4.2.1 Assessments of dietary intake
Four food records from four consecutive days including one weekend day were collected (Figure 9) and checked by a clinical nutritionist at return. The food records were analyzed by AivoDiet nutrient calculation software (v. 2.0.2.1, Aivo Finland, Turku, Finland) based on national and international analyses, and international food composition tables. Habitual fish consumption was assessed with fish consumption questionnaire prior to intervention. In addition, consumption records regarding the intakes of fish and CSO were collected during the study.
4.2.2 Fatty acid composition of blood lipid fractions
Blood samples for fatty acid analysis were taken into 9 ml Vacuette EDTA (455036) tubes. Plasma was separated by centrifugation at 1000g for 10 min (4 °C). Plasma samples were stored in –80 °C. Lipids were extracted from the fasting plasma sample with chloroform-methanol (2:1). Cholesteryl nonadecanoate (Nu Chek Prep Inc., Elysian, MA, USA), trinonadecanoin and phosphatidylcholine dinonadecanoyl (Larodan Fine Chemicals, Malmö, SWE) served as internal standards, and they were added to plasma samples before chloroform-methanol extraction. An aminopropyl column was used to separate the lipid fractions (209). Fatty acids in PL, CE and TG were transmethylated with 14% borontrifluoride in methanol at 100 °C for 1 h.
Erythrocytes were separated from EDTA-blood by centrifugation at 1000g for 10 min (4 °C) and hemolyzed in the tris–HCl buffer (pH 7.6, 10 mmol/l). EM were prepared by centrifugation of hemolysate at 30000g for 30 min at 4 °C. Membrane sediment was resuspended in 0.5 ml of distilled water. After mixing 0.1 ml of membrane suspension and 2 ml of methanol–toluene (4:1, v:v) in a glass tube, 0.2 ml of acetyl chloride was slowly added and this mixture was incubated at 100 °C for 1 h. After cooling in cold water 5 ml of 6% K2CO3 was carefully added and then vigorously shaken. Toluene was separated into an upper phase by centrifugation at 2000g for 5 min. Nonadecanoic acid methyl ester served as an internal standard and it was added to toluene extract. Finally, fatty acids in EM, PL, CE and TG were analyzed by 7890 A gas chromatograph (Agilent Technologies, Inc., Wilmington, DE, USA) equipped with a 25-m FFAP column (Agilent Technologies, Wilmington, DE). The fatty acid composition of blood lipid fractions are expressed as mol% of total fatty acids. In
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addition, serum concentration of DHA was determined using NMR spectroscopy (129,210).
4.2.3 Lipoprotein subclasses
Blood samples for lipoprotein subclass analysis were taken to 6 ml Vacuette Z serum (456089) tubes and kept in room temperature for at least 30 minutes. To separate serum, samples were then centrifuged at 2400g in room temperature for 10 min.
Lipoprotein particle concentration and size were measured from the serum samples using high-throughput proton NMR spectroscopy (129,210). Lipoprotein subclasses were defined by particle diameter as follows: chylomicrons and largest VLDL particles (average particle diameter at least 75 nm); five different VLDL subclasses:
very large VLDL (average particle diameter of 64.0 nm), large VLDL (53.6 nm), medium VLDL (44.5 nm), small VLDL (36.8 nm), and very small VLDL (31.3 nm);
IDL (28.6 nm); three LDL subclasses: large LDL (25.5 nm), medium LDL (23.0 nm), and small LDL (18.7 nm); and four HDL subclasses: very large HDL (14.3 nm), large HDL (12.1 nm), medium HDL (10.9 nm), and small HDL (8.7 nm). The following components of the lipoprotein particles were quantified: PL, TG, cholesterol, free cholesterol and CE. The mean size for VLDL, LDL and HDL particles were calculated by weighting the corresponding subclass diameters with their particle concentrations.
4.2.4 Binding of lipoproteins to proteoglycans
Proteoglycan binding was assessed in 96-well plates coated with proteoglycans isolated from human aortas as previously described (211). 1 µl of plasma from study subjects was added to the wells and incubated for 1 h. The amount of cholesterol bound to the wells was measured with Amplex Red cholesterol assay kit.
4.2.5 Isolation of LDL and HDL
LDL (d=1.019–1.063 g/ml) and HDL (d=1.063–1.210 g/ml) were prepared from isolated plasma of study subjects by sequential flotation ultracentrifugation using D2O for density adjustment (212).
4.2.6 LDL aggregation
LDL stability was determined by inducing LDL aggregation with human recombinant sphingomyelinase (182). LDL aggregation was followed by measuring the particle size using dynamic light scattering.
4.2.7 Endothelial cell activation by LDL
Human coronary artery endothelial cells (HCAECs) (PromoCell) were cultured in Endothelial Cell Growth Medium MV supplemented with 5% fetal calf serum, 0.4%
55 endothelial cell growth supplement, 10 ng/ml epidermal growth factor, 90 µg/ml heparin, 1 µg/ml hydrocortisone (PromoCell), 100 U/mL penicillin streptomycin solution, and 50 ng/ml amphotericin B to yield Complete Medium in T-75 flask according to the manufacturer's instructions. Confluent HCAECs were washed with 15 ml of PBS, trypsinized, and replated in complete medium, as described below.
Endothelial cell activation was measured in HCAECs between passages 4 and 7.
HCAECs were seeded at a density of 1-2 x 104 cells/well in 96- well plate and cultured for 2–3 days until the cells reached 90–95% confluency. To determine the effect of LDL on IL-8 secretion, HCAECs grown in Complete Medium were washed, placed in serum-free medium and incubated with LDL of the study subjects (50 µg protein/ml) for 6 h. The amount of IL-8 released into the media was measured by an IL-8 ELISA Kit (R & D Systems, Minneapolis, Minnesota, USA).
4.2.8 Cholesterol efflux from macrophage foam cells
Human monocytes were isolated from buffy coats (Finnish Red Cross Blood Transfusion Center, Helsinki, Finland) by centrifugation in Ficoll-Paque gradient as described (213). Washed cells were suspended in DMEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, counted, and seeded on 24 well-plates (1.5 million cells per well). After 1 h of incubation, non-adherent cells were removed, and the medium was replaced with macrophage-SFM medium (Gibco) supplemented with 1% penicillin-streptomycin and 10 ng/ml of granulocyte macrophage colony-stimulating factor (GM-CSF) (Biosite, San Diego, USA). The cells were cultured for 7 days in the presence of GM-CSF to allow them to differentiate into GM-CSF macrophages. The medium was then changed every 2 to 3 days throughout the culture period.
The monocyte-derived macrophages were incubated in DMEM (pH 7.4) containing 25 µg/ml of [3H]CE-acetyl-LDL for 24 h to induce the formation of foam cells. To measure cholesterol efflux, macrophages were washed with PBS, and fresh media containing the various HDL of study subjects (25 µg protein/ml) were added.
After incubation for 16 h, the media were collected, centrifuged at 300g for 10 min to remove cellular debris, and the radioactivity in the supernatants was determined by liquid scintillation counting. Cells were solubilized with 0.2 M NaOH and analyzed for radioactivity. Cholesterol efflux was expressed as the percentage radioactivity in the medium relative to the sum of total radioactivity present in the medium and the cells. Cholesterol efflux to the incubation medium in the absence of HDL was considered as basal efflux and was subtracted from the efflux values obtained in the presence of HDL. In an initial experiment using THP-1 cells (human monocytes), a small increase in cholesterol efflux was observed in the fatty fish group but not in other groups. Therefore, the fatty fish group was selected to determine cholesterol efflux capacity using human primary macrophages, a relevantly physiological cell type.
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4.2.9 SAA and apoE concentrations
Plasma apoE was assessed using ELISA Development Kit (catalog number 3712-1A-6; Mabtech, Nacka Strand, Sweden). SAA in serum was measured using Invitrogen SAA ELISA Kit (Cat nr. KHA0011, KHA0012 or KHA0011C; Thermo Fisher Scientific).
4.2.10 Other assessments
The subjects visited the study clinic at 0, 2, 4, 8 and 12 weeks. At each visit height and body weight were measured with an electronic scale while the subjects were in light clothing. BMI was calculated as body weight (kg) divided by body height squared (m2). At the same visits blood pressure was measured in sitting position after 5 min of rest. All anthropometric and blood pressure measures were performed by a study nurse. Data regarding physical activity, alcohol intake, smoking, body weight and use of medication were recorded by a questionnaire at week 0 and week 12.
Blood samples were drawn after a 10-hour overnight fasting from an antecubital vein. Blood samples for serum lipid analysis were taken to 6 ml Vacuette Z serum (456089) tubes and kept in room temperature for at least 30 minutes prior to centrifugation. To separate serum, samples were then centrifuged at 2400g in room temperature for 10 min. These samples were stored in +4 °C for up to one week. Blood samples for plasma glucose were taken to 3 ml Venosafe VF-053 SFC tubes and stored in –80 °C. OGTT with 75 g D-glucose, serum lipid profile and concentrations of apolipoproteins were analyzed as previously described (214).