THE LONG-CHAIN n-3 polyunsaturated

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1 ORIGINAL RESEARCH Marine n-3 Polyunsaturated Fatty Acids in Patients With End-stage Renal Failure and in Subjects Without Kidney Disease: A Comparative Study Trine Madsen, MD, PhD,* Jeppe H. Christensen, MD, DMSc, My Svensson, MD, PhD, Petra M. Witt, MD, PhD, Egon Toft, MD, DMSc, and Erik B. Schmidt, MD, DMSc* Objective: Patients with end-stage renal disease treated with chronic hemodialysis (HD) are reported to have low levels of marine n-3 polyunsaturated fatty acids (PUFA) in plasma and cell membranes compared with healthy subjects. The aim of this study was to investigate whether n-3 PUFA levels in plasma and cells are lower in HD patients as compared with subjects without kidney disease. Research Design: A comparative study was carried out. Setting: This study was carried out at the Departments of Nephrology and Cardiology, Aalborg Hospital, Aarhus University Hospital, Denmark. Participants: This study consisted of 2 study populations comprising HD patients and 5 study populations comprising subjects without kidney disease. Intervention: The fatty acid distribution in plasma phospholipids and platelet phospholipids was measured using gas chromatography. Main Outcome Measure: Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) levels in plasma or serum phospholipids and platelet phospholipids in HD patients were compared with n-3 PUFA levels in subjects without kidney disease. Results: EPA and DHA were lower and AA/EPA was higher in plasma/serum phospholipids in HD patients than in subjects without kidney disease. Similarly, higher AA and AA/EPA and lower EPA and DHA levels were found in platelet phospholipids of HD patients. Adjustment for gender, age, and habitual intake of fish and fish oil supplements did not change these results. Conclusion: HD patients have lower n-3 PUFA levels in plasma and cells compared with subjects without kidney disease. ª 2011 by the National Kidney Foundation, Inc. All rights reserved. *Department of Cardiology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark. Department of Nephrology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark. Department of Oncology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark. Department of Health Sciences and Technology, Aalborg University, Aalborg, Denmark. Address reprints request to Trine Madsen, MD, PhD, Department of Cardiology, Aalborg Hospital, Sdr. Skovvej 15, 9000 Aalborg, Denmark. trine.madsen@rn.dk ª 2011 by the National Kidney Foundation, Inc. All rights reserved /$36.00 doi: /j.jrn THE LONG-CHAIN n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6 n-3), obtained primarily from fatty fish and fish oil, may have beneficial cardiovascular effects. 1 Cardiovascular morbidity and mortality is high in patients with end-stage renal disease (ESRD) treated with chronic hemodialysis (HD), 2,3 and although currently there is a lack of documentation of such cases among HD patients, 4 some evidence does point toward the beneficial effects of n-3 PUFA on cardiovascular endpoints in these patients. Thus, in observational studies, fish intake as well as a higher cellular content of EPA and DHA Journal of Renal Nutrition, Vol 21, No 2 (March), 2011: pp

2 170 MADSEN ET AL Table 1. Selected Characteristics of the Study Populations Study Study Populations Study Year N Men/Women n/n Mean Age Years High Fish Intake n (%) Compartment Analyzed Index Hemodialysis patients / (48%) Plasma phospholipids and Platelet phospholipids A Hemodialysis patients / (55%) Serum phospholipids B Healthy subjects / (50%) Serum phospholipids C Healthy subjects / (72%) Serum phospholipids D Pre- and post-menopausal women / (53%) Platelet phospholipids E Coronary heart disease / (73%) Platelet phospholipids F Atrial fibrillation / (68%) Platelet phospholipids has been associated with a reduced mortality in HD patients. 5,6 Some authors have reported that HD patients have a low intake of fish and consequently low cellular levels of n-3 PUFA compared with healthy subjects. 7,8 However, other small studies have shown conflicting results. The aim of the present study was to test the hypothesis of low n-3 PUFA levels in HD patients. We measured the levels of marine n-3 PUFA and the n-6 PUFA, arachidonic acid (AA; 20:4n-6), in plasma phospholipids (reflecting intake in the last few days) and platelet phospholipids (reflecting intake in the last few weeks) in HD patients and compared with fatty acid levels in subjects without kidney disease. Methods Study Populations In Table 1, all 7 study populations included in the present comparative study are characterized with regard to study size, age, gender, fish intake, and the compartment in which fatty acids were measured. The index study population comprised 44 patients with ESRD treated with chronic HD who participated in a randomized placebo-controlled intervention study investigating the effect of intravenous n-3 PUFA infusion on fatty acid levels and noninvasive electrocardiographic markers of sudden cardiac death (unpublished data). The median duration of HD in this study population was 30 months (range, months), and 55% of the patients had documented cardiovascular disease (CVD). The fatty acid levels measured in plasma and platelet phospholipids in the index study were compared with fatty acid levels measured in previous studies (1 study in HD patients and 5 studies in subjects without kidney disease) conducted at our research laboratory where fatty acids were measured in either plasma or platelet phospholipids. Study A included stable ESRD patients treated with HD, all of whom had established CVD. 9 Studies B and C included healthy volunteers (unpublished data). Study D 9a included pre- and postmenopausal women who were healthy or had a stable and well-treated chronic disease (i.e., osteoporosis, asthma, and myxedema) (unpublished data). Study E included patients referred for coronary angiography because of clinically suspected coronary heart disease, of which 71% had significant coronary stenoses. 10 Study F included patients with atrial fibrillation (unpublished data). Studies B, C, E, and F were cross-sectional studies. Studies A and D were intervention studies and fatty acid levels measured at baseline were used in the present study. All the original studies were approved by the regional ethics committee, and informed consent had been obtained from all participants. Assessment of Self Reported Fish Intake Information on the intake of fish and fish oil supplements was recorded in all studies, but 3 different food frequency questionnaires were used. In these questionnaires, the frequency of fish consumption for lunch and/or dinner was recorded, and the questionnaires were recoded to extract the information Two or more fish serving per week or Less than two fish servings per week. The subjects were then classified as having a High fish intake (i.e., fish consumption at least twice weekly or daily intake of fish oil capsules) or a Low fish intake (i.e., fish consumption less than twice weekly and no use of fish oil capsules).

3 N-3 PUFA IN ESRD PATIENTS 171 Detailed information on serving size, type of fish (fatty or lean), and concentrations of n-3 PUFA in capsules was not available. Blood Sampling In the 2 HD studies (index study and study A), blood was drawn from the dialysis access immediately before the HD session. Some of the HD patients (17 patients in the index study and 73 patients in Study A) were nonfasting for blood sampling because they had undergone HD on the same afternoon. In the other studies, blood samples were drawn in the fasting state. Fatty Acid Analysis In the index study, the fatty acid composition was measured in plasma phospholipids and in platelet phospholipids, whereas in studies A, B, and C, fatty acids were measured in serum phospholipids. The serum phospholipids method has been described previously. 9 In studies D, E, and F, the fatty acid composition was measured in platelet phospholipids. Plasma Phospholipids For analysis of plasma phospholipids, ethylenediaminetetraacetic acid (EDTA) blood was centrifuged within 30 minutes. The plasma was flushed with nitrogen to avoid oxidation and was stored at 280 C until analysis. Extraction of total lipids in plasma and separation of the phospholipid fraction were done by modification of methods described by Folch et al. 11 and Burdge et al. 12 Briefly, plasma was mixed with chloroform-methanol (CHCl 3 - MeOH) (2:1 v/v) containing butylated hydroxytoluene (BHT) as an antioxidant. After gentle shaking, 0.9% saline was added, and the tubes were centrifuged. The lower organic phase was collected and the extraction procedure was repeated on the protein disk. The organic phases were combined and dried under nitrogen. Lipid extracts were dissolved in CHCl 3 and transferred to a Bond Elut NH 2 column (200 mg) (Varian, Middleburg, The Netherlands) preconditioned with hexane. The column was washed with CHCl 3 to remove triglycerides and cholesterol esters. Phospholipids were eluted with CHCl 3 -MeOH (3:2 v/v) and additional MeOH. The phospholipid fraction was dried under nitrogen and redissolved in heptane, and the fatty acids were methylated with potassium hydroxide in MeOH. Platelet Phospholipids Platelets were isolated from EDTA blood, washed with 0.9% saline, flushed with nitrogen to avoid oxidation, and stored at 280 C until analysis. The fatty acids were extracted according to van Kuijk et al., 13 redissolved in heptane, and methylated with potassium hydroxide in MeOH. Gas Chromatography Analysis The fatty acid composition was analyzed by gas chromatography using a Varian 3900 GC equipped with a CP-8400 auto sampler, a flame ionization detector, and a CP-sil m mm ID capillary column (Varian, Middleburg, the Netherlands). Split injection mode, temperature programming from 90 C to 210 C, and constant flow were used. Helium was used as carrier gas. Commercially available standards (Nu-chek-Prep Inc, Elysian, MN) were used to recognize individual fatty acids. The fatty acid composition was expressed as a percentage of the total fatty acids. Statistical Analysis Differences between studies in the distribution of gender, age, and percentage of subjects with a high intake of fish were assessed using chi-square test or one-way analysis of variance. Fatty acid levels in the index study were compared with each of the other studies using unpaired t-test. Adjustment for differences in gender, age, and percentage of subjects with a high fish intake was made using analysis of covariance. Bivariate correlation (Pearson) was used to determine the associations between continuous variables. A P value,.05 was considered statistically significant. Results Plasma Phospholipids In the index study, EPA and DHA levels were comparable with EPA and DHA levels in the other HD study (study A, Table 2). AA and AA/EPA were lower in study A than in the index study. However, AA/EPA was higher in study A than in studies B and C (P,.001 and P,.05, respectively). The gender distribution and the percentage of subjects with a high intake of fish did not differ between the individual studies (P 5.23 and P 5.12, respectively), but the age distribution differed (P,.001). Adjustments for these 3 variables generated P values that were similar to the P values in

4 172 MADSEN ET AL Table 2. Fatty Acid Composition (Percent of Total Fatty Acids) In Plasma/Serum Phospholipids Index Study (HD Patients) Study A (HD Patients) the crude analyses, or in some instances, increased the level of statistical significance (Table 2). Platelet Phospholipids In the index study, EPA and DHA levels were lower and AA/EPA ratio was higher compared with studies D, E, and F (Table 3). Results for AA were mixed, as AA levels were higher in study D and lower in studies E and F compared with the index study. Gender, age distribution, and the percentage of subjects with a high intake of fish differed between the individual studies (P,.001, P,.001, and P,.001, respectively). However, adjustments for gender, age, and the percentage of subjects with a high intake of fish generated P values that were similar to the P values in the crude analyses, or in some instances, increased the level of significance (Table 3). Low Versus High Intake of Fish In The Index Study Patients with a low intake of fish had lower EPA and DHA levels compared with patients with a high fish intake; however, the difference was statistically significant only for EPA in plasma and EPA in platelet phospholipids ( and , P,.05; and and , P,.05, respectively). Study B (Healthy Subjects) Study C (Healthy Subjects) AA *(*) (**) EPA **(***) **(***) DHA ***(***) *(***) AA/EPA *(*) ***(***) ***(***) HD, hemodialysis; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. Levels of significance after adjustment for gender, age, fish intake are shown in parenthesis. * P,.01; ** P,.05; *** P,.001 (unpaired t-test; versus index study). Relation Between Plasma and Platelet n-3 PUFA In the index study, there was a strong correlation between EPA 1 DHA content of plasma and platelet phospholipids (correlation coefficient, 0.853; P,.001). Discussion The content of marine n-3 PUFA in plasma and cell membranes is determined primarily by the dietary n-3 PUFA intake. In the 2 studies with HD patients presented here, relatively large percentages (48% and 55%) of the patients were classified as having a high intake of fish. Current recommendations are that individuals without CVD should eat fish at least twice weekly and patients with documented CVD should consume 1 g of EPA 1 DHA. 14 Although nutrition guidelines for HD patients 15 and guidelines for the management of CVD in HD patients 16 do not include fatty fish or fish oil, further research has been recommended. 16 Marine n-3 PUFA is incorporated into phospholipids in competition with n-6 PUFA (primarily AA). Lower EPA and DHA levels and higher AA/EPA ratio in HD patients were found compared with subjects without kidney disease. The differences in fatty acid composition were strengthened rather than weakened after adjustment for Table 3. Fatty Acid Composition (Percent of Total Fatty Acids) of Platelet Phospholipids Index Study (HD Patients) Study D (Women) Study E (Coronary Heart Disease) Study F (Atrial Fibrillation) AA *(*) *(*) *(*) EPA (**) *** ***(***) DHA ***(*) *(*) *(*) AA/EPA *(*) *(*) *(*) HD, hemodialysis; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. Levels of significance after adjustment for gender, age, fish intake are shown in parentheses. * P,.001; ** P,.05; *** P,.01 (unpaired t-test; versus index study).

5 N-3 PUFA IN ESRD PATIENTS 173 Table 4. Results From Published Studies Comparing n-3 PUFA Levels In HD Patients And Healthy Controls (Excluding Our Data) Authors Study Population Compartment Main Results* Friedman et al. 7 HD patients (n 5 75) Total plasma DHA lower and AA/EPA higher Healthy controls (n 5 25) RBC AA, DHA, and AA/EPA higher Koorts et al. 17 HD patients (n 5 14) RBC EPA lower Healthy controls (n 5 10) Peck et al. 18 HD patients (n 5 25) Total plasma AA and EPA lower Healthy controls (n 5 22) Ristic et al. 19 HD patients (n 5 37) Plasma phospholipids EPA and DHA lower Healthy controls (n 5 29) RBC EPA and DHA lower Dasgupta et al. 20 HD patients (n 5 9) Total plasma AA higher Healthy controls (n 5 9) An et al. 21 HD and PD patients (n 5 29) RBC AA higher Healthy controls (n 5 10) HD, hemodialysis; PD, peritoneal dialysis; RBC, red blood cells; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid. *Versus healthy controls (only statistically significant differences are mentioned). the number of patients with a high intake of fish in the individual studies. Low n-3 PUFA levels in HD patients may be explained by other factors including diarrhea, and malabsorption, disturbance of metabolism and incorporation of n-3 PUFA in a uremic milieu, inflammation, or loss of n-3 PUFA into the dialysate. Other studies have compared HD patients and healthy subjects with regard to n-3 PUFA levels, but most of the studies were relatively small and results conflicting (Table 4). 7,17 21 In the largest of these studies, Friedman et al 7 compared the fatty acid composition in total plasma and in red blood cells (RBC) in 75 HD patients and 25 matched healthy controls. In plasma, lower DHA and higher AA/EPA were seen in HD patients. Somewhat contradictory, the content of DHA measured in RBC was considerably higher in HD patients. In the index study, patients with a high intake of fish had higher n-3 PUFA levels compared with patients who had a low intake of fish. Similarly, supplementation of the diet with fish oil capsules has been shown to improve n-3 PUFA status in HD patients. In study A, 9 supplementation with 1.7 g/d n-3 PUFA for 3 months increased serum phospholipid EPA 1 DHA from 5.4% to 9.3%, and in another study, EPA 1 DHA in the RBC increased from 2.1% to 6.8% after supplementation with 1.3 g/d n-3 PUFA for 12 weeks. These increases are comparable with the changes reported in healthy subjects after n-3 PUFA supplementation. 22 There are several reasons why n-3 PUFA status in HD patients may be important. First, sudden cardiac death accounts for 30% of total mortality in HD patients, 23 and n-3 PUFA has been shown to protect against sudden cardiac death in healthy subjects and in patients with a previous myocardial infarction There is also some evidence that the intake of n-3 PUFA may improve survival in HD patients. In a follow-up study with 216 incident HD patients, the mortality rate observed in patients who reported to consume fish at baseline was approximately 50% lower compared with those not eating fish. 5 In a retrospective study in 93 HD patients, 6 an inverse association between baseline EPA 1 DHA in RBC and mortality was suggested, although not statistically significant, possibly because of the moderate study size. 6 In study A, a large randomized, placebo-controlled intervention study in 206 HD patients, 1.7 mg/d n-3 PUFA for 2 years reduced the number of myocardial infarctions, whereas there was no effect on the primary composite endpoint of cardiovascular events and death. 9 The second reason is that when AA/EPA is high, the proinflammatory leukotriene B5 derived from n-6 PUFA is produced in excess of the virtually inactive leukotriene B4 derived from n-3 PUFA. 27 It is well established that inflammatory processes promoted by HD treatment and the often co-existing malnutrition play an important role in the accelerated atherosclerosis in HD patients. 28 An increased n-3 PUFA intake may decrease inflammation and reduce CVD risk. Third, n-3 PUFA inhibit platelet aggregation by altering the balance between the proaggregatory thromboxane A2 derived from n-6 PUFA and the much less active thromboxane A3 derived from n-3 PUFA. 29 A small study showed improved

6 174 MADSEN ET AL patency of synthetic hemodialysis grafts, 30 whereas another showed no effect of n-3 PUFA. 31 Two large studies to address this issue are underway. 32,33 Finally, n-3 PUFA supplements have been shown to reduce hypertriglyceridemia 34 which are part of the atherogenic uremic dyslipidemia. 35 There are no studies that indicate which blood compartment or tissue provides the best marker of n-3 PUFA status in HD patients or whether a low n-3 PUFA status can in fact identify HD patients at a particularly high cardiovascular risk. We measured n-3 PUFA levels in plasma/serum phospholipids and platelets because these methods are well-established in our laboratory. Recently, the so-called omega-3 index (EPA 1 DHA in the RBC) has gained attention. 36 This index correlates with myocardial EPA 1 DHA 37 and has been shown to be inversely associated with CVD mortality. 36 It has been suggested that the omega-3 index should be implemented as a cardiovascular risk marker, with cut-off values indicating increasing levels of risk. 36 No equivalent cut-off values are established for n-3 PUFA in other blood compartments or tissues, but there is a strong correlation between omega-3 index and the content of EPA 1 DHA in plasma phospholipids, 36 and in the index study, EPA 1 DHA in plasma phospholipids and platelet phospholipids correlated well. However, RBC EPA 1 DHA may underestimate n-3 PUFA status in HD patients. Hemoglobin A1c underestimates glycemic control in HD patients with diabetes, because RBCs have a shorter life span in HD patients and erythropoietin treatment changes the proportion of young RBC to old RBC. 38,39 Similarly, little n-3 PUFA may be incorporated into RBC membranes because of the shorter life span of the RBC. Study Limitations The study populations compared in this article were derived from separate studies rather than being parts of the same research protocol. Thus, HD patients and controls were not matched, but differences between populations in age, gender, BMI, and fish intake were adjusted for in this article. The studies were conducted over a span of several years, and during that time the methods used for analyzing the fatty acid were gradually modified. Our laboratory has achieved very similar results when analyzing plasma and serum from the same samples (unpublished data), and all fatty analyses have been performed by the same laboratory technicians. We believe that the slightly different methods used do not affect the comparability of the fatty acid levels measured in the individual studies. Recall bias and the lack of information on serving size, type of fish, and n-3 PUFA concentrations in capsules make self-reported fish intake a rather crude measure of n-3 PUFA intake. Furthermore, 3 different food frequency questionnaires were used and a common variable (high vs. low intake of fish) was generated from the available data. Finally, in the 2 studies on HD, some of the patients were fasting whereas others were not for blood sampling, but the fasting state is not likely to affect the fatty acid distribution in phospholipids. Conclusion And Perspectives The present study showed that HD patients have lower n-3 PUFA levels in plasma and cells compared with subjects without kidney disease even after adjustment for fish intake. Other factors related to ESRD and HD treatment may be important but are not identified in this study. A low n-3 PUFA status may be associated with cardiovascular morbidity and mortality in HD patients. However, large intervention studies are needed before evidencebased recommendations can be made for an increased fish intake and/or n-3 PUFA supplementation in HD patients. Beside a possible therapeutic potential of n-3 PUFA in HD patients, it would be of interest to investigate whether n-3 PUFA status can be used as a prognostic marker to identify HD patients at particularly high risk of sudden cardiac death (SCD), but the best marker for n-3 PUFA status in HD patients has yet to be identified. Acknowledgments The study was supported by the Danish Kidney Foundation. The authors thank Lone Jeppesen Bjerregaard and Anne- Mette Haugaard for contributing data. References 1. Lavie CJ, Milani RV, Mehra MR, et al: Omega-3 polyunsaturated fatty acids and cardiovascular diseases. J Am Coll Cardiol 54: , Sarnak MJ, Levey AS, Schoolwerth AC, et al: Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Circulation 108: , 2003

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