Ficoll 400 was obtained from Pharmacia Biotech (Uppsala, Sweden). RPMI 1640 medium and glutamine were obtained from JRH Biosciences (Lenexa, KA). Sodium diatrizoate, DMSO, N-formyl-methionyl-L-leucyl-L-phenyalanine (fMLP), Rose Bengal stain (0.25 % w/v in phosphate-buffered saline (PBS) with Ca2+, Mg2+), zymosan and 3,3',5,5'-tetramethylbenzidine were purchased from Sigma (St Louis, MO). Angiograffin was obtained from Schering (Leverkusen, Germany). Agarose was purchased from Calbiochem (La Jolla, CA). Tumor necrosis factor (TNF) was obtained from the Ernst-Boehringer Ingelheim Institut (Vienna, Austria). The Staphylococcus aureus strain NCTC 6571, for measuring neutrophil bactericidal activity, was obtained from the National Centre for Tissue Cultures (Oxford, UK). 125I in the form of NaI and [H3]thymidine was purchased from Amersham International (Little Chalfont, UK). All monoclonal antibodies for the determination of lymphocyte subsets, IgG1 isotype control antibody and Simultest IMK kit were obtained from BD Biosciences (San Jose, CA). Phytohemagglutinin (PHA) was obtained from Murex Diagnostics (Dartford, UK). Anti-human interferon-γ (IFNγ) coating monoclonal antibody and anti-human interleukin-2 (IL-2) polyclonal antibody were purchased from Endogen (Rockford, IL). The biotin-labeled anti-human IFNγ and anti-human IL-2-detecting monoclonal antibodies, horseradish peroxidase, streptavidin and quality-control sample for IFNγ were also purchased from Endogen. The anti-human lymphotoxin (LT) coating and biotin-labeled anti-human LT-detecting monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN). The quality-control samples for LT and IL-2 and standards for IFNγ were obtained from the National Institute for Biological Standards and Control (South Mimms, UK). The standards for LT were purchased from Biosource International (Camarillo, CA). The standards for IL-2 were purchased from Hazelton Biotechnologies (Vienna, Austria).
A range of study foods including pancake mix, bread, milk, margarine, eggs, chocolate, soup mix, dips, instant oats, cheese spread, muffin mix, biscuits and salad dressing were provided by Goodman Fielder (Sydney, Australia). Foods were either enriched with n-3 LCPUFA from microencapsulated cod liver oil (Maritex, Aarhus, Denmark) (n-3 supplemented) or were devoid of n-3 LCPUFA (placebo). The fatty-acid composition of the study foods is described elsewhere [10, 11]. Each enriched food portion provided 125 mg EPA + DHA and subjects were asked to consume eight exchanges daily, to equal 1 g n-3 PUFA/day. Subjects were matched for gender and age then randomly allocated to treatment or control groups. Dietary interviews were conducted by trained dieticians using diet questionnaires and food records as described by Patch et al.  to score the acceptability and palatability of individual food items to ensure compliance with test foods. Subjects were encouraged to keep 'diet diaries' in order to monitor the amount of n-3 PUFA-rich foods consumed. The target macronutrients intakes (% of energy) were as follows: 20% protein, 50% carbohydrate, 30% fat (polyunsaturated: mono-unsaturated: saturated = 1:1:1) .
Subjects and study design
A double-blind, placebo-controlled dietary intervention study of 6 months duration was approved by the Human Research Ethics Committees of the University of Adelaide and the Commonwealth Scientific and Industrial Research Organization (CSIRO) (see Murphy et al.  for details). The trial commenced in 2003 and consent from the subjects recruited was obtained. Forty-four non-smoking volunteers aged 20 to 65 years, who were overweight (BMI > 25 kg/m2) and had fasting plasma triacylglycerol > 1.6 mmol/l, but were otherwise healthy, were recruited from the general community through media advertisements. Volunteers were excluded if they were taking non-steroidal anti-inflammatories, antihypertensives, lipid-lowering or other drugs affecting lipid metabolism. Other exclusion criteria include the consumption of more than one fish meal per week, regularly taking fish oil supplements, inability to consume test foods, recent (previous 3 months) diagnosis of diabetes, symptomatic heart disease, angina pectoris, history of myocardial infarction or stroke, peripheral vascular disease, liver or renal disease (plasma creatinine > 120 mmol/l), major surgery, blood pressure (BP) > 170/100 mmHg; or smokers and/or ex-smokers within the past two years.
Erythrocyte membrane fatty-acid composition
The fatty-acid composition was analyzed at 6 months as described by Murphy et al. . Erythrocytes were isolated, lysed and the membrane lipids extracted into methanol: toluene (4:1, by volume) according to the method of Lepage and Roy . Fatty-acid methyl esters were analyzed by flame-ionization gas chromatography (model GC-20A, Shimazdu Corporation, Kyoto, Japan) using a 50-m BPX70 capillary column (0.32 mm internal diameter and 0.25 μm film thickness (Scientific Glass Engineering, Melbourne, Australia). Individual fatty acids were identified by comparison with known fatty-acid standards (Nuchek Prep, Elysian, MN) and expressed as a percentage of total fatty acids quantified from peak areas.
Preparation of peripheral blood mononuclear cells and neutrophils
At the end of the 6-month trial, 10 ml blood was collected by venepuncture after a 12-h overnight fast into lithium heparin tubes. Mononuclear cells (MNL) and neutrophils were purified by the rapid one-step procedure according to Ferrante and Thong . Briefly, heparinized blood was layered onto a density separation medium consisting of Ficoll 400-Hypaque, density 1.114. Following centrifugation at 600 g for 35 min, the MNL and neutrophil bands were harvested and washed twice with RPMI 1640 medium by centrifugation (5 min × 600 g) and MNL resuspension in RPMI 1640 medium or neutrophils in Hanks Buffered Salt Solution (HBSS).
Neutrophil chemotaxis and chemokinesis
Neutrophils, 5 μl of 4 × 107cells/ml, were allowed to migrate under agarose for 90 min at 37°C/5% CO2-air incubator as previously described  in the presence of a chemotactic gradient generated by 5 μl 10-7 M of fMLP, or 5 μl of diluent DMSO (1% v/v in PBS). The distance migrated by the cells was expressed in mm/90 min. For chemokinesis, neutrophils (20 μl; 4 × 107/ml) were pre-incubated with 20 μl fMLP for 5 min at 37°C in a humidified CO2 (5% CO2 in air) incubator, centrifuged in a microcentrifuge (30 sec × 1 g force), the supernatant was removed and the cells resuspended in 20 μl HBSS with phenol. The random migration in four directions – top, bottom, left and right – was measured using an inverted Leitz microscope. Chemokinesis was expressed as the mean of the distance (in millimeters) traveled in the four directions.
Neutrophil adherence was assayed by measuring neutrophil adherence to plasma-coated plastic surfaces . To the 96-well microtiter plates was added 250 μl/well of 10% autologous plasma and the plates were incubated for 30 min 37°C in a humidified CO2 (5% CO2 in air) incubator. The plates were subsequently washed with HBSS and air-dried. Then, 100 μl of 5 × 106 neutrophils treated with either TNF or with HBSS as a control were loaded into the wells and incubated for 30 min at 37°C in a CO2 incubator. Non-adherent cells were removed by inversion of plates and the wells washed three times with HBSS. Adherent cells were stained with Rose Bengal (0.25 % w/v in PBS with Ca2+, Mg2+) , washed and the dye was then released by adding 50% ethanol and the absorbance was read at 570 nm using a plate reader (Dynatech MR7000, Dynatech Laboratories, Chantilly, VA). The degree of adherence was calculated by subtracting the mean absorbance values of blank wells from the mean of the test wells.
Neutrophil bactericidal activity
The ability of neutrophils to kill Staphylococcus aureus was assessed as described previously . Neutrophils (1 × 106 cells) were mixed with 1 × 106 S. aureus in HBSS and 10% (final) pooled human AB serum in tubes which were then gassed with 5% CO2-air mixture and incubated with end-to-end mixing at 37°C. Samples were taken at 0, 1 and 2 h, diluted in sterile distilled water and plated onto blood agar for colony growth and enumeration of the number of surviving bacteria.
Neutrophil iodination reaction
The quantitative neutrophil iodination reaction, which examines the ability to produce oxygen radicals and the release of myeloperoxidase enzyme, was determined by the method described by Thong and Ferrante . Briefly, 25 μl of 125I (200 μCi/ml) was added to 1 ml pooled human serum (1:4 dilution in HBSS) and 25 ml added to appropriate wells in a 96-well microtiter plate. Six wells were used for either pooled serum or autologous serum, three of which were stimulated with 50 μl zymosan. As a control, 50 μl of HBSS was added to the remaining three. The plates were incubated for 30 min at 37°C in a humidified CO2 (5% CO2 in air) and then 50 μl of 1 × 107 neutrophils/ml were added to appropriate wells. The plates were covered and incubated for 90 min. Finally, the cells were harvested using a cell harvester (Titertek Cell Harvester 550) and the quantity of bound 125I was expressed as picomoles/107 neutrophils. The amount of iodination due to stimulation was calculated by subtracting the basal iodination value (no zymosan added) from the stimulated iodination value (plus zymosan).
Lymphocyte phenotyping and leukocyte numbers
Lymphocyte subpopulations were determined using a lymphocyte kit and direct two-color immunofluorescence. The kit allows for determination of all T cells (CD3+/CD4+ and CD3+/CD8+), monocytes (CD16+), NK cells (CD3-/CD16+/56+) and B lymphocytes (CD19+) in one sample simultaneously. One hundred microliters of whole-blood samples were mixed with 2 ml of 1× lysing solution, vortexed and incubated for 10 min at room temperature in the dark. Immediately after incubation the tubes were centrifuged for 5 min at 300 g, the supernatant was removed and the pellet resuspended in 2 ml Isoton II. The cells were then centrifuged (300 g × 5 min), the supernatant was removed and the pellet fixed in formaldehyde (200 μl 1% solution). The fluorescence intensity was measured using a flow cytometer (FACScan, BD Biosciences, NSW, Australia). The data was processed with Lysis II software (BD Biosciences). To determine leukocyte and lymphocyte numbers, 130 μl of whole blood was aspirated and analyzed using a hematology analyzer CELL-DYN 3500SL (Abbott Diagnostics, North Chicago, IL).
Measurement of lymphoproliferation
Mononuclear cells in samples of 100 μl (2 × 105 cells) were cultured in medium supplemented with 4 mM L-glutamine solution, 100 U/ml penicillin, 100 μg/ml streptomycin, 5% human heat-inactivated AB serum and 100 μl PHA (2 μg/ml). The final volume of the culture was 200 μl, and all cultures were performed in triplicate as described previously . Proliferation was measured as the incorporation of [3H]thymidine over the final 6 h of a 72-h culture period.
Measurement of cytokine production
Mononuclear cells were cultured in concentrations and conditions as described above, the culture medium was removed after 72 h and stored at -70°C for cytokine (LT, IL-2 and IFNγ) analysis by enzyme-linked immunosorbent assay .
All statistical analyses were performed using GraphPad InStat software. Data were analyzed as comparisons between placebo and n-3 PUFA-supplemented groups, as well as correlations between the membrane fatty-acid levels and specific immunological parameters. The Kolmogorov-Smirnov test was used to determine normal distribution of data. Linear regression analyses were performed and statistical comparisons were carried out using Student's two-tailed t-test for paired or unpaired data and p < 0.05 was considered significant.