Advertisement

n-3 PUFAs improve erythrocyte fatty acid profile in patients with small AAA: a randomized controlled trial

Open AccessPublished:March 26, 2019DOI:https://doi.org/10.1194/jlr.P093013
      Abdominal aortic aneurysm (AAA) is an important cause of death in older adults, which has no current drug therapy. Inflammation and abnormal redox status are believed to be key pathogenic mechanisms for AAA. In light of evidence correlating inflammation with aberrant fatty acid profiles, this study compared erythrocyte fatty acid content in 43 AAA patients (diameter 3.0–4.5 cm) and 52 healthy controls. In addition, the effect of omega-3 PUFA (n-3 PUFA) supplementation on erythrocyte fatty acid content was examined in a cohort of 30 AAA patients as part of a 12 week randomized placebo-controlled clinical trial. Blood analyses identified associations between AAA and decreased linoleic acid (LA), and AAA and increased Δ6-desaturase activity and biosynthesis of arachidonic acid (AA) from LA. Omega-3 PUFA supplementation (1.5 g DHA + 0.3 g EPA/day) decreased red blood cell distribution width (14.8 ± 0.4% to 13.8 ± 0.2%; P = 0.003) and levels of pro-inflammatory n-6 PUFAs (AA, 12.46 ± 0.23% to 10.14 ± 0.3%, P < 0.001; adrenic acid, 2.12 ± 0.13% to 1.23 ± 0.09%; P < 0.001). In addition, Δ-4 desaturase activity increased (DHA/docosapentaenoic acid ratio, 1.85 ± 0.14 to 3.93 ± 0.17; P < 0.001) and elongase 2/5 activity decreased (adrenic acid/AA ratio, 0.17 ± 0.01 to 0.12 ± 0.01; P < 0.01) following supplementation. The findings suggest that n-3 PUFAs improve fatty acid profiles and ameliorate factors associated with inflammation in AAA patients.
      Abdominal aortic aneurysm (AAA) is a full-thickness dilation of the infrarenal aorta that can lead to lethal artery rupture (
      • Wang Q.
      • Ding Y.
      • Song P.
      • Zhu H.
      • Okon I.S.
      • Ding N-Y.
      • Chen H.
      • Liu D.
      • Zou M-H.
      Tryptophan-derived 3-hydroxyanthranilic acid contributes to angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo.
      ). The global prevalence of AAA in adults over the age of 65 years is estimated to be between 1% and 3% (
      • Sampson U.K.
      • Norman P.E.
      • Fowkes F.G.R.
      • Aboyans V.
      • Song Y.
      • Harrell Jr., F.E.
      • Forouzanfar M.H.
      • Naghavi M.
      • Denenberg J.O.
      • McDermott M.M.
      Estimation of global and regional incidence and prevalence of abdominal aortic aneurysms 1990 to 2010.
      ). While chronic aortic inflammation, oxidative stress, and enhanced proteolytic enzyme activity are implicated in the destructive remodeling of aortic connective tissue that occurs in AAA (

      MA3RS Study Investigators. 2017. Aortic wall inflammation predicts abdominal aortic aneurysm expansion, rupture, and need for surgical repair. Circulation. 136: 787–797.

      ,
      • Meital L.T.
      • Sandow S.L.
      • Calder P.C.
      • Russell F.D.
      Abdominal aortic aneurysm and omega-3 polyunsaturated fatty acids: mechanisms, animal models, and potential treatment.
      ), no recognized drug therapies exist to halt or reverse these aberrant molecular processes or their injurious mechanical sequelae.
      Technical advances in MS methods have resulted in the availability of tools to accurately profile the human lipidome (
      • Orešič M.
      • Hänninen V.A.
      • Vidal-Puig A.
      Lipidomics: a new window to biomedical frontiers.
      ) and, more specifically, quantify the accumulation of omega-3 PUFAs (n-3 PUFAs) in red blood cell membranes. Animal data have suggested the potential of n-3 PUFA supplementation to limit small AAA growth through positive impacts on oxidative stress status (
      • Wales K.M.
      • Kavazos K.
      • Nataatmadja M.
      • Brooks P.R.
      • Williams C.
      • Russell F.D.
      N-3 PUFAs protect against aortic inflammation and oxidative stress in angiotensin II-infused apolipoprotein E−/−mice.
      ,
      • Kugo H.
      • Zaima N.
      • Mouri Y.
      • Tanaka H.
      • Yanagimoto K.
      • Urano T.
      • Unno N.
      • Moriyama T.
      The preventive effect of fish oil on abdominal aortic aneurysm development.
      ) and interference with inflammatory cascades (
      • Pope N.H.
      • Salmon M.
      • Davis J.P.
      • Chatterjee A.
      • Su G.
      • Conte M.S.
      • Ailawadi G.
      • Upchurch G.R.
      D-series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization.
      ,
      • Yoshihara T.
      • Shimada K.
      • Fukao K.
      • Sai E.
      • Sato-Okabayashi Y.
      • Matsumori R.
      • Shiozawa T.
      • Alshahi H.
      • Miyazaki T.
      • Tada N.
      • et al.
      Omega 3 polyunsaturated fatty acids suppress the development of aortic aneurysms through the inhibition of macrophage-mediated inflammation.
      ,
      • Kavazos K.
      • Nataatmadja M.
      • Wales K.M.
      • Hartland E.
      • Williams C.
      • Russell F.D.
      Dietary supplementation with omega-3 polyunsaturated fatty acids modulate matrix metalloproteinase immunoreactivity in a mouse model of pre-abdominal aortic aneurysm.
      ). While clinical evidence in AAA patients is limited, low serum EPA levels have been reported to be associated with larger aneurysm size and faster growth rate in a Japanese patient population (
      • Aikawa T.
      • Miyazaki T.
      • Shimada K.
      • Sugita Y.
      • Shimizu M.
      • Ouchi S.
      • Kadoguchi T.
      • Yokoyama Y.
      • Shiozawa T.
      • Hiki M.
      • et al.
      Low serum levels of epa are associated with the size and growth rate of abdominal aortic aneurysm.
      ). A separate study involving a Danish cohort of patients, however, found no association between n-3 PUFA blood status and the development or growth of AAA (
      • Lindholt J.S.
      • Kristensen K.L.
      • Burillo E.
      • Martinez-Lopez D.
      • Calvo C.
      • Ros E.
      • Martín-Ventura J.L.
      • Sala-Vila A.
      Arachidonic acid, but not omega-3 index, relates to the prevalence and progression of abdominal aortic aneurysm in a population-based study of Danish men.
      ). Omega-3 PUFA blood status is typically evaluated by omega-3 index measurement (
      • Arnold C.
      • Konkel A.
      • Fischer R.
      • Schunck W-H.
      Cytochrome P450-dependent metabolism of ω-6 and ω-3 long-chain polyunsaturated fatty acids.
      ). The omega-3 index represents the ratio of EPA + DHA to all other fatty acids incorporated in red blood cell membrane phospholipids, and substantial published research suggests that this measurement is a reliable predictor of cardiovascular event risk (
      • von Schacky C.
      Use of red blood cell fatty-acid profiles as biomarkers in cardiac disease.
      ,
      • Del Gobbo L.C.
      • Imamura F.
      • Aslibekyan S.
      • Marklund M.
      • Virtanen J.K.
      • Wennberg M.
      • Yakoob M.Y.
      • Chiuve S.E.
      • Dela Cruz L.
      • Frazier-Wood A.C.
      • et al.
      ω-3 polyunsaturated fatty acid biomarkers and coronary heart disease: pooling project of 19 cohort studies.
      ,
      • Kleber M.E.
      • Delgado G.E.
      • Lorkowski S.
      • März W.
      • von Schacky C.
      Omega-3 fatty acids and mortality in patients referred for coronary angiography. The Ludwigshafen Risk and Cardiovascular Health Study.
      ,
      • Harris W.S.
      • Tintle N.L.
      • Etherton M.R.
      • Vasan R.S.
      Erythrocyte long-chain omega-3 fatty acid levels are inversely associated with mortality and with incident cardiovascular disease: the Framingham Heart Study.
      ). In light of this and evidence correlating specific disease states with distinct fatty acid profiles (
      • Goozee K.
      • Chatterjee P.
      • James I.
      • Shen K.
      • Sohrabi H.R.
      • Asih P.R.
      • Dave P.
      • Ball B.
      • ManYan C.
      • Taddei K.
      • et al.
      Alterations in erythrocyte fatty acid composition in preclinical Alzheimer's disease.
      ,
      • Park Y.
      • Lim J.
      • Lee J.
      • Kim S.G.
      Erythrocyte fatty acid profiles can predict acute non-fatal myocardial infarction.
      ,
      • Bannikoppa P.
      • Dhayanand J.
      • Madhukumar R.
      • Padmanabhan A.
      • Bafna U.
      • Vijayakumar M.
      • Devi K.U.
      • Pramod K.
      • Thomas T.
      • Jayshree R.
      • et al.
      Fatty acid intake and erythrocyte fatty acid profile in women with breast, ovarian and cervical cancers.
      ), the present study was designed to compare full blood count data, baseline omega-3 index values, and fatty acid profiles of erythrocytes obtained from AAA patients with those of a healthy control cohort as part of an ongoing clinical trial. In addition, the impact of physiologically appropriate n-3 PUFA supplementation on full blood count values, the omega-3 index, and erythrocyte membrane fatty acid composition was examined in a cohort of AAA patients as part of a 12 week randomized double-blind placebo-controlled clinical trial. It was hypothesized that comparative analysis of erythrocyte fatty acid composition would yield a distinct AAA fatty acid profile that could direct future progress toward identification of novel therapeutic targets.

      MATERIALS AND METHODS

      Observational (case-control) study

      AAA patients were recruited from the Sunshine Coast University Hospital and a private clinic (Sunshine Vascular) and healthy control participants were recruited from the general population of the Sunshine Coast, Queensland, Australia with approval from the University of the Sunshine Coast (A/13/473 and A/16/833) and the Prince Charles Hospital Human Research Ethics Committees (HREC/16/QPCH/114 and HREC/12/QPCH/13). The study was conducted in accordance with the Declaration of Helsinki (1964). The patient group included 43 men with small AAA (diameter 3.0–4.5 cm), and the control group included 52 men without a documented AAA. It has previously been reported that there are significant differences in omega-3 index between men and women (
      • Itomura M.
      • Fujioka S.
      • Hamazaki K.
      • Kobayashi K.
      • Nagasawa T.
      • Sawazaki S.
      • Kirihara Y.
      • Hamazaki T.
      Factors influencing EPA+DHA levels in red blood cells in Japan.
      ), and AAA is known to be much more common in men than women (
      • Golledge J.
      • Muller J.
      • Daugherty A.
      • Norman P.
      Abdominal aortic aneurysm: pathogenesis and implications for management.
      ). In view of this, only men were included in the current study. Written informed consent was obtained for each participant. Maximum AAA diameter was measured by ultrasound prior to study entry. Exclusion criteria included age below 60 years or above 86 years, BMI above 39 kg·m−2, uncontrolled hypertension, cardiac arrhythmia, heart failure, symptomatic aortic stenosis, chronic obstructive pulmonary disease, chronic inflammatory disease, and regular use of prescription anti-inflammatory medication. A family history of AAA or known aneurysmal disease served as additional exclusion criteria for control participants. All participants refrained from nonprescribed anti-inflammatory medications 72 h prior to blood collection and abstained from alcohol and caffeine for the 12 h leading up to their study visit.

      Omega-3 clinical trial

      The impact of n-3 PUFA supplementation on erythrocyte fatty acid composition was investigated in AAA patients as part of a parallel-design double-blind placebo-controlled trial (ANZCTR12616000483459), carried out between June 2017 and June 2018. Patients with small AAA (3.0–4.9 cm) (n = 32) were recruited from Nambour General Hospital, Sunshine Coast University Hospital, and a private clinic (Sunshine Vascular) and randomized (Fig. 1) to receive either active fatty acids (three Blackmores Omega Brain capsules each containing 500 mg DHA and 100 mg EPA and delivering a total of 1.8 g n-3 PUFAs per day) or placebo fatty acids (three capsules each containing 490 mg corn oil, 490 mg olive oil, and 20 mg fish oil) for a period of 12 weeks. Capsules were of similar appearance and flavor. Participants were supplied with capsules at day 0, week 3, and week 8, with instruction to take three each morning. The final study visit was at week 12. Substantial research literature suggests that the intervention dose and duration selected for the clinical trial will be sufficient to raise the omega-3 index of supplemented subjects to protective levels associated with positive cardiovascular outcomes (
      • Harris W.S.
      • Von Schacky C.
      The omega-3 index: a new risk factor for death from coronary heart disease?.
      ,
      • Jackson K.H.
      • Polreis J.M.
      • Tintle N.L.
      • Kris-Etherton P.M.
      • Harris W.S.
      Association of reported fish intake and supplementation status with the omega-3 index.
      ). Exclusion criteria for the clinical trial were: age below 55 years or above 86 years, consumption of three or more fish meals per week, the use of fish oil or krill oil supplements, and the use of anti-inflammatory medications. Written informed consent was obtained from each participant and information regarding medical history, physical activity, and dietary intake of n-3 PUFAs was collected. The protocol was approved by the University of the Sunshine Coast (Ethics approval number A/16/833) and the Prince Charles Hospital Human Research Ethics Committees (HREC/16/QPCH/114). The study was conducted in accordance with the Declaration of Helsinki (1964).
      Figure thumbnail gr1
      Fig. 1Study flowchart. The intervention study included 32 AAA patients, 30 of which completed the protocol.

      Randomization and evaluation of compliance

      An Excel block randomization algorithm was used to assign participants to either an active or a placebo treatment protocol. Random selection of block size 4 or 6 during the computerized sort avoided selection bias (
      • Efird J.
      Blocked randomization with randomly selected block sizes.
      ). Sequentially numbered containers were used to implement the random allocation of capsules. The randomization allocation sequence was generated by the corresponding author. Compliance with treatment protocols was evaluated by monitoring capsule returns and by GC-MS measurement of red blood cell omega-3 index.

      Blood sample collection

      Fasting blood samples were collected from AAA patients and healthy control participants into EDTA-containing tubes and serum separator tubes. Erythrocytes from EDTA tubes were sedimented by centrifugation (1,500 g, 15 min, 15°C); the plasma and buffy coat fractions were removed and the packed red blood cells were stored at −80°C until analysis. Blood collected into serum separator tubes was allowed to clot at 22°C for 30 min prior to centrifugation (1,500 g, 15 min, 15°C). The serum was collected and centrifuged (4,000 g, 5 min, 4°C) and the supernatant was stored at −80°C until processing and analysis. For the omega-3 clinical trial, fasted blood samples were collected from AAA patients at day 0 (prior to initiation of the study), at day 21 (following 3 weeks of supplementation), and at day 84 (following 12 weeks of supplementation). Erythrocytes from EDTA tubes were processed and stored as described above.

      Erythrocyte fatty acid analysis

      Choice of erythrocytes as the preferred matrix for assessment of n-3 PUFA status was based on evidence suggesting that: 1) measurement of erythrocyte fatty acid levels assesses long-term dietary fatty acid intake; 2) erythrocyte fatty acid levels are less sensitive than plasma to day-to-day n-3 PUFA intake; 3) erythrocyte n-3 PUFA levels display one-fourth of the within-person variability that occurs with plasma; and 4) erythrocyte n-3 PUFA levels are highly correlated with those in a variety of tissues (
      • Harris W.S.
      • Del Gobbo L.
      • Tintle N.L.
      The omega-3 index and relative risk for coronary heart disease mortality: estimation from 10 cohort studies.
      ). Erythrocyte fatty acid composition was determined using GC-MS as previously described (
      • Bürgin-Maunder C.S.
      • Brooks P.R.
      • Hitchen-Holmes D.
      • Russell F.D.
      Moderate dietary supplementation with omega-3 fatty acids does not impact plasma von Willebrand factor profile in mildly hypertensive subjects.
      ). Briefly, a 600 μl aliquot of methanol containing butylated hydroxytoluene (BHT; 20 mg/100 ml) as an antioxidant was added to erythrocyte samples (300 μl). The cells were homogenized with glass rods (1 min), flushed with nitrogen gas, and incubated on ice for 30 min. A 600 μl aliquot of chloroform was added to the suspension and cells were homogenized, flushed, and incubated as before. The preparation was centrifuged (3,000 g, 4°C, 5 min), and the supernatant was withdrawn, flushed with nitrogen gas, and stored on ice. This procedure was repeated twice with 300 μl volumes of methanol/BHT and chloroform and incubation periods of 10 min. In a final extraction step, 1 ml of pooled lipid supernatant was combined with chloroform (800 μl) and KCl (0.05 M; 460 μl), the solution was mixed by vortex, flushed with nitrogen gas, and centrifuged (3,000 g, 4°C, 10 min). The supernatant was discarded and the fraction containing lipids was dried under nitrogen gas. The extracted lipids were hydrolyzed in the presence of 500 μl of 9 M HCl:water:acetonitrile (1:1:18 containing 25 mg/50 ml BHT) during overnight incubation at 65°C. The hydrolyzed samples were dried under nitrogen gas and, following a 10 min incubation at −80°C, were freeze-dried (Thermo Savant Modulyo freeze dryer system, Thermo Fisher Scientific) for 30 min. Samples were derivatized in 250 μl of hexane containing 10 μl 1-tert-butyldimethylsilylimidazole during a 2 h incubation at 37°C. Fatty acids were separated and analyzed with a Perkin Elmer Clarus 580 gas chromatograph coupled to a Perkin Elmer Clarus SQ85 mass spectrometer using a Perkin Elmer Elite 5MS column (30 m × 0.25 mm internal diameter × 0.25 mm film). The GC method included a split ratio of 30:1 with injector at 300°C and temperature programming of 170°C (initial) ramped to 310°C at a rate of 6°C/min followed by a 5 min hold period. The MS method included ionization at 70 eV with a scan range of m/z 45–450 for 4.0–28.3 min. Peaks were identified by comparison with known standards or the National Institute of Standards and Technology Library (NIST 2008 Library). Omega-3 indices were expressed as the combined percentage of DHA and EPA integrated peak areas divided by the total fatty acid integrated peak areas.

      Enzyme activity

      Product to precursor erythrocyte phospholipid fatty acid ratios served as indices of activity levels of enzymes involved in fatty acid metabolism. The index of Δ-9 desaturase activity was calculated as the ratio of oleic acid to stearic acid (
      • Fuhrman B.J.
      • Barba M.
      • Krogh V.
      • Micheli A.
      • Pala V.
      • Lauria R.
      • Chajes V.
      • Riboli E.
      • Sieri S.
      • Berrino F.
      • et al.
      Erythrocyte membrane phospholipid composition as a biomarker of dietary fat.
      ), and the index of Δ-4 desaturase activity was calculated as the ratio of DHA to docosapentaenoic acid (DPA) (
      • Dasilva G.
      • Pazos M.
      • García-Egido E.
      • Gallardo J.M.
      • Ramos-Romero S.
      • Torres J.L.
      • Romeu M.
      • Nogués M-R.
      • Medina I.
      A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.
      ). The index of Δ-6 desaturase was calculated in two ways: 1) as a ratio of dihomo-γ-linolenic acid to linoleic acid (LA) (
      • Dasilva G.
      • Pazos M.
      • García-Egido E.
      • Gallardo J.M.
      • Ramos-Romero S.
      • Torres J.L.
      • Romeu M.
      • Nogués M-R.
      • Medina I.
      A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.
      ) (this calculation includes the activities of the enzymes Δ-6 desaturase and elongase 5); and 2) as a ratio of γ-linolenic acid to LA (
      • Fuhrman B.J.
      • Barba M.
      • Krogh V.
      • Micheli A.
      • Pala V.
      • Lauria R.
      • Chajes V.
      • Riboli E.
      • Sieri S.
      • Berrino F.
      • et al.
      Erythrocyte membrane phospholipid composition as a biomarker of dietary fat.
      ) (this calculation includes the activity of Δ-6 desaturase only). The ratio of arachidonic acid (AA) to LA served as an index of desaturase/elongase-mediated LA → AA biosynthesis (
      • McNamara R.K.
      • Jandacek R.
      • Rider T.
      • Tso P.
      • Cole-Strauss A.
      • Lipton J.W.
      Omega-3 fatty acid deficiency increases constitutive pro-inflammatory cytokine production in rats: relationship with central serotonin turnover.
      ). The index of stearoyl-CoA desaturase activity was calculated as the ratio of palmitoleic acid to palmitic acid (
      • Dasilva G.
      • Pazos M.
      • García-Egido E.
      • Gallardo J.M.
      • Ramos-Romero S.
      • Torres J.L.
      • Romeu M.
      • Nogués M-R.
      • Medina I.
      A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.
      ), and the index of elongase 2/5 activity was calculated as the ratio of adrenic acid to AA (
      • McNamara R.K.
      • Jandacek R.
      • Rider T.
      • Tso P.
      • Dwivedi Y.
      • Pandey G.N.
      Selective deficits in erythrocyte docosahexaenoic acid composition in adult patients with bipolar disorder and major depressive disorder.
      ).

      Full blood count analysis

      Whole blood from fasted participants was collected into EDTA tubes, and full blood count analyses were conducted within 10 min of the collection using a Coulter Ac·T diff™. Data regarding red blood cell indices [hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red blood cell distribution width (RDW)], white blood cell counts (lymphocytes, monocytes, and granulocytes), and platelet counts and indices (mean platelet volume, plateletcrit, and platelet distribution width) were collected. Instrument calibration was verified prior to measurement of each sample using appropriate quality controls.

      Data analysis

      Observational (case-control) study.

      Erythrocyte fatty acid content and full blood count analysis have not been investigated previously in small AAA and healthy control participants. Group size estimates for the observational study were based on RDW values reported in healthy controls (RDW, 13.1 ± 1.3, n = 40) and patients with coronary artery ectasia (RDW, 14.8 ± 1.6, n = 54) (
      • Dogdu O.
      • Koc F.
      • Kalay N.
      • Yarlioglues M.
      • Elcik D.
      • Karayakali M.
      • Ozbek K.
      • Kaya M.G.
      Assessment of red cell distribution width (RDW) in patients with coronary artery ectasia.
      ). A group size estimate of 14 was calculated with 85% power (α level of 0.05) using power/sample size (University British Columbia) and pooled variance (Solvers statistics) calculators.

      Omega-3 clinical trial.

      The group size estimate for the omega-3 clinical trial was based on fatty acid values obtained from a 5 month omega-3 fatty acid supplementation trial (1.8 g/day) in a healthy cohort (
      • Flock M.R.
      • Skulas-Ray A.C.
      • Harris W.S.
      • Gaugler T.L.
      • Fleming J.A.
      • Kris-Etherton P.M.
      Effects of supplemental long-chain omega-3 fatty acids and erythrocyte membrane fatty acid content on circulating inflammatory markers in a randomized controlled trial of healthy adults.
      ). Baseline erythrocyte levels (n = 23) were: AA (16.1 ± 1.2%), adrenic acid (4.1 ± 0.5%), EPA (0.5 ± 0.4%), and DHA (3.9 ± 0.8%). End-of-study levels (n = 24) were: AA (13.6 ± 1.2%), adrenic acid (2.6 ± 0.5%), EPA (2.5 ± 0.4%), and DHA (7.0 ± 0.8%). Group size estimates were between one and four (85% power; α level of 0.05).
      Continuous demographic data for AAA patients and control participants were compared using a Student's t-test and are presented as mean ± SD. Categorical demographic variables were compared using a Fisher's exact test. Experimental data are presented as mean ± SEM, and between group differences were examined by Student's t-test analysis. The association between independent variables identified as being significantly different (see Table 1) and AAA was assessed using linear regression analysis with adjustment for covariates shown to be imbalanced between groups (hypertension, diabetes mellitus, coronary heart disease (CHD), dyslipidemia, use of statins or low-dose aspirin, current and previous smoking history, and age). All variables were introduced in one step in order of decreasing tolerance. Data were analyzed with Prism (GraphPad Software, La Jolla, CA). IBM SPSS Statistics Version 24 was used for multi-variable regression analysis and statistical significance was set at P < 0.05.
      TABLE 1Independent associations between AAA diagnosis and assessed variables using multi-variable regression analysis
      VariableαStandard Error95% CIP
      Margaric acid0.5090.0290.025–0.1440.007
      LA−0.5220.541−2.640 to −0.4460.007
      DPA0.7150.1150.260–0.725<0.001
      Δ6-Desaturase (1)0.5380.0180.017–0.0890.005
      LA → AA biosynthesis0.5160.1430.110-0.6890.008
      n-6/n-3 Ratio−0.4030.334−1.1364 to −0.0100.047
      Data were adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age. No independent associations were observed for RDW, omega-3 index, adrenic acid, AA, myristic acid, pentadecanoic acid, palmitoleic acid, elaidic acid, γ-linolenic acid, DHA, lignoceric acid, nervonic acid, tricosanoic acid, stearoyl-CoA desaturase, elonase-2/5, or Δ6-desaturase (2).

      RESULTS

      Observational (case-control) study

      Baseline characteristics.

      Baseline characteristics of the case-control study are shown in Table 2. AAA patients were older and were characterized by a higher prevalence of hypertension, diabetes mellitus, dyslipidemia, CHD, and smoking history. AAA patients were more commonly prescribed statins and anti-platelet drugs.
      TABLE 2Demographic, biometric, and medical characteristics of AAA patients and control participants
      VariableObservational StudyOmega-3 Clinical Trial
      Control Participants (n = 52)AAA Patients (n = 43)AAA n-3 Cohort (n = 15)AAA Placebo Cohort (n = 15)
      Gender (male/female)52/043/015/015/0
      Age (years)71.2 ± 5.974.6 ± 5.6
      AAA significantly different to control (Student's t-test).
      73.6 ± 5.075.1 ± 5.7
      AAA size (mm)38.8 ± 5.439.3 ± 5.739.2 ± 5.0
      align=“left” Smoking
       Never21 (40%)9 (21%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      1 (7%)2 (13%)
       Past28 (54%)30 (70%)12 (80%)12 (80%)
       Current3 (6%)4 (9%)2 (13%)1 (7%)
      BMI (kg/m2)25.9 ± 4.228.6 ± 4.829.4 ± 4.129.0 ± 5.0
      SBP (mmHg)137.3 ± 13.9137.9 ± 16.2135.8 ± 16.3144.0 ± 19.6
      DBP (mmHg)79.3 ± 8.278.2 ± 8.877.6 ± 7.680.0 ± 10.7
      Hypertension20 (38%)27 (63%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      6 (40%)11 (73%)
      Diabetes1 (2%)6 (14%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      3 (20%)1 (7%)
      Dyslipidemia19 (37%)30 (70%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      7 (47%)12 (80%)
      CHD5 (10%)18 (42%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      5 (33%)4 (27%)
      Medication
       Anti-hypertensive agents
       Beta blockers7 (13%)13 (30%)4 (27%)3 (20%)
       AT II receptor antagonists7 (13%)13 (30%)3 (20%)5 (33%)
       ACE inhibitors7 (13%)6 (14%)3 (20%)1 (7%)
       Ca
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      2+ channel blockers
      5 (10%)6 (14%)1 (7%)5 (33%)
       Diuretics3 (6%)5 (12%)2 (13%)1 (7%)
       Anti-platelet drugs4 (8%)25 (58%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      11 (73%)7 (47%)
       NSAIDs4 (8%)6 (14%)1 (7%)2 (13%)
       Statins18 (35%)32 (74%)
      AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).
      8 (53%)c13 (87%)
      Continuous demographic data are presented as mean ± SD; categorical demographic data are presented as number (percentage). AT II, angiotensin II; Ca2+, calcium; NSAIDs, nonsteroidal anti-inflammatory drugs; SBP, systolic blood pressure; DBP, diastolic blood pressure.
      a AAA significantly different to control (Student's t-test).
      b AAA significantly different to control (Fisher's exact test, P < 0.05).cAAA omega cohort significantly different to placebo cohort (Fisher's exact test, P < 0.05).

      Fatty acid methyl ester analysis.

      Twenty-three fatty acid methyl esters were consistently identified in erythrocyte membranes obtained from AAA patients and healthy control participants (see supplemental Table S1).

      Erythrocyte fatty acid profiles.

      Mean omega-3 index values were similar in cases and controls, while the mean omega-6:omega-3 (n-6/n-3) ratio was significantly lower than the corresponding value obtained for the control cohort (P = 0.047; Table 3). Levels of the saturated fatty acid, margaric acid (C17:0), were significantly higher in AAA patients compared with healthy control participants (P = 0.007), while levels of the n-6 fatty acid, LA (C18:2), were significantly lower (P = 0.007). The n-3 PUFA, DPA (C22:5), was significantly higher in erythrocytes from AAA patients compared with control participants (P < 0.001). Data was adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age (Table 1).
      TABLE 3Erythrocyte fatty acid profiles of control participants and AAA patients
      Fatty AcidControlAAA
      Saturated fatty acids
       C14:00.46 ± 0.030.61 ± 0.06
       C15:00.17 ± 0.010.26 ± 0.04
       C16:024.03 ± 0.2423.40 ± 0.22
       C17:00.28 ± 0.010.34 ± 0.01
      P < 0.01, AAA significantly different to control.
       C18:014.23 ± 0.1314.23 ± 0.13
       C20:00.42 ± 0.050.46 ± 0.01
       C22:03.16 ± 0.143.17 ± 0.14
      Monounsaturated fatty acids
       C16:1n70.37 ± 0.030.48 ± 0.04
       C18:1n-914.56 ± 0.2014.44 ± 0.23
      Trans fatty acids
       C18:1t1.31 ± 0.031.42 ± 0.04
      n-6 PUFAs
       C18:29.27 ± 0.217.57 ± 0.17
      P < 0.01, AAA significantly different to control.
       C18:30.05 ± 0.0030.06 ± 0.003
       C20:20.15 ± 0.010.16 ± 0.01
       C20:31.28 ± 0.041.34 ± 0.05
       C20:412.52 ± 0.1812.89 ± 0.22
       C22:41.80 ± 0.062.05 ± 0.07
      n-3 PUFAs
       C20:50.94 ± 0.050.98 ± 0.05
       C22:51.84 ± 0.031.96 ± 0.05
      P < 0.001, AAA significantly different to control.
       C22:63.13 ± 0.103.46 ± 0.11
      Very long chain fatty acids
       C24:04.56 ± 0.095.05 ± 0.11
       C24:1n94.70 ± 0.105.27 ± 0.1
       C23:00.22 ± 0.010.24 ± 0.01
      Other fatty acids
       C20:1n-70.18 ± 0.010.16 ± 0.01
      Ratio n-6/n-34.08 ± 0.133.61 ± 0.11
      P < 0.05, AAA significantly different to control.
      n-3 Index4.07 ± 0.134.42 ± 0.12
      Data are expressed as mean percent ± SEM. Data were adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age.
      a P < 0.01, AAA significantly different to control.
      b P < 0.001, AAA significantly different to control.
      c P < 0.05, AAA significantly different to control.

      Enzyme activity.

      The indices of Δ-4 (DPA → DHA) and Δ-9 desaturase (stearic → oleic) activity in erythrocytes from AAA patients were comparable to the control cohort (Table 4) as were the index of stearoyl-CoA desaturase (palmitic → palmitoleic acid) activity and the index of elongase 2/5 (AA → adrenic acid) activity. The index of Δ-6 desaturase (1) activity was significantly higher in erythrocytes from AAA patients compared with control participants (P = 0.005), as was the index of desaturase/elongase-mediated LA → AA biosynthesis (P = 0.008). Data were adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age (Table 1).
      TABLE 4Erythrocyte fatty acid ratios as indices of enzyme activities in control participants and AAA patients
      Index of Enzyme ActivityControlAAA
      Δ-4 desaturase (DHA/DPA)1.72 ± 0.061.81 ± 0.07
      Δ-9 desaturase (oleic acid/stearic acid)1.03 ± 0.021.02 ± 0.02
      Δ-6 desaturase (1) (dihomo-γ-linolenic/linoleic)0.14 ± 0.0050.18 ± 0.01
      P < 0.01, AAA significantly different to control.
      Δ-6 desaturase (2) (γ-linolenic/linoleic)0.006 ± 0.00040.008 ± 0.0005
      LA → AA biosynthesis (AA/LA)1.37 ± 0.061.75 ± 0.06
      P < 0.01, AAA significantly different to control.
      Stearoyl-CoA desaturase (palmitoleic acid/palmitic acid)0.015 ± 0.0010.02 ± 0.002
      Elongase 2/5 (adrenic acid/AA)0.14 ± 0.0050.16 ± 0.03
      Data are expressed as mean ± SEM. Data are adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age.
      a P < 0.01, AAA significantly different to control.

      Red blood cell indices, white blood cell counts, and platelet counts and indices.

      RDW and other red blood cell indices were similar in cases and controls, as were white blood cell counts and platelet counts and indices following adjustment for covariates shown to be imbalanced between groups (Table 5).
      TABLE 5Red blood cell indices, white blood cell counts, and platelet counts and indices for control participants and AAA patients
      Blood ParameterControlAAA
      WBCs (×109/liter)6.1 ± 0.26.7 ± 0.3
      Lymphocytes (×109/liter)1.9 ± 0.12.1 ± 0.1
      Monocytes (×109/liter)0.4 ± 0.020.4 ± 0.1
      Granulocytes (×109/liter)3.8 ± 0.24.2 ± 0.2
      RBCs (×1012/liter)4.7 ± 0.14.8 ± 0.1
      Hemoglobin (g/l)142.2 ± 2.3143.1 ± 2.6
      Hematocrit (%)43.5 ± 0.643.7 ± 0.7
      MCV (fl)91.8 ± 0.892.1 ± 0.7
      MCH (pg)30.0 ± 0.330.1 ± 0.3
      MCHC (g/l)326.9 ± 1.5327.2 ± 1.8
      RDW (%)13.9 ± 0.214.7 ± 0.3
      Platelet (×109/liter)240.6 ± 10.8199.9 ± 10.8
      MPV (fl)7.8 ± 0.28.0 ± 0.2
      Plateletcrit (%)0.19 ± 0.010.16 ± 0.01
      PDW17.1 ± 0.117.0 ± 0.2
      Data are expressed as mean ± SEM. Data are adjusted for hypertension, diabetes mellitus, CHD, low-dose aspirin and statin use, active or previous smoking history, and age. WBCs, white blood cells; RBCs, red blood cells; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MPV, mean platelet volume; PDW, platelet distribution width.

      Omega-3 clinical trial

      Baseline characteristics.

      Baseline characteristics were similar between groups with the exception of statin use (Table 2).

      Participant compliance and tolerability.

      A high level of adherence to supplement intake was identified by return capsule counts (placebo: 96.4 ± 1.2%, 96.8 ± 2.1%, and 95.0 ± 2.9% at weeks 3, 8, and 12, respectively; omega-3 fatty acid group: 97.2 ± 0.9%, 94.0 ± 1.7%, and 95.0 ± 1.6% at weeks 3, 8, and 12, respectively). This was supported by GC-MS analysis of fatty acid incorporation in red blood cell membrane phospholipids. The omega-3 index was markedly increased in all participants in the omega-3 fatty acid group over the 12 week trial period (Table 6). No change in omega-3 index was observed in participants who were randomized to receive placebo capsules. Two participants in the placebo group withdrew from the trial citing gastrointestinal disturbances (Fig. 1). Among participants who completed the 12 week trial, three in the placebo group reported burping, with one of them also experiencing nausea. In the omega-3 fatty acid group, three participants reported burping or reflux, and one participant reported flatulence.
      TABLE 6Erythrocyte fatty acid profiles of AAA patients
      Fatty AcidAAA, Fish Oil CohortAAA, Placebo Cohort
      BaselineWeek 3Week 12BaselineWeek 3Week 12
      Saturated fatty acids
       C14:00.71 ± 0.120.71 ± 0.060.51 ± 0.060.73 ± 0.090.67 ± 0.080.58 ± 0.06
       C15:00.36 ± 0.080.39 ± 0.080.24 ± 0.050.29 ± 0.070.44 ± 0.120.18 ± 0.01
       C16:022.71 ± 0.2223.65 ± 0.3223.49 ± 0.3622.63 ± 0.3022.73 ± 0.2323.22 ± 0.43
       C17:00.35 ± 0.010.36 ± 0.020.36 ± 0.030.39 ± 0.020.37 ± 0.020.34 ± 0.02
       C18:014.15 ± 0.2514.39 ± 0.2514.98 ± 0.4314.44 ± 0.2314.08 ± 0.2214.62 ± 0.26
       C20:00.46 ± 0.020.48 ± 0.090.65 ± 0.130.51 ± 0.020.49 ± 0.030.59 ± 0.11
       C22:03.27 ± 0.282.69 ± 0.292.98 ± 0.283.04 ± 0.283.02 ± 0.313.20 ± 0.25
      Monounsaturated fatty acids
       C16:1n70.58 ± 0.100.56 ± 0.070.54 ± 0.090.51 ± 0.060.56 ± 0.070.52 ± 0.07
       C18:1n-914.03 ± 0.3213.94 ± 0.2514.46 ± 0.3813.79 ± 0.3014.15 ± 0.4114.31 ± 0.41
      Trans fatty acids
       C18:1t1.40 ± 0.081.38 ± 0.081.70 ± 0.211.41 ± 0.071.38 ± 0.051.56 ± 0.13
      n-6 PUFAs
       C18:27.57 ± 0.267.55 ± 0.337.43 ± 0.297.60 ± 0.318.21 ± 0.367.39 ± 0.21
       C18:30.06 ± 0.0050.09 ± 0.040.05 ± 0.010.06 ± 0.0050.06 ± 0.0040.05 ± 0.01
       C20:20.17 ± 0.020.17 ± 0.020.14 ± 0.010.18 ± 0.010.19 ± 0.020.25 ± 0.11
       C20:31.32 ± 0.051.14 ± 0.05
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      1.04 ± 0.06
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      1.51 ± 0.081.52 ± 0.09
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      1.38 ± 0.09
      P < 0.01, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
       C20:412.46 ± 0.2311.72 ± 0.22
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      10.14 ± 0.3
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      13.09 ± 0.3812.57 ± 0.4012.81 ± 0.41
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
       C22:42.12 ± 0.131.92 ± 0.121.23 ± 0.09
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      2.09 ± 0.092.02 ± 0.122.05 ± 0.12
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      n-3 PUFAs
       C20:50.99 ± 0.071.35 ± 0.06
      P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      1.72 ± 0.11
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      0.89 ± 0.060.91 ± 0.08
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      0.95 ± 0.09
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
       C22:51.97 ± 0.091.87 ± 0.081.61 ± 0.08
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      2.03 ± 0.091.94 ± 0.091.89 ± 0.10
       C22:63.54 ± 0.224.72 ± 0.19
      P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      6.21 ± 0.20
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      3.48 ± 0.173.53 ± 0.21
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      3.31 ± 0.24
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      Very long chain fatty acids
       C24:05.25 ± 0.195.04 ± 0.174.92 ± 0.245.44 ± 0.125.34 ± 0.165.23 ± 0.20
       C24:1n95.67 ± 0.145.51 ± 0.164.93 ± 0.2
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      5.42 ± 0.125.38 ± 0.175.11 ± 0.22
       C23:00.24 ± 0.020.26 ± 0.010.24 ± 0.020.28 ± 0.010.29 ± 0.010.25 ± 0.02
      Other fatty acids
       C20:1n-70.13 ± 0.020.14 ± 0.020.16 ± 0.020.17 ± 0.020.14 ± 0.020.21 ± 0.01
      P < 0.05, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      Ratio n-6/n-33.48 ± 0.162.69 ± 0.08
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      2.00 ± 0.08
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      3.66 ± 0.203.68 ± 0.22
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      3.73 ± 0.21
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      n-3 Index4.53 ± 0.226.07 ± 0.19
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      8.03 ± 0.20
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      4.37 ± 0.204.43 ± 0.25
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      4.26 ± 0.28
      P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      Data are expressed as mean percent ± SEM.
      a P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      b P < 0.001, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      c P < 0.01, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.
      d P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      e P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      f P < 0.05, fish oil cohort week 3/week 12 significantly different to placebo cohort week 3/week 12.

      Erythrocyte fatty acid profiles in AAA patients following n-3 PUFA supplementation.

      Significant increases in DHA (76%, P < 0.001) and EPA (69%, P < 0.001) levels were observed in erythrocytes from AAA patients following 12 weeks of n-3 PUFA supplementation (Table 6). This effect was accompanied by significant decreases in the n-6 fatty acids, AA (C20:4, P < 0.001), adrenic acid (C22:4, P < 0.001), and dihomo-γ-linolenic acid (C20:3, P = 0.003), from baseline to week 12. Additional fatty acids altered by n-3 PUFA supplementation included the n-3 PUFA, DPA (C22:5), and the very long chain fatty acid, nervonic acid (C24:1), both of which decreased at week 12 (P = 0.004 and P = 0.009, respectively). The mean omega-3 index value among AAA patients increased at week 3 (33%, P < 0.001) and week 12 (74%, P < 0.001), while the mean n-6/n-3 ratio decreased at these time points (22%, P < 0.001 and 42%, P < 0.001, respectively). No changes in any fatty acid levels were observed for the placebo group from baseline to week 3 or from baseline to week 12.

      Enzyme activity.

      Twelve week n-3 PUFA supplementation significantly increased the index of Δ-4 desaturase (DPA → DHA) activity in erythrocytes from AAA patients (P < 0.001), while the index of Δ-6 desaturase (LA → DGLA and LA → GLA) activity, the index of Δ-9 desaturase (stearic → oleic) activity, and the index of stearoyl-CoA desaturase (palmitic → palmitoleic acid) activity remained unchanged (Table 7). Omega-3 PUFA supplementation of AAA patients lowered the index of desaturase/elongase-mediated LA → AA biosynthesis and the index of elongase 2/5 (AA → adrenic acid) activity to levels that were comparable to the control cohort (P = 0.031 and P = 0.002, respectively). No changes in any enzyme activity levels were observed for the placebo cohort.
      TABLE 7Erythrocyte fatty acid ratios as indices of enzyme activities in AAA patients
      Index of Enzyme ActivityFish Oil Cohort
      BaselineWeek 3Week 12
      Δ-4 desaturase (DHA/DPA)1.85 ± 0.142.60 ± 0.16
      P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      3.93 ± 0.17
      P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      Δ-9 desaturase (Oleic acid/stearic acid)1.00 ± 0.030.97 ± 0.030.98 ± 0.04
      Δ-6 desaturase (1) (dihomo-γ-linolenic/linoleic)0.18 ± 0.010.15 ± 0.010.15 ± 0.01
      Δ-6 desaturase (2) (γ-linolenic/linoleic)0.01 ± 0.0010.01 ± 0.0010.01 ± 0.001
      LA → AA biosynthesis (AA/LA)1.68 ± 0.081.60 ± 0.091.40 ± 0.07
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      Stearoyl-CoA desaturase (palmitoleic acid/palmitic acid)0.03 ± 0.0040.02 ± 0.0030.02 ± 0.003
      Elongase 2/5(adrenic acid/AA)0.17 ± 0.010.16 ± 0.010.12 ± 0.01
      P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      Data are expressed as mean ± SEM.
      a P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      b P < 0.001, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      c P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.

      Omega-3 fatty acid supplementation decreases RDW.

      Significant decreases in RDW (P = 0.003), red blood cell counts (P = 0.019), hematocrit percentage (P = 0.016), platelet counts (P = 0.042), and plateletcrit percentage (P = 0.021) were observed for AAA patients following 12 weeks of n-3 PUFA supplementation (Table 8).
      TABLE 8Red blood cell indices, white blood cell counts and platelet counts and indices for AAA patients (mean±SEM)
      Blood ParameterFish Oil CohortPlacebo Cohort
      BaselineWeek 3Week 12BaselineWeek 3Week 12
      WBCs (×109/liter)6.9 ± 0.56.3 ± 0.56.6 ± 0.46.6 ± 0.56.5 ± 0.46.7 ± 0.5
      Lymphocytes (×109/liter)2.1 ± 0.12.0 ± 0.22.1 ± 0.22.1 ± 0.21.8 ± 0.21.9 ± 0.2
      Monocytes (×109/liter)0.4 ± 0.10.4 ± 0.10.4 ± 0.10.4 ± 0.050.3 ± 0.020.5 ± 0.1
      Granulocytes (×109/liter)4.4 ± 0.43.8 ± 0.34.0 ± 0.34.1 ± 0.34.3 ± 0.34.4 ± 0.4
      RBCs (×1012/liter)4.7 ± 0.14.6 ± 0.14.6 ± 0.1
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      4.8 ± 0.14.7 ± 0.14.8 ± 0.2
      Hemoglobin (g/l)144.1 ± 3.5142.5 ± 4.0139.2 ± 3.6143.8 ± 3.9142.2 ± 4.5143.7 ± 5.1
      Hematocrit (%)44.1 ± 1.043.5 ± 1.142.5 ± 1.1
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      43.7 ± 1.143.5 ± 1.343.8 ± 1.5
      MCV (fl)93.5 ± 0.994.6 ± 1.293.6 ± 1.291.0 ± 0.991.8 ± 1.091.9 ± 1.2
      MCH (pg)30.5 ± 0.331.0 ± 0.530.7 ± 0.530.0 ± 0.430.0 ± 0.430.2 ± 0.6
      MCHC (g/l)326.6 ± 2.8327.1 ± 3.2328.3 ± 2.2328.8 ± 2.4326.6 ± 1.9328.0 ± 2.3
      RDW (%)14.8 ± 0.414.5 ± 0.413.8 ± 0.2
      P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      14.4 ± 0.314.7 ± 0.314.0 ± 0.2
      Platelet (×109/liter)207.9 ± 16.0205.5 ± 21.1198.3 ± 14.1
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      192.8 ± 16.3194.4 ± 19.2191.3 ± 24.1
      MPV (fl)8.0 ± 0.37.5 ± 0.47.7 ± 0.27.7 ± 0.27.8 ± 0.28.0 ± 0.2
      Plateletcrit (%)0.17 ± 0.010.15 ± 0.020.15 ± 0.01
      P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      0.15 ± 0.010.15 ± 0.020.15 ± 0.02
      PDW16.8 ± 0.217.0 ± 0.216.9 ± 0.217.3 ± 0.217.0 ± 0.317.3 ± 0.3
      Data are expressed as mean ± SEM. WBCs, white blood cells; RBCs, red blood cells; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MPV, mean platelet volume; PDW, platelet distribution width.
      a P < 0.05, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.
      b P < 0.01, fish oil cohort week 3/week 12 significantly different to fish oil cohort baseline.

      DISCUSSION

      Observational (case-control) study

      Analysis of erythrocyte fatty acid profiles among AAA patients highlighted a mean omega-3 index value that was comparable to the control cohort in conjunction with an n-6/n-3 fatty acid ratio that was significantly lower. Among individual fatty acids measured, levels of the saturated fatty acid, margaric acid, and the omega-3 fatty acid, DPA, were significantly higher in erythrocytes from AAA patients, and levels of the omega-6 fatty acid, LA, were significantly lower compared with control. The lower levels of LA in conjunction with higher levels of adrenic acid in the AAA cohort are of note. A meta-analysis of prospective cohort studies (n = 310,602 participants, n = 12,479 cases) indicated that the highest levels of dietary LA intake were associated with a 15% lower risk of CHD events and a 21% lower risk of CHD deaths when compared with the lowest levels of LA intake (
      • Farvid M.S.
      • Ding M.
      • Pan A.
      • Sun Q.
      • Chiuve S.E.
      • Steffen L.M.
      • Willett W.C.
      • Hu F.B.
      Dietary linoleic acid and risk of coronary heart disease: a systematic review and meta-analysis of prospective cohort studies.
      ). In addition, a higher level of circulating LA in a community-based US cohort (n = 2,792) was associated with lower total mortality risk (extreme-quintile hazard ratio = 0.87; P = 0.005) and a lower risk of cardiovascular disease mortality (22% lower risk in the highest versus the lowest quintile; P = 0.02) (
      • Wu J.H.
      • Lemaitre R.N.
      • King I.B.
      • Song X.
      • Psaty B.M.
      • Siscovick D.S.
      • Mozaffarian D.
      Circulating omega-6 polyunsaturated fatty acids and total and cause-specific mortality: the Cardiovascular Health Study.
      ). It has been suggested that the observed cardio-protective effects of LA are related to its competition with pro-inflammatory AA for reacylation into membrane phospholipids (
      • Rett B.S.
      • Whelan J.
      Increasing dietary linoleic acid does not increase tissue arachidonic acid content in adults consuming Western-type diets: a systematic review.
      ). In line with this, a strong inverse association has been reported between serum LA levels and high-sensitivity C-reactive protein (hsCRP), a key marker of inflammation (
      • Virtanen J.K.
      • Mursu J.
      • Voutilainen S.
      • Tuomainen T-P.
      The associations of serum n-6 polyunsaturated fatty acids with serum C-reactive protein in men: the Kuopio Ischaemic Heart Disease Risk Factor Study.
      ). These findings suggest that the altered n-6 fatty acid profile in AAA skews cellular responses toward pro-inflammatory eicosanoid production and upregulated inflammatory pathways.
      Desaturase enzymes catalyze the rate-limiting steps in long chain fatty acid synthetic pathways and their activities influence erythrocyte phospholipid fatty acid composition (
      • Fuhrman B.J.
      • Barba M.
      • Krogh V.
      • Micheli A.
      • Pala V.
      • Lauria R.
      • Chajes V.
      • Riboli E.
      • Sieri S.
      • Berrino F.
      • et al.
      Erythrocyte membrane phospholipid composition as a biomarker of dietary fat.
      ). In this study, Δ-4 and Δ-9 desaturase activity levels in erythrocytes from AAA patients were comparable to those of a healthy control cohort. In contrast, the index of desaturase/elongase-mediated LA → AA biosynthesis was significantly higher in erythrocytes from AAA patients compared with control participants, as was the index of Δ-6 desaturase activity. The higher index of Δ-6 desaturase activity suggests that anti-inflammatory n-3 PUFA formation is repressed in favor of pro-inflammatory n-6 PUFA biosynthesis. It is of note that the higher index of desaturase/elongase-mediated LA → AA biosynthesis did not translate into higher levels of AA in this cohort. AA is a known substrate for inflammatory eicosanoid synthesis and, while it is possible that the absence of an increase in AA levels in AAA patients may be due to excessive shunting of this fatty acid to an alternative eicosanoid biosynthetic pathway, we have previously reported lower levels of prostaglandin E2 (a product of AA metabolism) in this AAA cohort compared with healthy control participants. It is thus possible that the higher conversion of LA to AA in AAA patients led to increased flux through the n-6 biosynthetic pathway beyond AA to adrenic acid resulting in the observed higher levels of the latter. The anomalies observed in enzyme activity indices among AAA patients suggest a predisposition toward reactions that favor increased production of n-6 PUFAs with high pro-inflammatory potential and repressed production of their anti-inflammatory n-3 PUFA counterparts.

      Omega-3 clinical trial

      Twelve week n-3 PUFA supplementation decreased levels of the n-6 fatty acids, AA and adrenic acid, and increased levels of the n-3 fatty acids, DHA and EPA, in AAA patient erythrocytes, while concomitantly raising the mean omega-3 index to a value almost double that at study entry (8.03%). Importantly, the postsupplementation omega-3 index value was within the 8–12% range that affords cardioprotection (
      • Harris W.S.
      • Del Gobbo L.
      • Tintle N.L.
      The omega-3 index and relative risk for coronary heart disease mortality: estimation from 10 cohort studies.
      ). The large reduction in AA and adrenic acid levels with n-3 PUFA supplementation likely reflects competition of DHA and EPA for incorporation into existing erythrocyte membranes and/or greater availability of n-3 PUFAs for integration into newly synthesized membranes (
      • Saifullah A.
      • Watkins B.A.
      • Saha C.
      • Li Y.
      • Moe S.M.
      • Friedman A.N.
      Oral fish oil supplementation raises blood omega-3 levels and lowers C-reactive protein in haemodialysis patients–a pilot study.
      ). The anti-inflammatory and immunomodulatory properties of increased cellular phospholipid EPA and DHA levels are likely to favorably impact AAA disease. Higher n-3 PUFA intake results in partial substitution of AA for EPA and DHA, a net decrease in pro-inflammatory eicosanoid production, and favorable impacts on inflammatory responses (
      • Massaro M.
      • Scoditti E.
      • Carluccio M.A.
      • De Caterina R.
      Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease.
      ). The latter is due to competition of n-3 PUFAs with AA for the same metabolic enzymes, resulting in production of an alternate series of less biologically potent eicosanoids with weaker pro-inflammatory, platelet-aggregating, and vasoconstrictive activities (
      • Massaro M.
      • Scoditti E.
      • Carluccio M.A.
      • De Caterina R.
      Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease.
      ,
      • Yates C.M.
      • Calder P.C.
      • Rainger G.E.
      Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in chronic inflammatory disease.
      ,
      • Calder P.C.
      Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance.
      ). Omega-3 PUFAs are, in addition, substrates of cytochrome P450 enzymes, and increasing the levels of these fatty acids results in enhanced production of DHA- and EPA-derived metabolites at the expense of AA-derived metabolite production (
      • Schunck W-H.
      • Konkel A.
      • Fischer R.
      • Weylandt K-H.
      Therapeutic potential of omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflammatory diseases.
      ). The lower n-6/n-3 ratio, a value reflecting the balance between precursor PUFAs giving rise to downstream pro- and anti-inflammatory eicosanoids, respectively, supports an n-3 PUFA-driven switch toward a more favorable eicosanoid profile.
      Twelve week n-3 PUFA supplementation significantly increased the index of Δ-4 desaturase activity in erythrocytes from AAA patients and lowered the index of desaturase/elongase-mediated LA → AA biosynthesis and the index of elongase 2/5 activity to levels that were comparable to the control cohort (see supplemental Fig. S1 for a summary). The index of Δ-6 desaturase activity was unaffected by n-3 PUFA supplementation. Δ-4 desaturase and Δ-6 desaturase each form part of a distinct biochemical pathway that yields DHA as a biosynthetic product. Δ-6 desaturase forms part of a coupled microsomal-peroxisomal pathway that produces DHA through sequential desaturations and elongations, while Δ-4 desaturase forms part of an alternative pathway that yields DHA through a single Δ4 desaturation step (
      • Park H.G.
      • Park W.J.
      • Kothapalli K.S.
      • Brenna J.T.
      The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells.
      ). As expected with DHA supplementation, the ratio of DHA:DPA increased. Determination of the contribution of Δ-4 desaturase, if any, to this result would require direct measurement of the activity of this enzyme. The decreases in enzyme activities leading to pro-inflammatory n-6 PUFA production suggest that n-3 PUFA supplementation alters fatty acid metabolism in a manner that is likely to improve the inflammatory status of AAA patients.
      Twelve week supplementation with n-3 PUFAs (1.8 g/day) significantly decreased RDW, red blood cell counts, hematocrit percentage, and platelet count and platelet percentage in AAA patients. RDW is a numerical measure reflecting size variability or heterogeneity of volume among circulating erythrocytes (
      • Salvagno G.L.
      • Sanchis-Gomar F.
      • Picanza A.
      • Lippi G.
      Red blood cell distribution width: A simple parameter with multiple clinical applications.
      ). It is well-documented that RDW correlates with inflammatory biomarkers in a multitude of clinical settings (
      • Vayá A.
      • Sarnago A.
      • Fuster O.
      • Alis R.
      • Romagnoli M.
      Influence of inflammatory and lipidic parameters on red blood cell distribution width in a healthy population.
      ,
      • Song C.S.
      • Park D.I.
      • Yoon M.Y.
      • Seok H.S.
      • Park J.H.
      • Kim H.J.
      • Cho Y.K.
      • Sohn C.I.
      • Jeon W.K.
      • Kim B.I.
      Association between red cell distribution width and disease activity in patients with inflammatory bowel disease.
      ,
      • Fornal M.
      • Wizner B.
      • Cwynar M.
      • Królczyk J.
      • Kwater A.
      • Korbut R.A.
      • Grodzicki T.
      Association of red blood cell distribution width, inflammation markers and morphological as well as rheological erythrocyte parameters with target organ damage in hypertension.
      ), and a large cohort study has supported the existence of a strong graded relationship between RDW and high-sensitivity C-reactive protein and RDW and erythrocyte sedimentation rate, independent of confounding factors (
      • Lippi G.
      • Targher G.
      • Montagnana M.
      • Salvagno G.L.
      • Zoppini G.
      • Guidi G.C.
      Relation between red blood cell distribution width and inflammatory biomarkers in a large cohort of unselected outpatients.
      ). In light of this and evidence suggesting the existence of extensive cross-talk between the pathways of inflammation and coagulation (
      • van der Poll T.
      • de Boer J.D.
      • Levi M.
      The effect of inflammation on coagulation and vice versa.
      ,
      • Levi M.
      • van der Poll T.
      Inflammation and coagulation.
      ), it is likely that the observed changes reflect n-3 PUFA-mediated improvements in inflammatory status in AAA patients who received this supplement.
      The study, while limited by small sample size, was characterized by multiple strengths that included a placebo-controlled double-blind study design and use of a validated biomarker of erythrocyte membrane fatty acid content. Although it is clear that n-3 PUFA supplementation improves fatty acid status among AAA patients, further studies will be required to determine whether this improvement translates into positive alterations in the histopathologic appearance of AAA disease at the level of the aorta. In addition, while an aberrant fatty acid profile was observed in AAA patients compared with healthy control participants, it is not yet known why this occurs. It is possible that expression of fatty acid metabolizing enzymes is dysregulated in AAA patients, resulting in the observed aberrant profile of enzyme activity and erythrocyte fatty acid proportions. It is noteworthy that participants were screened at entry for dietary intake of fish and seafood, with all participants consuming no more than two oily fish meals per week. It is therefore unlikely that the observed differences in fatty acid profile are attributed to differences in diet between the two cohorts.
      Taken together, the results presented here indicate that erythrocytes from AAA patients demonstrate a distinct fatty acid profile that is characterized by aberrant proportions of n-6 fatty acids with high inflammatory potential, while full blood count parameters among these patients are characterized by alterations that reflect the influence of systemic inflammation and oxidative stress. Improvements in inflammatory parameters and n-6 fatty acid status following n-3 PUFA supplementation suggest that dietary fatty acids represent a viable therapeutic intervention in AAA.

      Acknowledgments

      The authors thank Blackmores Pty. Ltd. for the supply of omega-3 marine triglyceride capsules (Blackmores Omega Brain) and placebo capsules for the trial. The authors also thank Digby Krastins for assistance with blood collection, Lucia Pemble, Suzanne Ryan, Sandra Allen, and Jill Webber for assistance with AAA patient recruitment, and Dr. Chaim Meital and Moffat Beach Family Medical Practice for assistance with recruitment of control participants.

      REFERENCES

        • Wang Q.
        • Ding Y.
        • Song P.
        • Zhu H.
        • Okon I.S.
        • Ding N-Y.
        • Chen H.
        • Liu D.
        • Zou M-H.
        Tryptophan-derived 3-hydroxyanthranilic acid contributes to angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo.
        Circulation. 2017; 136: 2271-2283
        • Sampson U.K.
        • Norman P.E.
        • Fowkes F.G.R.
        • Aboyans V.
        • Song Y.
        • Harrell Jr., F.E.
        • Forouzanfar M.H.
        • Naghavi M.
        • Denenberg J.O.
        • McDermott M.M.
        Estimation of global and regional incidence and prevalence of abdominal aortic aneurysms 1990 to 2010.
        Glob. Heart. 2014; 9: 159-170
      1. MA3RS Study Investigators. 2017. Aortic wall inflammation predicts abdominal aortic aneurysm expansion, rupture, and need for surgical repair. Circulation. 136: 787–797.

        • Meital L.T.
        • Sandow S.L.
        • Calder P.C.
        • Russell F.D.
        Abdominal aortic aneurysm and omega-3 polyunsaturated fatty acids: mechanisms, animal models, and potential treatment.
        Prostaglandins Leukot. Essent. Fatty Acids. 2017; 118: 1-9
        • Orešič M.
        • Hänninen V.A.
        • Vidal-Puig A.
        Lipidomics: a new window to biomedical frontiers.
        Trends Biotechnol. 2008; 26: 647-652
        • Wales K.M.
        • Kavazos K.
        • Nataatmadja M.
        • Brooks P.R.
        • Williams C.
        • Russell F.D.
        N-3 PUFAs protect against aortic inflammation and oxidative stress in angiotensin II-infused apolipoprotein E−/−mice.
        PLoS One. 2014; 9: e112816
        • Kugo H.
        • Zaima N.
        • Mouri Y.
        • Tanaka H.
        • Yanagimoto K.
        • Urano T.
        • Unno N.
        • Moriyama T.
        The preventive effect of fish oil on abdominal aortic aneurysm development.
        Biosci. Biotechnol. Biochem. 2016; 80: 1186-1191
        • Pope N.H.
        • Salmon M.
        • Davis J.P.
        • Chatterjee A.
        • Su G.
        • Conte M.S.
        • Ailawadi G.
        • Upchurch G.R.
        D-series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization.
        FASEB J. 2016; 30: 4192-4201
        • Yoshihara T.
        • Shimada K.
        • Fukao K.
        • Sai E.
        • Sato-Okabayashi Y.
        • Matsumori R.
        • Shiozawa T.
        • Alshahi H.
        • Miyazaki T.
        • Tada N.
        • et al.
        Omega 3 polyunsaturated fatty acids suppress the development of aortic aneurysms through the inhibition of macrophage-mediated inflammation.
        Circ. J. 2015; 79: 1470-1478
        • Kavazos K.
        • Nataatmadja M.
        • Wales K.M.
        • Hartland E.
        • Williams C.
        • Russell F.D.
        Dietary supplementation with omega-3 polyunsaturated fatty acids modulate matrix metalloproteinase immunoreactivity in a mouse model of pre-abdominal aortic aneurysm.
        Heart Lung Circ. 2015; 24: 377-385
        • Aikawa T.
        • Miyazaki T.
        • Shimada K.
        • Sugita Y.
        • Shimizu M.
        • Ouchi S.
        • Kadoguchi T.
        • Yokoyama Y.
        • Shiozawa T.
        • Hiki M.
        • et al.
        Low serum levels of epa are associated with the size and growth rate of abdominal aortic aneurysm.
        J. Atheroscler. Thromb. 2017; 24: 912-920
        • Lindholt J.S.
        • Kristensen K.L.
        • Burillo E.
        • Martinez-Lopez D.
        • Calvo C.
        • Ros E.
        • Martín-Ventura J.L.
        • Sala-Vila A.
        Arachidonic acid, but not omega-3 index, relates to the prevalence and progression of abdominal aortic aneurysm in a population-based study of Danish men.
        J. Am. Heart Assoc. 2018; 7: e007790
        • Arnold C.
        • Konkel A.
        • Fischer R.
        • Schunck W-H.
        Cytochrome P450-dependent metabolism of ω-6 and ω-3 long-chain polyunsaturated fatty acids.
        Pharmacol. Rep. 2010; 62: 536-547
        • von Schacky C.
        Use of red blood cell fatty-acid profiles as biomarkers in cardiac disease.
        Biomark. Med. 2009; 3: 25-32
        • Del Gobbo L.C.
        • Imamura F.
        • Aslibekyan S.
        • Marklund M.
        • Virtanen J.K.
        • Wennberg M.
        • Yakoob M.Y.
        • Chiuve S.E.
        • Dela Cruz L.
        • Frazier-Wood A.C.
        • et al.
        ω-3 polyunsaturated fatty acid biomarkers and coronary heart disease: pooling project of 19 cohort studies.
        JAMA Intern. Med. 2016; 176: 1155-1166
        • Kleber M.E.
        • Delgado G.E.
        • Lorkowski S.
        • März W.
        • von Schacky C.
        Omega-3 fatty acids and mortality in patients referred for coronary angiography. The Ludwigshafen Risk and Cardiovascular Health Study.
        Atherosclerosis. 2016; 252: 175-181
        • Harris W.S.
        • Tintle N.L.
        • Etherton M.R.
        • Vasan R.S.
        Erythrocyte long-chain omega-3 fatty acid levels are inversely associated with mortality and with incident cardiovascular disease: the Framingham Heart Study.
        J. Clin. Lipidol. 2018; 12: 718-727.e6
        • Goozee K.
        • Chatterjee P.
        • James I.
        • Shen K.
        • Sohrabi H.R.
        • Asih P.R.
        • Dave P.
        • Ball B.
        • ManYan C.
        • Taddei K.
        • et al.
        Alterations in erythrocyte fatty acid composition in preclinical Alzheimer's disease.
        Sci. Rep. 2017; 7: 676
        • Park Y.
        • Lim J.
        • Lee J.
        • Kim S.G.
        Erythrocyte fatty acid profiles can predict acute non-fatal myocardial infarction.
        Br. J. Nutr. 2009; 102: 1355-1361
        • Bannikoppa P.
        • Dhayanand J.
        • Madhukumar R.
        • Padmanabhan A.
        • Bafna U.
        • Vijayakumar M.
        • Devi K.U.
        • Pramod K.
        • Thomas T.
        • Jayshree R.
        • et al.
        Fatty acid intake and erythrocyte fatty acid profile in women with breast, ovarian and cervical cancers.
        Clin. Nutr. ESPEN. 2017; 19: 59-63
        • Itomura M.
        • Fujioka S.
        • Hamazaki K.
        • Kobayashi K.
        • Nagasawa T.
        • Sawazaki S.
        • Kirihara Y.
        • Hamazaki T.
        Factors influencing EPA+DHA levels in red blood cells in Japan.
        In Vivo. 2008; 22: 131-135
        • Golledge J.
        • Muller J.
        • Daugherty A.
        • Norman P.
        Abdominal aortic aneurysm: pathogenesis and implications for management.
        Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2605-2613
        • Harris W.S.
        • Von Schacky C.
        The omega-3 index: a new risk factor for death from coronary heart disease?.
        Prev. Med. 2004; 39: 212-220
        • Jackson K.H.
        • Polreis J.M.
        • Tintle N.L.
        • Kris-Etherton P.M.
        • Harris W.S.
        Association of reported fish intake and supplementation status with the omega-3 index.
        Prostaglandins Leukot. Essent. Fatty Acids. 2019; 142: 4-10
        • Efird J.
        Blocked randomization with randomly selected block sizes.
        Int. J. Environ. Res. Public Health. 2011; 8: 15-20
        • Harris W.S.
        • Del Gobbo L.
        • Tintle N.L.
        The omega-3 index and relative risk for coronary heart disease mortality: estimation from 10 cohort studies.
        Atherosclerosis. 2017; 262: 51-54
        • Bürgin-Maunder C.S.
        • Brooks P.R.
        • Hitchen-Holmes D.
        • Russell F.D.
        Moderate dietary supplementation with omega-3 fatty acids does not impact plasma von Willebrand factor profile in mildly hypertensive subjects.
        BioMed Res. Int. 2015; 2015: 394871
        • Fuhrman B.J.
        • Barba M.
        • Krogh V.
        • Micheli A.
        • Pala V.
        • Lauria R.
        • Chajes V.
        • Riboli E.
        • Sieri S.
        • Berrino F.
        • et al.
        Erythrocyte membrane phospholipid composition as a biomarker of dietary fat.
        Ann. Nutr. Metab. 2006; 50: 95-102
        • Dasilva G.
        • Pazos M.
        • García-Egido E.
        • Gallardo J.M.
        • Ramos-Romero S.
        • Torres J.L.
        • Romeu M.
        • Nogués M-R.
        • Medina I.
        A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.
        J. Nutr. Biochem. 2017; 43: 53-67
        • McNamara R.K.
        • Jandacek R.
        • Rider T.
        • Tso P.
        • Cole-Strauss A.
        • Lipton J.W.
        Omega-3 fatty acid deficiency increases constitutive pro-inflammatory cytokine production in rats: relationship with central serotonin turnover.
        Prostaglandins Leukot. Essent. Fatty Acids. 2010; 83: 185-191
        • McNamara R.K.
        • Jandacek R.
        • Rider T.
        • Tso P.
        • Dwivedi Y.
        • Pandey G.N.
        Selective deficits in erythrocyte docosahexaenoic acid composition in adult patients with bipolar disorder and major depressive disorder.
        J. Affect. Disord. 2010; 126: 303-311
        • Dogdu O.
        • Koc F.
        • Kalay N.
        • Yarlioglues M.
        • Elcik D.
        • Karayakali M.
        • Ozbek K.
        • Kaya M.G.
        Assessment of red cell distribution width (RDW) in patients with coronary artery ectasia.
        Clin. Appl. Thromb. Hemost. 2012; 18: 211-214
        • Flock M.R.
        • Skulas-Ray A.C.
        • Harris W.S.
        • Gaugler T.L.
        • Fleming J.A.
        • Kris-Etherton P.M.
        Effects of supplemental long-chain omega-3 fatty acids and erythrocyte membrane fatty acid content on circulating inflammatory markers in a randomized controlled trial of healthy adults.
        Prostaglandins Leukot. Essent. Fatty Acids. 2014; 91: 161-168
        • Farvid M.S.
        • Ding M.
        • Pan A.
        • Sun Q.
        • Chiuve S.E.
        • Steffen L.M.
        • Willett W.C.
        • Hu F.B.
        Dietary linoleic acid and risk of coronary heart disease: a systematic review and meta-analysis of prospective cohort studies.
        Circulation. 2014; 130: 1568-1578
        • Wu J.H.
        • Lemaitre R.N.
        • King I.B.
        • Song X.
        • Psaty B.M.
        • Siscovick D.S.
        • Mozaffarian D.
        Circulating omega-6 polyunsaturated fatty acids and total and cause-specific mortality: the Cardiovascular Health Study.
        Circulation. 2014; 130: 1245-1253
        • Rett B.S.
        • Whelan J.
        Increasing dietary linoleic acid does not increase tissue arachidonic acid content in adults consuming Western-type diets: a systematic review.
        Nutr. Metab. (Lond.). 2011; 8: 36
        • Virtanen J.K.
        • Mursu J.
        • Voutilainen S.
        • Tuomainen T-P.
        The associations of serum n-6 polyunsaturated fatty acids with serum C-reactive protein in men: the Kuopio Ischaemic Heart Disease Risk Factor Study.
        Eur. J. Clin. Nutr. 2018; 72: 342-348
        • Saifullah A.
        • Watkins B.A.
        • Saha C.
        • Li Y.
        • Moe S.M.
        • Friedman A.N.
        Oral fish oil supplementation raises blood omega-3 levels and lowers C-reactive protein in haemodialysis patients–a pilot study.
        Nephrol. Dial. Transplant. 2007; 22: 3561-3567
        • Massaro M.
        • Scoditti E.
        • Carluccio M.A.
        • De Caterina R.
        Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease.
        Prostaglandins Leukot. Essent. Fatty Acids. 2008; 79: 109-115
        • Yates C.M.
        • Calder P.C.
        • Rainger G.E.
        Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in chronic inflammatory disease.
        Pharmacol. Ther. 2014; 141: 272-282
        • Calder P.C.
        Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance.
        Biochim. Biophys. Acta. 2015; 1851: 469-484
        • Schunck W-H.
        • Konkel A.
        • Fischer R.
        • Weylandt K-H.
        Therapeutic potential of omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflammatory diseases.
        Pharmacol. Ther. 2018; 183: 177-204
        • Park H.G.
        • Park W.J.
        • Kothapalli K.S.
        • Brenna J.T.
        The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells.
        FASEB J. 2015; 29: 3911-3919
        • Salvagno G.L.
        • Sanchis-Gomar F.
        • Picanza A.
        • Lippi G.
        Red blood cell distribution width: A simple parameter with multiple clinical applications.
        Crit. Rev. Clin. Lab. Sci. 2015; 52: 86-105
        • Vayá A.
        • Sarnago A.
        • Fuster O.
        • Alis R.
        • Romagnoli M.
        Influence of inflammatory and lipidic parameters on red blood cell distribution width in a healthy population.
        Clin. Hemorheol. Microcirc. 2015; 59: 379-385
        • Song C.S.
        • Park D.I.
        • Yoon M.Y.
        • Seok H.S.
        • Park J.H.
        • Kim H.J.
        • Cho Y.K.
        • Sohn C.I.
        • Jeon W.K.
        • Kim B.I.
        Association between red cell distribution width and disease activity in patients with inflammatory bowel disease.
        Dig. Dis. Sci. 2012; 57: 1033-1038
        • Fornal M.
        • Wizner B.
        • Cwynar M.
        • Królczyk J.
        • Kwater A.
        • Korbut R.A.
        • Grodzicki T.
        Association of red blood cell distribution width, inflammation markers and morphological as well as rheological erythrocyte parameters with target organ damage in hypertension.
        Clin. Hemorheol. Microcirc. 2014; 56: 325-335
        • Lippi G.
        • Targher G.
        • Montagnana M.
        • Salvagno G.L.
        • Zoppini G.
        • Guidi G.C.
        Relation between red blood cell distribution width and inflammatory biomarkers in a large cohort of unselected outpatients.
        Arch. Pathol. Lab. Med. 2009; 133: 628-632
        • van der Poll T.
        • de Boer J.D.
        • Levi M.
        The effect of inflammation on coagulation and vice versa.
        Curr. Opin. Infect. Dis. 2011; 24: 273-278
        • Levi M.
        • van der Poll T.
        Inflammation and coagulation.
        Crit. Care Med. 2010; 38: S26-S34