Prostaglandins, Leukotrienes and Essential Fatty Acids 89 (2013) 189–194

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Prostaglandins, Leukotrienes and EssentialFatty Acids

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Inflammatory response following heart surgery and associationwith n�3 and n�6 long-chain polyunsaturated fatty acids in plasmaand red blood cell membrane lipids

L. Bjorgvinsdottir a, O.S. Indridason b, R. Heidarsdottir a, K. Skogstrand c, D.O. Arnar a,b,d,B. Torfason a,e, D.M. Hougaard c, R. Palsson a,b, G.V. Skuladottir a,n

a Faculty of Medicine, School of Health Sciences, University of Iceland, Vatnsmyrarvegur 16, IS-101 Reykjavik, Icelandb Internal Medicine Services, Landspitali—The National University Hospital of Iceland, Hringbraut, IS-101 Reykjavik, Icelandc Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmarkd Cardiovascular Research Center, Landspitali—The National University Hospital of Iceland, Hringbraut, IS-101 Reykjavik, Icelande Landspitali—The National University Hospital of Iceland, Hringbraut, IS-101 Reykjavik, Iceland

a r t i c l e i n f o

Article history:Received 8 April 2013Received in revised form12 July 2013Accepted 26 July 2013

Keywords:Heart surgeryInflammatory mediatorsInflammatory responsen�3 long-chain polyunsaturated fatty acidsPostoperative periodRed blood cell membranes

78/$ - see front matter & 2013 Elsevier Ltd. Ax.doi.org/10.1016/j.plefa.2013.07.007

espondence to: Department of Physiology, FaSciences, University of Iceland, VatnsmyrarTel.: +354 525 4825; fax: +354 525 4886.ail address: gudrunvs@hi.is (G.V. Skuladottir).

a b s t r a c t

Background: Open heart surgery is associated with a systemic inflammatory response. The n�3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA), and the n�6 LC-PUFA arachidonic acid (AA) may contribute to modulation of the inflammatoryresponse.Objective: We investigated whether the preoperative levels of EPA, DHA and AA in plasma phospholipids(PL) and red blood cell (RBC) membrane lipids in patients (n¼168) undergoing open heart surgery wereassociated with changes in the plasma concentration of selected inflammatory mediators in theimmediate postoperative period.Results and conclusions: The postoperative concentration of TNF-β was lower (Po0.05) and thoseof hs-CRP, IL-6, IL-8, IL-18 and IL-10 higher (Po0.05) than the respective preoperative concentrations.We observed that the preoperative levels of EPA and AA in plasma PL and RBC membrane lipids wereassociated with changes in the concentration of pro-inflammatory and anti-inflammatory mediators,suggesting a complex role in the postoperative inflammatory process.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Heart surgery provokes a vigorous inflammatory response thatpropagates within the injured tissue to initiate the healing process[1–4]. However, excessive systemic inflammation may result inadverse outcomes during the postoperative period [5]. The inflam-matory response is modulated by a balance between pro-inflammatory and anti-inflammatory mediators secreted by a vari-ety of cell types, including activated monocytes, tissue macrophages,lymphocytes and endothelial cells [5–7].

The n�3 long-chain polyunsaturated fatty acids (LC-PUFA), eico-sapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beenshown to evoke anti-inflammatory responses [8], while the n�6 LC-PUFA aracidonic acid (AA) generally induce a more pronouncedpro-inflammatory effect [9,10]. The fatty acid composition of plasma

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culty of Medicine, School ofvegur 16, IS-101 Reykjavik,

phospholipids (PL), which are merely transporters of circulating fattyacids, is believed to reflect short-term dietary n�3 LC-PUFA con-sumption [11]. In contrast, the fatty acid composition of red blood cell(RBC) membrane lipids is considered a good indicator of a long-termdietary n�3 LC-PUFA consumption [11]. Furthermore, fatty acidcomposition of RBC membrane lipids has been shown to reflect thefatty acid composition of other cell membrane lipids, including thoseof cardiac myocytes in the intraventricular septum [12], and atria [13].

Several pro-inflammatory and anti-inflammatory mediators, aswell as the acute-phase reactant C-reactive protein (CRP) havebeen implicated in the inflammatory response early in the post-operative course following open heart surgery [6,7,14–16]. Humanstudies have demonstrated that dietary EPA and DHA may attenu-ate postoperative concentrations of circulating pro-inflammatorymediators [17,18]. To investigate the role of EPA, DHA and AA inthe inflammatory response following open heart surgery, weexamined the association between the preoperative levels ofEPA, DHA and AA in plasma PL and RBC membrane lipids andthe postoperative changes in plasma concentrations of 12 selectedinflammatory mediators.

L. Bjorgvinsdottir et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 89 (2013) 189–194190

2. Patients and methods

2.1. Subjects

This study was based on data collected as part of a prospective,randomized, double-blinded, placebo-controlled clinical trial onthe use of n�3 LC-PUFA therapy for one week prior to open heartsurgery for the prevention of postoperative atrial fibrillation. Thefatty acid analysis was pre-specified in the study protocol whereasthe measurement of the inflammatory markers other than CRPwas added post-hoc. This study was approved by the BioethicsCommittee of Landspitali—The National University Hospital ofIceland (62/2004), and the Icelandic Data Protection Authority.The details of the study design have been published previously[19,20]. In brief, 168 patients scheduled for elective or semi-emergent open heart surgery were included in this study. Patientsyounger than 40 years of age, those with a history of any form ofsupraventricular arrhythmias or using the antiarrhythmic medica-tions amiodarone and/or sotalol, and patients undergoing emer-gency surgery were excluded. Prior to surgery, all participantsanswered a questionnaire on lifestyle and health-releated issues,including consumption of fish, intake of liquid cod liver oil andn�3 LC-PUFA capsules, smoking habit, height, body weight, andmedication use. All patients participating in the study gave writteninformed consent. One week prior to the surgical procedure, thepatients were randomly assigned to one of two groups initiatingthe study treatment and were asked to discontinue intake of liquidcod liver oil and supplemental n�3 LC-PUFA capsules, but wereotherwise advised to remain on their usual diet. The n�3 LC-PUFAtreatment consisted of 1240 mg of EPA and 1000 mg of DHA in theform of ethyl esters administered once daily, while the identicalplacebo capsules contained 2000 mg of olive oil, also administeredonce daily. The n�3 LC-PUFA capsules are commercially availablein Iceland (Omega-3 Forte, Lysi Inc, Reykjavík, Iceland).

2.2. Blood plasma and red blood cells

Venous blood samples were obtained from the patients beforeinitiating the study medication (baseline), immediately before thesurgery (preoperatively) and on the third postoperative day (post-operatively). The blood samples were collected into disodiumEDTA tubes and the plasma separated from RBC by immediatecentrifugation at 1000g for 10 min. The RBC were washed threetimes with an isotonic saline solution and the antioxidant buty-lated hydroxytoluene (BHT), dissolved in methanol, was added tothe cells at a final concentration of 50 mg/L. The plasma and RBCsamples were frozen at �76 1C and stored until the analysis of theinflammatory mediators and the fatty acids was carried out.

2.3. Inflammatory mediators

The plasma samples were analysed for inflammatory mediatorstumor necrosis factor-α (TNF-α), TNF-β, interleukin-1β (IL-1β), IL-6,IL-8, IL-10, IL-12, IL-18, interferon-γ (IFN-γ), macrophage inflam-matory protein-1α (MIP-1α) and transforming growth factor-β(TGF-β) as previously described [21], and determined using theLuminex 100™ platform (Luminex Corp, TX, USA). These media-tors were selected based on their potential role in the acuteinflammatory response following major surgery (6,7,14,15,16).The samples were measured in duplicate and standard curveswere fitted with a five parameter logistic equation (Logistic-5PL)using BioPlex™ Manager 5.0 (Bio-Rad Laboratories, CA, USA).

Plasma CRP concentration was determined using a commer-cially available high sensitivity (hs)-CRP latex-enhanced immuno-turbidimetric assay (Roche Diagnostics, Mannheim, Germany) andHitachi 911 analyser. The lower detection limit of the assay is0.1 mg/L. The total coefficient of variation for hs-CRP measure-ments of internal controls was 1.1% at a concentration of 3.73 mg/Land 1.9% at a concentration of 0.68 mg/L.

2.4. Fatty acid composition of plasma phospholipids and RBCmembranes lipids

Total lipids were extracted from plasma using the Folch method[22], and the phospholipid (PL) fraction isolated using thin layerchromatography. The lipid fraction was extracted from RBC mem-brane lipids using a method described by Bligh and Dyer [23]except that isopropanol was used instead of methanol (isopropa-nol/chloroform 2:1, v/v). BHT (50 mg/L) was added to the extrac-tion medium. Fatty acid methyl esters (FAME) of plasma PL andRBC membrane lipids were formed using 14% boron trifluoride/methanol (Sigma Chemical Co., St. Louis, MO, USA) at 110 1C for45 min. The FAME of plasma PL were analysed by gas–liquidchromatography as previously described [19], but those of theRBC membrane lipids were analysed by gas chromatography(Agilent 6890N, Agilent, Palo Alto, CA, USA) using a ChrompackCP-SIL 8CB column (25 m�250 mm i.d. �0.12 mm film thickness).The oven was programmed to provide an initial temperature of150 1C for 4 min, then increasing temperature by 4 1C/min to230 1C and then by 20 1C/min to 280 1C, and finally the oven washeld isothermal for 4 min. The injector and detector temperatureswere maintained at 280 1C and 300 1C, respectively. Hydrogen wasused as the carrier gas. The FAME peaks were identified andcalibrated against commercial standards (Sigma Chemical Co.; Nu-Chek-Prep, Elysian, MN, USA). Fatty acid values in plasma PL andRBC membrane lipids are presented as % weight of total fatty acidswith chain length from C14 to C24. Instrumental control and datahandling was performed using HP 3365 Chemstation, VersionA.02.12. (Hewlett Packard Co., Palo Alto, CA, USA).

2.5. Statistical analysis

The main objective was to examine the relationship betweenfatty acid levels in plasma PL and RBC membrane lipids preopera-tively and the changes in concentrations of inflammatory media-tors from immediately prior to surgery to the third postoperativeday. Independent samples t-test was used to compare groups withrespect to the levels of EPA, DHA and AA in plasma PL and RBCmembrane lipids at baseline and preoperatively, and paired t-testto examine the significance of changes between time points withingroups. Due to non-normal distribution, Wilcoxon signed rank testwas used to compare the difference in median concentrations ofinflammatory mediators between time points. Spearman's corre-lation coefficient was employed to examine the relationshipbetween continuous variables. Multivariable linear regressionwas used to assess the relationship between preoperative levelsof fatty acids in plasma PL and RBC membrane lipids and changesin the concentrations of inflammatory mediators following sur-gery, adjusting for age, body mass index (BMI) and smoking, asthese variables were a priori assumed to have a potential con-founding effect on the relationship.

Data are presented as median and range, percentages or mean7standard error of the mean (SEM). A two-sided P valueo0.05 wasconsidered statistically significant. All statistical analyses were carriedout using SPSS software (version 17.0, IBM Corporation, Somers,NY, USA).

Table 1Baseline and surgical characteristics of the patients.

Characteristic Value (n¼168)

Age (years) 67 (43–82)Gender (% men) 79.2BMI (kg/m2) 27.4 (17.2–41.3)Diabetes (%) 14.9Smoking (%) 19.0Fish intake (%, 4once a week) 72.0Liquid cod liver oil (%) 54.8n�3 LC-PUFA capsules (%) 26.8Use of statins (%) 80.4ECC time (min) 96 (0–261)On-pump surgery (%) 88.1Aortic cross-clamp time (min) 48 (0–208)Blood volume in drains (mL) 765 (96–4980)

Data are presented as median (range) or percentage. BMI,body mass index; LC-PUFA, long-chain polyunsaturatedfatty acids; ECC, extracorporeal circulation.

Table 2Fatty acid levels (% of total fatty acids) in plasma PL and RBC membrane lipids atbaseline and preoperatively after one week of placebo or n�3 LC-PUFA treatment.

Fatty acids Placebo (n¼79) n�3 LC-PUFA (n¼80)

Plasma PLEPA (20:5n�3)Baseline 2.7570.20 2.5470.15Preoperative 2.3470.12a 4.4370.16a,b

DHA (22:6n�3)Baseline 6.0670.16 6.1770.16Preoperative 5.9870.14 6.9870.13 a,b

AA (20:4n�6)Baseline 8.8570.26 8.6070.25Preoperative 9.0770.25 a 8.9370.24 a

RBC membrane lipidsEPA (20:5n�3)Baseline 1.9670.09 1.9270.10Preoperative 1.8870.08 a 2.3270.09 a,b

DHA (22:6n�3)Baseline 7.3170.13 7.4870.14Preoperative 7.3570.13 7.5870.14 a,b

AA (20:4n�6)Baseline 12.2270.19 12.0770.20Preoperative 12.1670.18 12.0870.20

Data are expressed as mean7SEM. PL, phospholipids; RBC, red blood cell;LC-PUFA, long-chain polyunsaturated fatty acids; EPA, eicosapentaenoic acid;DHA, docosahexaenoic acid; AA, arachidonic acid.

a Po0.05, compared with the baseline levels within groups. Paired t-test.b Po0.05, compared with the placebo group at the same time period.

Independent samples t-test.

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3. Results

3.1. Characteristics of the patients

The median plasma concentrations of the inflammatory med-iators did not differ between the groups of patients receiving n�3LC-PUFA or placebo (P40.05; data not shown). Thus, all patientswere combined for analysis of the association between preopera-tive levels of fatty acids in plasma PL and RBC membrane lipidsand postoperative changes in plasma concentrations of inflamma-tory mediators. Baseline and surgical characteristics of the patients(n¼168) are outlined in Table 1. The median age of the patientswas 67 (range, 43–82) years, 79.2% were men, and their medianBMI was 27.4 (range, 17.2–41.3) kg/m2. Seventy-two percent of thepatients consumed fish once or more each week, 55% used codliver oil and one-quarter n�3 LC-PUFA capsules as dailysupplements.

3.2. EPA, DHA and AA levels of plasma PL and RBC membrane lipids

As shown in Table 2, our patients had relatively high baselinelevels of EPA and DHA in plasma PL and RBC membrane lipids.Table 2 also demonstrates how the one-week n�3 LC-PUFA orplacebo treatment affected the levels of EPA, DHA and AA inplasma PL and RBC membrane lipids of our patients, i.e. thepreoperative levels.

3.3. Preoperative and postoperative plasma concentrations ofinflammatory mediators

Fig. 1 shows the plasma concentrations of the inflammatorymediators in the patients preoperatively and on postoperative daythree. The postoperative concentration of the pro-inflammatorycytokine TNF-β was significantly lower (Po0.05), and those ofthe pro-inflammatory mediators hs-CRP, IL-6, IL-8 and IL-18 andthe anti-inflammatory cytokine IL-10 were significantly higher(Po0.05) than the preoperative concentrations. No changes wereobserved in the concentrations of the pro-inflammatory mediatorsTNF-α, IL-1β, IL-12, IFN-γ, MIP-1α, and the anti-inflammatorycytokine TGF-β.

3.4. Relationship between preoperative levels of EPA, DHA, and AA inplasma PL and RBC membrane lipids and postoperative changes ininflammatory mediators

A separate multivariable linear regression model was used toassess the relationship between the preoperative levels of AA, EPAand DHA in plasma PL and RBC membrane lipids and the intra-individual change in the plasma concentration of each inflamma-tory mediator, observed on the third postoperative day. Thechanges in the plasma concentrations of TNF-β, IL-1β, IL-10,IFN-γ and TGF-β were significantly (Po0.05) associated with thelevels of one or more of the fatty acids in plasma PL and/or in RBCmembrane lipids when adjusted for age, BMI and smoking(Table 3). A more conspicuous decrease in the concentration ofTNF-β was associated with a higher AA level (β¼�0.240), and asmaller decrease with a higher ratios of DHA/AA and EPA+DHA/AA(β¼0.196 and 0.170, respectively) in RBC membrane lipids. A morepronounced increase in the IL-1β concentration was associatedwith higher ratios of DHA/AA and EPA+DHA/AA in plasma PL(β¼0.221 and 0.191, respectively), and with a higher level of EPA,as well as with higher ratios of EPA/AA, DHA/AA and EPA+DHA/AAin RBC membrane lipids. A greater increase in IL-10 was associatedwith a higher AA level, a lower EPA level and lower ratios of EPA/AA and EPA+DHA/AA in plasma PL. A greater increase in IFN-γ wasassociated with a higher EPA level, and with a higher ratio of EPA/AA in RBC membrane lipids. A smaller increase in TGF-β wasassociated with a higher level of AA, and a greater increase wasassociated with higher ratios of DHA/AA and EPA+DHA/AA inplasma PL. Furthermore, a smaller increase in TGF-β was asso-ciated with a higher AA level, and a greater increase with higherEPA and DHA levels, and higher ratios of EPA/AA, DHA/AA and EPA+DHA/AA in RBC membrane lipids. The relationship between post-operative changes in the plasma concentrations of hs-CRP, IL-6, IL-8,

Fig. 1. Median plasma concentrations of the inflammatory mediators TNF-β, IL-6, hs-CRP, IL-8, IL-10, IL-18, TNF-α, IL-1β, IL-12, IFN-γ, MIP-1α, and TGF-β preoperatively and onthe third postoperative day in patients undergoing open heart surgery. nPo0.02 and nnPo0.001 compared with preoperative concentrations. Wilcoxon signed rank test.

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IL-12, IL-18, TNF-α and MIP-1α and the preoperative levels of AA, EPAand DHA in plasma PL or RBC membrane lipids was not statisticallysignificant.

4. Discussion and conclusions

In this study we observed a vigorous systemic inflammatoryresponse following open heart surgery. More importantly, wefound this response to be related to the preoperative fatty acidcomposition of plasma PL and/or RBC membrane lipids.

During cardiac surgery, tissue injury and other factors induce arobust systemic inflammatory reaction [5]. Animal and humanstudies have demonstrated that the inflammatory response can bemodulated through changes in dietary intake of n�3 LC-PUFA,which predominantly have anti-inflammatory properties [8].Moreover, it has been demonstrated that patients undergoing

major non-cardiac surgery who received fish oil parenterally for7 days postoperatively, had lower serum concentrations of thepro-inflammatory mediators IL-1β, IL-8 and IFN-γ on postoperativeday 4 compared with patients receiving soybean oil-based lipidemulsion, which is rich in the AA precursor linoleic acid [17].In the present study, we observed marked postoperative changesin the concentrations of several pro-inflammatory and anti-inflammatory mediators, consistent with previous reports[14,24], although no difference was observed between the n�3LC-PUFA- and placebo-treated patients. The relatively high base-line levels of EPA and DHA in plasma PL and RBC membrane lipids,reflect the daily use of cod liver oil and/or n�3 LC-PUFA supple-ments by a large proportion of patients in our study. The one-weekcourse of n�3 LC-PUFA treatment resulted in modest changes inthe levels of plasma PL EPA and DHA and almost no change in thelevels of RBC membrane EPA and DHA compared with the baselinelevels. Thus, short-term supplementation with moderate doses of

Table 3Relationship between preoperative levels of fatty acids in plasma phospholipids (PL) or red blood cell (RBC) membrane lipids and the postoperative change in the plasmaconcentrations of selected inflammatory mediators, measured immediately before surgery and on the third postoperative day.

dTNF-β (pg/mL) dIL-1β (pg/mL) dIL-10 (pg/mL) dIFN-γ (pg/mL) dTGF-β (pg/mL)

Betaa P R2 Beta P R2 Beta P R2 Beta P R2 Beta P R2

Plasma PLAA �0.013 0.868 0.029 �0.101 0.214 0.031 0.163 0.044 0.047 0.000 0.997 0.022 �0.217 0.007 0.065EPA �0.016 0.847 0.029 0.058 0.478 0.025 �0.191 0.018 0.057 0.127 0.116 0.037 �0.013 0.873 0.021DHA 0.130 0.118 0.044 0.159 0.055 0.045 �0.034 0.683 0.023 0.158 0.057 0.044 0.101 0.226 0.030EPA/AA ratio �0.359 0.991 0.029 0.129 0.117 0.037 �0.195 0.017 0.057 0.116 0.158 0.034 0.072 0.384 0.025DHA/AAratio 0.096 0.255 0.037 0.221 0.008 0.065 �0.132 0.117 0.037 0.107 0.204 0.032 0.234 0.005 0.069EPA+DHA/AA ratio 0.052 0.532 0.031 0.191 0.022 0.054 �0.177 0.034 0.050 0.122 0.147 0.035 0.167 0.046 0.045

RBClipidsAA �0.240 0.003 0.091 �0.154 0.061 0.042 0.072 0.383 0.026 �0.110 0.181 0.030 �0.264 0.001 0.080EPA 0.072 0.385 0.042 0.221 0.008 0.063 �0.064 0.447 0.025 0.182 0.030 0.049 0.198 0.018 0.050DHA 0.144 0.102 0.054 0.121 0.176 0.031 �0.035 0.693 0.022 0.011 0.907 0.019 0.165 0.064 0.037EPA/AA ratio 0.107 0.198 0.048 0.227 0.006 0.066 �0.062 0.457 0.025 0.168 0.044 0.045 0.219 0.009 0.058DHA/AA ratio 0.196 0.021 0.070 0.175 0.041 0.046 �0.064 0.459 0.000 0.077 0.368 0.024 0.237 0.005 0.063EPA+DHA/AA ratio 0.170 0.044 0.062 0.203 0.017 0.055 �0.063 0.463 0.025 0.144 0.180 0.030 0.239 0.005 0.065

a Standardized beta coefficient (β) for the fatty acid from each regression model. The analysis of the relationship between arachidonic acid (AA), eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) and selected inflammatory mediators is adjusted for age, body mass index (BMI) and smoking. Bold P values indicate significantrelationship by multivariable linear regression analysis. R2 represents the proportion of the variability in the change in an inflammatory mediator which is explained bythe model.

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n�3 LC-PUFA did not have an effect on the inflammatory responseas measured by the changes in the concentration of inflammatorymediators on postoperative day 3 in the present study.

A more detailed analysis of fatty acid levels in plasma PL and RBCmembrane lipids suggested a potential role for EPA, DHA and/or AAin the inflammatory process. We found the alterations in the plasmaconcentrations of the pro-inflammatory mediators TNF-β, IL-1β,IFN-γ, and the anti-inflammatory mediators IL-10 and TGF-β inresponse to surgery to be significantly, albeit weakly, associatedwith the preoperative levels of AA, EPA, and the ratios of EPA/AA,DHA/AA and EPA+DHA/AA in either plasma PL and RBC membranelipids or both, whereas no such association was found with DHA. Itis known that EPA can compete with AA, as the same enzymes,cyclooxygenase and lipoxygenase, are involved in the metabolism ofthese fatty acids, and that eicosanoids derived from EPA haveweaker inflammatory effects than those derived from AA [9,10].Existing evidence also suggesting that during an acute inflamma-tory response, macrophages generate the potent anti-inflammatoryand proresolving lipid mediator lipoxin A4 from AA, which in turnstimulates the production of the anti-inflammatory mediator IL-10[25]. Consistent with this notion, we observed that higher AA leveland lower EPA level in plasma PL were associated with a greaterincrease in IL-10. In contrast, we did not find a significant associa-tion between AA or EPA in RBC membrane lipids with the changesin IL-10, suggesting that the content of these fatty acids in plasmaPL may play a greater role in this process.

However, higher levels of AA and lower ratios of DHA/AA andEPA+DHA/AA in RBC membrane lipids were associated with agreater decline in TNF-β concentration, again suggesting a poten-tial anti-inflammatory effect of AA. While the average concentra-tions of the other three mediators, IL-1β, IFN-γ and TGF-β, did notchange following surgery, the intra-individual variability inchanges of their concentration associated significantly with pre-operative fatty acid levels. This association was most consistent forTGF-β as lower AA levels and higher ratios of DHA/AA and EPA+DHA/AA in both plasma PL and RBC membrane lipids as well as ahigher EPA level in RBC membrane lipids were associated with anincrease in the concentration of this anti-inflammatory cytokine. Ithas been demonstrated that TGF-β is produced by numerous celltypes and is one of the most potent chemoattractant for mono-cytes and other cell types within wounds [26]. TGF-β also down-regulates the production of pro-inflammatory mediators [5],

inhibits cell proliferation and induces apoptosis [27]. Though thesefindings would suggest a possible anti-inflammatory effect of EPA,the relationship observed between EPA in RBC membrane lipidsand IL-1β implies a pro-inflammatory effect. Taken together, ourfindings suggest that the involvement of circulating and mem-brane EPA and AA in the production of inflammatory mediatorsfollowing open heart surgery is greater than that of DHA. More-over, the role of these fatty acids seems to be complex since EPAand AA associate with both pro-inflammatory and anti-inflammatory mediators.

Despite a careful design of the present study, there were severalnotable limitations. First, it should be emphasized that the findingsare largely confined to elderly patients undergoing open heartsurgery who have relatively high baseline levels of n�3 LC-PUFAin plasma PL and RBC membrane lipids and may not be representa-tive for other populations. It should also be noted that we did onlymeasure the concentrations of inflammatory mediators immedi-ately prior to surgery and on the third postoperative day. Systemicinflammatory response following cardiac surgery is complex andtime-dependent, as it involves multiple cell types and a largenetwork of mediators. Earlier work has demonstrated that cardiacmyocytes are capable of synthesizing inflammatory mediators [1,2],and that epicardial adipose tissue is a source of several such agents[4]. Thus, the LC-PUFA EPA, DHA and AA may have local inflamma-tory effects that are not necessarily reflected by circulating inflam-matory mediators or the plasma PL or RBC membrane fatty acidcomposition although the latter has been shown to correlate wellwith the fatty acid composition of atrial myocytes [13]. However, itmight be considered a limitation that we did not measure the fattyacid levels of cells in pericardial or atrial tissues or in other celltypes, e.g. white blood cells. In addition, it has been well documen-ted that the inflammatory cascade is initiated immediately aftertissue injury and, therefore, many inflammatory mediators maypeak very early in the postoperative course [6,7,16]. Due to multiplecomparisons our analysis may by chance show a statisticallysignificant association between the changes in inflammatory med-iators concentrations and the preoperative levels of the fatty acids inplasma PL and RBC membrane lipids. We did not adjust for multipletesting because the design of our study was exploratory in natureand primarily hypothesis generating. Therefore, additional studiesare needed to examine how EPA, DHA and AA may be associatedwith inflammatory mediators in the first hours and 24–48 h

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following open heart surgery in both young and elderly patients.Finally, we only examined a limited number of inflammatorymediators so that additional studies will be required to explorethe role of other mediators of inflammation.

In conclusion, our findings support the notion that the levels ofEPA and AA in plasma PL and/or cell membrane lipids may affectthe inflammatory response following open heart surgery. How-ever, both these fatty acids associate with pro-inflammatory andanti-inflammatory mediators, suggesting a complex role in theinflammatory process that occurs during the postoperative period.Additional studies are needed to better characterize the influenceof n�3 LC-PUFA on the postoperative inflammatory response,which may be better elucidated by measuring the concentrationsof inflammatory mediators earlier following surgery and at multi-ple time points.

Acknowledgements

The contribution to this work by the patients, the staff atLandspitali—The National University Hospital of Iceland, and LiljaG. Steinsdottir, Laboratory Assistant at the University of Iceland, isgreatly appreciated.

Sources of support: Supported by grants from the IcelandicResearch Fund (RANNIS, Grant No. 080411021), the University ofIceland Research Fund, and the Landspitali—The National Univer-sity Hospital of Iceland Research Fund.

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