Blood samples were taken from the antecubital vein into suitable tubes (Vacuette, Greiner) using minimal stasis after a 12-hour fast while the subjects were seated (after a 15-min rest). Samples for the isolation of lipoproteins were taken into EDTA-containing tubes, immediately placed in ice, and plasma was separated after centrifugation (Heraeus, 2000 × g, + 4°C). EDTA-plasmas were supplemented with sucrose (0.6% w/v final concentration). All samples were kept frozen at -70°C until analyzed. Fasting blood glucose concentration was determined from capillary blood using Hemocue Glucose Analyzer (Hemocue, Ängelholm Sweden).
Plasma cholesterol, HDL cholesterol, triacylglycerol, apoA-I and apoB concentrations were measured with Cobas Integra 700 automatic analyzer using reagents and calibrators as recommended by the manufacturer (Roche Diagnostic, Basel, Schwitzerland). LDL cholesterol was calculated according to Friedewald.
For the accurate assessment of serum MMP-9, aliquots of sera were removed and stored at -70°C in a freezer that was not in daily use until analysis. Quantification of immunoreactive MMP-9 was carried out by enzyme-linked immunosorbent assay (ELISA) (Diabor Ltd, Oulu, Finland). ELISAs were performed on 96-well microtiter plates using standard protocols. Recombinant MMP-9 was used as standard. The microtiter plate was coated with the monoclonal antibody (code GE-213). The bound proteins from serum and standards were detected with a secondary polyclonal antibody produced in chicken against MMP-9. A peroxidase-labeled anti-chicken-IgG (Chemicon, USA) was used for detection of the bound secondary antibody. O-phenylenediamine (OPD) was used to visualize the peroxidase label. The color formation was measured at 450 nm (Anhos 2000 microplate reader) and calculations were done using a Multicalc program (Wallac, Turku, Finland). The monoclonal antibody recognized both the free MMP-9 and that bound to its inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) [2, 18].
Lipoproteins were fractionated by isopycnic density gradient ultracentifugation. Two ml of plasma was mixed with 4.0 ml of d 1.35 g/l NaCl/KBr solution in a 14 × 95 mm tube (Beckman, Palo Alto, USA) and then successively overlayered with 4.5 ml of a d 1.006 salt solution and 1.0 ml of distilled water. For centrifugation a Beckman SW40 Ti rotor, at 36000 rpm for 40 hours in a Beckman L60 centrifuge at 10°C was used. After ultracentrifugation the contents of the tubes were fractionated with an Isco gradient fractionator (Model 640, Lincoln, USA). The 280 nm absorbance of the effluent was continuously monitored with an Isco UA-5 absorbance detector. The different lipoproteins were well separated by the resulting slightly curving salt gradient. The fractions belonging to LDL, HDL2 and HDL3 were pooled on the basis of the absorbance curve.
The total fatty acid compositions of the ultracentrifugally isolated LDL, HDL2 and HDL3 particles were analyzed by gas-liquid chromatography. Lipids were extracted with chloroform/methanol, partitioned and the chloroform phase was dried under N2. The lipids were then hydrolyzed and transesterified with H2SO4 in dry methanol at 85°C for 2 h under N2. Following the addition of water, methyl esters of the fatty acids were extracted with petroleum ether and analyzed in a Shimadzu GC-14A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) with a flame ionization detector using a Supelco SP 2560 capillary column (100 m, 0.25 mm I.D., 0.20 μm film thickness). The carrier gas was helium. The column temperature was 180°C for 15 min, then programmed to increase at 3°C/min to 230°C and held for 40 min. The individual fatty acids were identified with the aid of a standard mixtures of methyl esters (Lipid standards 189-15 and 189-17, Sigma). The areas were measured with a Shimadzu C-R4A Chromatopac Integrator and the results expressed as percentages of the sum of all fatty acids from 14:0 to 22:6n-3. As a control sample we used a pool of isolated HDL that was suitably diluted and kept frozen at -70°C. The inter assay coefficient of variation for the percentage of different fatty acids ranged from 0.3 to 4.4 %.