Jackie Madden

Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish

Background: Estimation of the intake of oily fish at a population level is difficult. The measurement of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in biological samples may provide a useful biomarker of intake. Objective: We identified the most appropriate biomarkers for the assessment of habitual oily fish intake and changes in intake by elucidating the dose- and time-dependent response of EPA and DHA incorporation into various biological samples that represent roles in fatty acid transport, function, and storage. Design: This was a double-blind, randomized, controlled intervention trial in 204 men and women that lasted 12 mo. EPA and DHA capsules were provided in a manner to reflect sporadic consumption of oily fish (ie, 1, 2, or 4 times/wk). EPA and DHA were assessed at 9 time points over 12 mo in 9 sample types (red blood cells, mononuclear cells, platelets, buccal cells, adipose tissue, plasma phosphatidylcholine, triglycerides, cholesteryl esters, and nonesterified fatty acids). Results: A dose response (P < 0.05) was observed for EPA and DHA in all pools except for red blood cell EPA (P = 0.057). EPA and DHA measures in plasma phosphatidylcholine and platelets were best for the discrimination between different intakes (P < 0.0001). The rate of incorporation varied between sample types, with the time to maximal incorporation ranging from days (plasma phosphatidylcholine) to months (mononuclear cells) to >12 mo (adipose tissue). Conclusions: Plasma phosphatidylcholine EPA plus DHA was identified as the most suitable biomarker of acute changes in EPA and DHA intake, and platelet and mononuclear cell EPA plus DHA were the most suitable biomarkers of habitual intake. This trial was registered at Current Controlled Trials (www.controlled-trials.com) as ISRCTN48398526.

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells.

Docosahexaenoic Acid Inhibits the Adhesion of Flowing Neutrophils to Cytokine Stimulated Human Umbilical Vein Endothelial Cells

The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

The pattern of fatty acids displaced by EPA and DHA following 12 Months supplementation varies between blood cell and plasma fractions

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are increased in plasma lipids and blood cell membranes in response to supplementation. Whilst arachidonic acid (AA) is correspondingly decreased, the effect on other fatty acids (FA) is less well described and there may be site-specific differences. In response to 12 months EPA + DHA supplementation in doses equivalent to 0–4 portions of oily fish/week (1 portion: 3.27 g EPA+DHA) multinomial regression analysis was used to identify important FA changes for plasma phosphatidylcholine (PC), cholesteryl ester (CE) and triglyceride (TAG) and for blood mononuclear cells (MNC), red blood cells (RBC) and platelets (PLAT). Dose-dependent increases in EPA + DHA were matched by decreases in several n-6 polyunsaturated fatty acids (PUFA) in PC, CE, RBC and PLAT, but were predominantly compensated for by oleic acid in TAG. Changes were observed for all FA classes in MNC. Consequently the n-6:n-3 PUFA ratio was reduced in a dose-dependent manner in all pools after 12 months (37%–64% of placebo in the four portions group). We conclude that the profile of the FA decreased in exchange for the increase in EPA + DHA following supplementation differs by FA pool with implications for understanding the impact of n-3 PUFA on blood lipid and blood cell biology.

Compared with Daily, Weekly n–3 PUFA Intake Affects the Incorporation of Eicosapentaenoic Acid and Docosahexaenoic Acid into Platelets and Mononuclear Cells in Humans

Consumption of oily fish is sporadic, whereas controlled intervention studies of n–3 (ω-3) fatty acids usually provide capsules containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as a daily dose. This methodologic study explored whether there are differences in the short-, medium-, and long-term incorporation of EPA and DHA into blood plasma and cells with the provision of identical amounts of EPA and DHA, equivalent to 2 oily fish servings per week (or 6.54 g/wk EPA and DHA), either intermittently (i.e., 1 portion twice per week) or continuously (i.e., divided into daily amounts). The study was part of a randomized, double-blind controlled intervention lasting 12 mo, with participants stratified by age and sex. There were 5 intervention groups, 2 of which are reported here: the 2 intermittent portions (2I) and 2 continuous portions (2C) groups. EPA and DHA were measured in plasma phosphatidylcholine, platelets, and blood mononuclear cells (MNCs) at 9 time points. Sixty-five participants completed the study (2I group, n = 30, mean age of 49.2 y; 2C group, n = 35, mean age of 50.6 y). The incorporation pattern over the 12-mo intervention was different between the 2 groups in all samples (P < 0.0001, time × treatment interaction). At the end of the 12-mo intervention, the 2C group had higher EPA, DHA, and EPA + DHA in platelets (all P < 0.01) and higher EPA and EPA + DHA in MNCs (both P < 0.05) compared with the 2I group. No significant differences were shown for plasma phosphatidylcholine EPA (P = 0.1), DHA (P = 0.15), EPA + DHA (P = 0.07), or MNC DHA (P = 0.06). In conclusion, the pattern of consumption does affect the incorporation of EPA and DHA into cells used as biomarkers of intake. The differences identified here need to be considered in the design of studies and when extrapolating results from continuous capsule-based intervention studies to dietary guidelines for oily fish consumption. This trial was registered at www.controlled-trials.com as ISRCTN48398526.

Fatty acid profile of plasma NEFA does not reflect adipose tissue fatty acid profile

Adipose tissue (AT) fatty acid (FA) composition partly reflects habitual dietary intake. Circulating NEFA are mobilised from AT and might act as a minimally invasive surrogate marker of AT FA profile. Agreement between twenty-eight FA in AT and plasma NEFA was assessed using concordance coefficients in 204 male and female participants in a 12-month intervention using supplements to increase the intake of EPA and DHA. Concordance coefficients generally showed very poor agreement between AT FA and plasma NEFA at baseline SFA: 0·07; MUFA: 0·03; n-6 PUFA: 0·28; n-3 PUFA: 0·01). Participants were randomly divided into training (70 %) and validation (30 %) data sets, and models to predict AT and dietary FA were fitted using data from the training set, and their predictive ability was assessed using data from the validation set. AT n-6 PUFA and SFA were predicted from plasma NEFA with moderate accuracy (mean absolute percentage error n-6 PUFA: 11 % and SFA: 8 %), but predicted values were unable to distinguish between low, medium and high FA values, with only 25 % of n-6 PUFA and 33 % of SFA predicted values correctly assigned to the appropriate tertile group. Despite an association between AT and plasma NEFA EPA (P=0·001) and DHA (P=0·01) at baseline, there was no association after the intervention. To conclude, plasma NEFA are not a suitable surrogate for AT FA.

Electrospray ionisation mass spectrometry analysis of differential turnover of phosphatidylcholine by human blood leukocytes

Synthesis and turnover of membrane phospholipids is essential for cell growth and function, and hydrolysis of membrane phospholipid is central to many intracellular signalling mechanisms. Hydrolysis of phosphatidylcholine (PC) is a major signalling mechanism of neutrophils, leukocytes that phagocytose and kill bacteria as part of the innate immune response, generating phosphatidic acid, diacylglycerol and arachidonate-derived lipid second messengers. We describe here the application of tandem MS/MS electrospray ionisation mass spectrometry to the analysis of molecular patterns of PC synthesis by blood neutrophil and lymphocyte cells from healthy volunteers. This technique combined incorporation of the headgroup choline, methyl-labelled with deuterium (methyl-d9-choline), with precursor scans of diagnostic labelled and native fragment ions. The technique was very sensitive, permitting detection of d9 enrichment <0.01%. Results showed that the two different cell types maintained distinct molecular species compositions of PC, even though they were exposed to the same nutrient supply in blood. Moreover, while the pattern of lymphocyte PC synthesis directly mirrored composition, the fractional synthesis of arachidonoyl (C20∶4,n-6)-containing PC species in neutrophils was greatly enhanced compared with composition. This increased turnover of arachidonoyl species in neutrophils may be related to the active synthesis of eicosanoids and other arachidonoyl-derived mediators in this cell type.

Incorporation of n –3 polyunsaturated fatty acids into human peripheral blood mononuclear cells according to intakes representing consumption of oily fish one, two or four times per week over 12 months

There is evidence that the n–3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in seafood and in marine oils, influence immune cell function, and the mechanisms of action of these fatty acids are being increasingly understood. The functional effects of these fatty acids appear to be closely linked with their appearance in cell membrane phospholipids. Maximal incorporation of EPA and DHA into immune cell phospholipids occurs within a few weeks of beginning a regimen of increased consumption and occurs in a linear dose-response fashion. However, studies performed to date investigating the incorporation of EPA and DHA into human immune cells have used marine oils consumed daily. Thus, although much is known about the effect of very frequent intake of EPA and DHA, this situation does not mimic what occurs with less frequent intake of these fatty acids such as when oily fish are consumed just a few times per week. Therefore, the current study set out to describe the effect of increased n–3 PUFA intake mimicking the consumption of one, two or four oily fish meals per week on the fatty acid composition of human peripheral blood mononuclear cells (PBMC). Healthy male and female volunteers (n = 204) aged 20–80 years were recruited into a double blind, placebo controlled trial and were randomly assigned to one of five groups (n = 40–42/group) each comprising equal gender and age distributions. Subjects consumed 6 · 1 g capsules per day for 12 months; capsules contained either placebo oil (an oil mix replicating the fatty acid composition of the average UK adult diet) or fish oil. Subjects in the placebo group consumed six placebo capsules/d; subjects in the ‘1 portion of oily fish/ week’ group consumed six fish oil capsules (providing 2.8 g EPA +DHA) on one day of each week and six placebo capsules on the other 6 days of each week; subjects in the ‘2 portions of oily fish/week’ group consumed six fish oil capsules on two days of each week (i.e. a total of 5.6 g EPA+DHA) and six placebo capsules on the other 5 days of each week; subjects in the ‘4 portions of oily fish/week’ group consumed six fish oil capsules on 4 days of each week (i.e. a total of 11.2 g EPA+DHA) and six placebo capsules on the other 3 days of each week; the fifth group consumed two fish oil capsules (providing 0.8 g EPA +DHA) and four placebo capsules/d. Blood was collected in the fasting state on nine occasions (at 0, 1, 2 and 4 weeks and 2, 3, 6, 9 and 12 months) and PBMC prepared using standard techniques. PBMC fatty acid composition was determined by gas chromatography. One hundred and sixty three subjects completed the study (n = 30–35 per group). Preliminary dose-response and time course data for the appearance of EPA and DHA in PBMC lipids over 3 months are reported here. Both EPA and DHA were significantly higher in PBMC in the ‘4 portions/week’ group than in the control group at 2 and 4 weeks and at 2 and 3 months. PBMC arachidonic acid was lower in the ‘4 portions/week’ group than in the control group at 2 weeks and at 2 months. There were few other differences in PBMC EPA, DHA and arachidonic acid among groups over this time period. In conclusion, providing EPA +DHA at the level of 11.2 g/week spread over 4 days/week can affect PBMC fatty acid composition and may influence PBMC function.