Healthy male subjects with plasma HDL cholesterol levels < 10th percentile (low HDL group, n = 10) and healthy age-matched male subjects with plasma HDL cholesterol levels > 90th percentile (high HDL group, n = 10), were recruited for endotoxin challenge . In addition, normolipemic males (n = 4; HDL cholesterol levels 1.4 +/- 0.3 mM) were recruited as controls for a vehicle bolus. Written informed consent was obtained from all participants and the study protocol was approved by the institutional review board of the Academic Medical Center.
The study was performed as described . In brief, study participants were required to refrain from alcohol and caffeine-containing beverages at least 24 hours before the study. On the morning of the study day at 7:30 AM after an overnight fast, study participants were admitted to the research unit. At 7.45 AM a catheter was inserted in an antecubital vein of each arm. At 8.00 AM (time [t] 0), blood was drawn for baseline measurements. Subsequently, subjects received a bolus infusion of 1 ng/kg body weight of endotoxin (Escherichia coli lipopolysaccharide, catalog number 1235503, lot G2B274; United States Pharmacopeial Convention Inc, Rockville, MD) in the antecubital vein of the contralateral arm. Healthy controls received a vehicle bolus to control for any diurnal effects. Blood samples were collected at t = 0, 1, 2.5, 4, 6, and 8 hours after endotoxin challenge. The next morning at 8.00 AM, 24 hours after endotoxin infusion, study participants returned after an overnight fast for a final blood withdrawal.
Blood was collected in EDTA or heparin, kept on ice, and centrifuged at 2000g for 20 minutes at 4°C. Plasma was snap-frozen and stored at -80°C until analysis.
Inflammatory marker protein measurements
Serum amyloid A (SAA) ELISA was carried out using a kit from Anogen biotech Laboratories (Mississauga, Ontario, Canada). PON-1 activity measurements were performed in heparinized plasma as described previously .
Sample preparation and HDL capture
SELDI-TOF MS analysis was performed as described . Complete time series of plasma of each participant was used for analyses on separate spots on PS20 chips (in triplicate). In short, native HDL was directly captured from 100 μL diluted EDTA plasma (1:2 diluted with Tris Buffered Saline pH 7.4) which was incubated on chips coated with antibodies against apo A-I for 2 hours at room temperature on a horizontal shaker (600 rpm). The chips were subsequently washed 4 times with TBS for 10 minutes, followed by a 5 minute TBS-Tween (0.005%) rinse and a final wash step with Hepes solution (5 mM). All spots were allowed to dry and subsequently 1.2 μL sinapinic acid (10 mg/ml in 50/49.9/0.1% acetonitril/H2O/TFA) was applied to each spot.
SELDI-TOF analysis and data preprocessing
Spectra were recorded at two laser intensities of 203 to 210 relative units and the focus mass was set to 28 kDa. Spectra were generated with approximately 100 shots at 13 positions per SELDI spot. Analysis was carried out using a PBS IIc protein chip reader (Ciphergen Biosystems, Fremont, CA, USA) using an automated data collection protocol of the Protein-Chip Software (version 3.1.1.). Data were collected up to 150,000 kDa. Calibration was performed using a protein calibration chip (Bio-Rad). All spectra were automatically corrected for baseline values and spot to spot correction was assessed by the Ciphergen protein chip software. Finally, since the HDL-capture design was based on a 100% chip saturation with HDL particles, all spectra were normalized on apo A-I signal.
HDL isolation for two-dimensional gel electrophoresis (2-DE), protein quantification and identification
HDL isolation of six subjects, three representing the high HDL and three the low HDL groups, was performed before (t = 0) and after (t = 8) LPS challenge as described previously . In brief, 5 mL of EDTA-plasma, adjusted to a density of 1.24 g/mL with solid KBr (0.3816 g/mL) was layered in the bottom of a centrifuge tube. The plasma fraction was then overlaid with KBr/phosphate solution (0.083 4 g/mL, density 1.063 g/mL). Ultracentrifugation was performed in a Beckman XL-90 equipped with a Ti 70 rotor at 290 000*g for 4 hours at 15°C. The fractions corresponding to HDL2 and HDL3 were collected with a syringe and a second ultracentrifugation step (KBr/phosphate solution, density of 1.24 g/mL) was performed for 2 hours to eliminate the presence of plasma contaminants. HDL proteins were desalted using PD-10 columns and protein concentration was measured using Bio-Rad protein assay. 2-DE was performed using IPGphor and Multiphor, Amersham Biosciences. HDL proteins (300 μg) were applied by in gel rehydration for 12 hours at low voltage (30 V) in pH 3-10NL IPGs strips. Proteins were then focused at 53000 Vh at max voltage of 8000 V. The second dimension was performed by transferring the proteins to a homogenous gel (T = 14%, C = 1.5%) running at 40-800 V, 10°C, 20-40 mA overnight. Separated proteins were stained by Sypro Ruby (Molecular Probes, USA). Images of the protein patterns were analyzed by a CCD camera in a UV-scanning illumination mode (Flour-S Multi-Imager, Bio-Rad, USA) combined with a computerized imaging 12-bit system (PDQuest 2-D gel analysis software, Bio-Rad).
Proteins were quantified as fluorescence intensity (counts) per total fluorescence on the 2-D gels, expressed in %. Proteins were identified by peptide mass fingerprinting of tryptic peptides using Matrix Assisted Laser Desorption/Ionization Time of Flight MS (MALDI TOF MS) (Voyager DE Pro, Applied Biosystems, USA) prior to database searches as described previously.
Bioinformatics and statistics
Prior to statistical analysis all generated spectra were preprocessed as described before . In brief, each individual m/z spectrum was divided into non-overlapping intervals whose sizes are increasing proportionally with the mass over charge (m/z) values. The size of an interval starting at 3000 (m/z) is computed as m.r with r fixed at 0.5% in all our results below. The intensity associated with each interval was taken as the sum of the intensities over the interval. Discretisation of the m/z axis leads to a reduction of the number of variables down to 704 m/z intervals.
Subsequently, a statistical test was performed for removal of the m/z intervals over which the intensity did not significantly change over time. For that purpose, the non-parametric Kruskal-Wallis one-way analysis of variance method was used for testing the equality of population medians among the 7 time points; 104 m/z intervals at the low laser intensity and 77 m/z intervals at the high laser intensity showed a p-value lower than 0.05. Raw p-values were then adjusted for multiple testing using Benjamini and Hochberg's method. This reduced the number of significant intervals down to 21 and 14, respectively. Since the high laser intensity showed less sensitivity, we restricted our analysis to the low laser intensity.
Hierarchical clustering was performed using, for each individual, the mean (of the triplicates) dynamics of all 21 significant markers. Hierarchical clustering was computed using an agglomerative approach with complete linkage and one minus the correlation (1-r) as a distance measure. All analyses were performed with Matlab® (The MathWorks Inc., Natick, MA, USA).