Methods

We enrolled 96 consecutive patients with HF with reduced left ventricular ejection fraction (LVEF) followed at the Ahmanson-UCLA Cardiomyopathy Center who met the inclusion criteria. The protocol was reviewed by the Institutional Review Board at this institution, and informed consent approved by the Institutional Review Board was obtained from all participating patients. The inclusion criteria for enrollment were the presence of (1) symptomatic HF for 3 months (New York Heart Association classes II to IV) or asymptomatic HF with a history of symptoms within the past year, (2) LVEF <40%, as documented by echocardiography, radionuclide ventriculography, gatedsingle-photon emission computed tomography, or contrast ventriculography within the past 6 months as performed as part of routine care, and (3) age ≥18 years. Any patients with major infection or cardiovascular procedure within 3 months of enrollment were excluded from the study. Twenty-one healthy subjects (9 women and 12 men, aged 47.7 ± 3.2 years) served as controls. Subjects exhibiting acute or chronic infection or acute intercurrent illnesses were excluded. Medical records of each participating patients were reviewed by a physician, and medical history was recorded at the time of enrollment. Serum total cholesterol, low-density lipoprotein (LDL) cholesterol, HDL cholesterol, and triglyceride (TG) levels as well as other laboratory tests including chemistry and those for biomarkers beta natriuretic peptide and high-sensitivity CRP were performed.

For determination of HDL inflammatory index (HII), fasting blood was collected in heparinized tubes (Becton Dickinson, Franklin Lanes, New Jersey). Sucrose solution (50%) was added (to protect LDL and HDL from freeze-thaw) at a ratio of 1 volume sucrose to 4 volumes of plasma, thoroughly mixed, divided into aliquots, and kept frozen at −80°C until use. HII was measured for 96 patients with HF and 21 control subjects. All personal identifiers were removed. The lipoproteins were isolated by fast-performance liquid chromatography (FPLC), and culture of human aortic endothelial cells, and monocytes were prepared as described previously. Briefly, monolayers of human aortic endothelial cells were treated with LDL (100 μg/ml) in the absence or presence of the subjects’ HDL (50 μg/ml) for 18 hours. The cultures were subsequently washed and a fresh culture medium without LDL or HDL was added and incubated for 8 additional hours. The collected culture supernatants were assayed for monocyte chemotactic activity (MCA) as described previously. Briefly, monocytes were isolated from the blood of healthy donors. The culture supernatant to be tested for MCA was placed in a unit in a well below a 5-micron pore membrane in a chemotaxis chamber. Monocytes were added to the well above the membrane and the unit was incubated at 37°C for 1 hour. Monocytes that migrated to the lower aspect of the membrane were enumerated in high-power fields using an image processing system. Induction of MCA by a standard control LDL was determined in the absence or presence of the subject’s HDL. The value obtained in the absence of HDL was taken as 1.0. With HDL present together with LDL, values that were >1.0 indicated the presence of proinflammatory HDL and values <1.0 indicated the HDL as being anti-inflammatory.

LDL inflammatory index (LII) was obtained in the plasma samples from 96 patients with HF and 21 controls. The subject’s LDL from plasma was added to the human aortic endothelial cell cultures and incubated and processed as described previously for determination of HII. Briefly, LDL added to artery wall cells at 100 μg of LDL cholesterol per milliliter in the absence of HDL underwent lipid oxidation and stimulated the induction of MCA. The culture supernatant was assayed for MCA and the resulting value was divided by the MCA obtained for the control internal standard LDL used at 100 μg LDL cholesterol/ml. Plasma inflammatory index was determined using plasma samples diluted with saline solution to 2,500-fold. Diluted plasma was added to artery wall cell cultures in a manner similar to that used for LDL and the plasma inflammatory index was determined.

HDL mimetic peptide 4F is an 18–amino acid peptide that was made to mimic the properties of apoA-I, the main protein of HDL, and was made more effective by having 4 phenylalanines on the hydrophobic surface, and thus it is called 4F. It has been demonstrated that oral 4F improves the HII in patients with documented coronary atherosclerosis. To determine if the HDL mimetic peptide is capable of improving the patients’ LDL or HDL in vitro, additional aliquots of the plasma samples were treated with 1 μg/ml of 4F or with saline solution for 15 minutes at 37°C and spin filtered to eliminate the peptide followed by isolation of LDL and HDL using FPLC fractionation and determination of LII and HII. Serum amyloid A (SAA) levels were determined using a human SAA enzyme-linked immunosorbent assay kit (KHA0012; Invitrogen, Carlsbad, California).

Twenty-one plasma samples from controls and twenty-one randomly selected patient plasma samples were subjected to FPLC, and the HDL-containing fractions were used for determination of paraoxonase (PON) activity. The HDL samples were assayed for PON activity using paraoxon as the substrate. The cuvette contained 1.0 mM of paraoxon in 20 mM tris-HCl, pH 8.0. The reaction was initiated by the addition of the plasma or lipoprotein sample, and the increase in the absorbance at 405 nm was recorded over a 90-second period.

The content of free 5-hydroxyeicosatetraenoic acid (HETE), 9-HETE, 12-HETE, 9-hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, and other lipid mediators were determined by LC-ESI-MS/MS. Briefly, 50 μl of HDL or albumin solution obtained by FPLC containing 50 μg of protein was transferred to a 13 × 100 mm glass tube, 1.9 ml of pH 2.3 water was added and spiked with 50 μl of internal standards mixture (including 5(S)-HETE-d8, 12(S)-HETE-d8, 15(S)-HETE-d8, 13(S)-hydroxyoctadecadienoic acid-d4, PGD2-d4, 8(9)-EET-d11, and arachidonic acid-d8, 10 ng/ml each) in methanol. The resulting sample was loaded onto a preconditioned 1-ml Oasis HLB solid-phase extraction cartridge on a vacuum manifold (Waters, Milford, Massachusetts). The solid-phase extraction cartridge was equilibrated with 1-ml methanol followed by 1-ml water before loading the sample. Analytes were subsequently eluted with 1-ml methanol. The eluate was evaporated to dryness under a stream of argon. One hundred microliter of methanol was added to the dried extract, vortexed for 30 seconds, and the reconstituted extract was centrifuged at 13,200 rpm for 20 minutes at 4°C to remove any precipitate that could clog the LC-MS/MS instrument. The resulting supernatants were transferred to autosampler vials and processed for LC-MS/MS analysis.

For statistical analysis, the subjects with HF were divided into lower- and higher-HII groups, with the median HII (1.65) used as criteria for cutoff. Independent samples t test, Pearson chi-square, and nonparametric tests as appropriated were used to detect significant differences in baseline characteristics among lower- and higher-HII groups. The same methods were used to assess differences between patients with HF and controls without HF. Stepwise linear regression analysis was performed to assess whether New York Heart Association functional class and LVEF was associated with the HII level. The variables included in the stepwise regression analysis were lipids levels (total cholesterol, LDL, HDL, and TG) and age. The model coefficients (β) for different variables are included in Supplementary Table 1 . For Tables 1 and 2 , independent samples t tests were performed for continuous parametric variables, chi-square tests were performed for categorical variables, and independent samples Mann-Whitney U tests were performed for nonparametric variables.

Table 2

Baseline characteristics of normal subjects versus patients with heart failure

Variable	Group	Mean	SD	p
Age (yrs)	Control	47.7	3.2	<0.0001
	HF	54.9	13.6
Total cholesterol (mg/dl)	Control	170.1	18.8	0.82
	HF	171.7	48.5
HDL (mg/dl)	Control	58.5	10.3	<0.0001
	HF	42.9	11.9
LDL (mg/dl)	Control	95.7	16.9	0.93
	HF	96.1	39.4
TG (mg/dl)	Control	80.5	30.6	<0.0001
	HF	187.8	154.4

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