity and elimination of free radicals 38 . Finally, O3FA was associated with lower levels of TC and LDL, whereas elevated HDL level in patients with BMI was greater than 25.0. The reason for these could be that overweight participants had significant higher levels of TC and LDL than normal weight individuals, and O3FA supplementation showed reductions in BMI due to 03FA could promote fat oxidation and impair adipogenesis 39 .
A few advantages of the present meta-analysis were as follows: only RCTs were included for evaluation; the effect of O3FA supplementation in patients with ESRD was quantitatively determined using large pooled sample size; and the study provided evidence supporting the effects of O3FA supplementation on serum lipids and vascular inflammation markers such as TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin.
The present meta-analysis has certain limitations. First, a plenty of substantial heterogeneity among the included trials was identified in view of patients with different baseline characteristics. Second, publication bias could not be avoided when meta-analyzing published studies. Finally, more detailed relevant analysis and more comprehensive results could be restricted by conducting analysis using pooled data instead of individual data.
In summary, the findings of this meta-analysis suggested that O3FA supplementation was associated with lower serum TG, LDL, and CRP levels. Furthermore, sensitivity or subgroup analysis suggested that O3FA might play an important role on regulating TC levels. However, there was no significant difference between the effects of O3FA and control on HDL, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin levels. Future trials should focus on specific disease status and benefits of O3FA treatment for patients with ESRD.

Methods
Data Sources, Search Strategy, and Selection Criteria. The present meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocol, 2009 40 . The RCTs designed to evaluate the influence of omega-3 supplementation on serum lipids and vascular inflammation markers were included in our study, regardless of language and publication status. Meanwhile, the impact of omega-3 supplementation on TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, systolic blood pressure (SBP), diastolic blood pressure (DBP), glucose, lipoprotein(a) and ferritin were examined. The relevant RCTs to be included in this meta-analysis were identified as follows: (1) Screening of electronic databases: PubMed, Embase, and the Cochrane Central Register of Controlled Trials were searched for studies from their inception until April 2016, using ("linolenic acid" OR "timnodonic acid" OR "ALA" OR "δ -amino linolenic acid" OR "EPA" OR "eicosapentaenoic Acid" OR "docosahexaenoic acid" OR "DHA" OR "docosahexaenoic acid" OR "omega-3 fatty acid" OR "fish oil" OR "n-3 fatty acids" OR "fatty acid" OR "omega-3" OR "α -linolenic acid" OR "eicosapentanoic acid") AND ("kidney failure" OR "chronic renal failure" OR "dialysis" OR "hemodialysis" OR "peritoneal dialysis") as the search terms.
(2)Other sources: ongoing (completed but not published) RCTs were identified from the metaRegister of Controlled Trials. Data pertaining to registered RCTs was obtained from the website http://clinicaltrials.gov/ (US NIH). Besides, manual searches were carried out from the reference lists within the entire relevant original and review articles in order to identify the additional eligible trials.
Two authors followed a standardized approach for conducting literature research. In case of disagreements between the two authors, mutual consensus was arrived after discussion. Since observational studies were susceptible to confounding variables and bias, the present systemic review was limited to RCTs. Subsequently, eligible studies were identified based on the following criteria: (1) patients with ESRD; (2) RCTs; (3) omega-3 fatty acid supplementation; and (4) at least one of the following variables: TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin. Studies were excluded if: (1) patients were diagnosed with other diseases; (2) the study was an observational study; (3) the study with inappropriate control; and (4) the mean difference was not obtained or calculated.

Data Collection and Quality Assessment.
A standardized protocol was adopted by two authors to extract all the data from included trials. The data including first author, publication year, country, sample size, mean patient's age, sex ratio of participants, disease condition, intervention, control, outcomes, and follow-up duration were collected. In addition, data of serum lipids and vascular inflammation markers including TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin were collected. Simultaneously, the quality of included RCTs were assessed using Jadad score 34 , ranging from 0 to 5, on the basis of parameters including randomization, blinding, allocation concealment, withdrawals and dropouts, and use of intention-to-treat analysis.
Statistical Analysis. The results of each RCT was considered as continuous data, and standard mean difference (SMD) and 95% confidence intervals (CIs) of each individual study were calculated from mean, standard deviation, and sample size in each group in individual RCT. Furthermore, SMD with 95%CIs were calculated for serum lipids and vascular inflammation markers, including TG, TC, LDL, HDL, CRP, albumin, hemoglobin, homocysteine, SBP, DBP, glucose, lipoprotein(a) and ferritin in patients with ESRD receiving omega-3 supplementation. The pooled SMDs of O3FA supplementation and control were compared using the fixed-effect  (Mantel-Haenszel method) and random-effect models (DerSimonian-Laird method) 41,42 . In addition, to investigate the potential heterogeneity exist between RCTs, a subgroup analysis was performed based on the country, control, follow-up duration, and study quality. Besides, each individual trial was removed for carrying out a sensitivity analysis in the meta-analysis 43 . The heterogeneity of the treatment effects among RCTs was assessed using Cochrane Q-test; meanwhile, a P value of less than 0.10 was considered statistically significant 44,45 . P value for heterogeneity between subgroups were calculated by using Chi-square test 46 . Visual inspections of funnel plots for TG, TC, LDL, and HDL were conducted. The publication bias for TG, TC, LDL, and HDL parameters was also statistically assessed using Egger 47 and Begg 48 tests, and P values less than 0.05 was considered to have a significant publication bias. If significant publication bias was detected, trim and fill method were used to adjustment for publication bias 49 . STATA software (Version 10.0; StataCorp, Texas, United States of America) was used to perform the statistical analyses.