Female patients with stable AP (n=22) were selected using the following criteria: the presence of chest or arm discomfort that is rarely described as pain, but is reproducibly associated with physical exertion or stress and relieved within 5–10 min of rest and/or administration of sublingual nitroglycerin. The diagnosis was confirmed with a treadmill exercise test and coronary angiography in all patients. Patients did not take any medications, except for statins, prior to hospitalization. Age- and gender-matched reference subjects (n=20) were recruited from healthy volunteers who underwent regular health evaluations at the Health Center of Yeungnam University Hospital (Daegu, Korea). They had unremarkable medical records without a history of endocrinological disorders. Heavy alcohol consumers (>30 g EtOH/day) and those who had taken prescribed drugs to treat hyperlipidemia, diabetes mellitus, or hypertension were excluded. Informed consent was obtained from all patients and the control group prior to enrollment in the study. The Institutional Review Board at the Medical Center of Yeungnam University approved the protocol.

After overnight fasting, blood was collected using a vacutainer (BD Bio Sciences, Franklin Lakes, NJ, USA) containing EDTA (final concentration, 1 mM). Plasma was isolated by low-speed centrifugation and stored at −80˚C until analysis.

Very low-density lipoproteins (VLDL, d<1.019 g/ml), LDL (1.019<d<1.063), HDL₂ (1.063<d<1.125) and HDL₃ (1.125<d<1.225) were isolated from individual patient and control sera via sequential ultracentrifugation (16), with the density adjusted by the addition of NaCl and NaBr in accordance with standard protocols. Samples were centrifuged for 24 h at 10˚C at 100,000 × g using a Himac CP 90α (Hitachi, Tokyo, Japan) at the Instrumental Analysis Center of Yeungnam University.

For each of the lipoproteins which were individually purified, total cholesterol (TC) and TG measurements were obtained using commercially available kits (cholesterol, T-CHO, and TG, Cleantech TS-S; Wako Pure Chemical, Osaka, Japan). The protein concentrations of lipoproteins were determined via the Lowry protein assay, as modified by Markwell et al (17) using the Bradford assay reagent (Bio-Rad, Seoul, South Korea) with bovine serum albumin (BSA) as a standard. To assess the degree of oxidation of individual LDL, the concentration of oxidized species in LDL was determined by the thiobarbituric acid reactive substances (TBARS) method using malondialdehyde (MDA) as a standard (18).

The ferric reducing ability of plasma (FRAP) was determined using the method described by Benzie and Strain (19) with a slight modification, as described previously (20). The antioxidant activities of the individual HDL fractions (20 μg each) were then estimated by measuring the increase in absorbance induced by the generated ferrous ions.

Cholesteryl ester conversion was performed via lecithin:cholesterol acyltransferase (LCAT) assays, as previously described (21). An equal amount of individual lipoproteins (in 50 μl) from each patient was utilized as the enzyme source. ApoA-I-rHDL containing radiolabeled cholesterol (1 μCi of [¹⁴C]-4-cholesterol/69 μg of cholesterol/1 mg of apoA-I) was used as a substrate, and the apoA-I was then expressed using an E. coli expression system, as described previously (21). Discoidal rHDL was prepared via the sodium cholate dialysis method using initial molar ratios of palmitoyloleoyl phosphatidylcholine (POPC)-cholesterol-apoA-I-sodium cholate at a ratio of 95:5:1:150 (wt/wt/wt/wt). The reaction was initiated by the addition of individual serum, and the mixture was then incubated for 1 h at 37˚C. Next, the esterified cholesterol and free cholesterol were separated via thin layer chromatography, and the activity was expressed as the percentage conversion rate of cholesteryl ester from free cholesterol.

An rHDL-containing apoA-I and cholesteryl oleate was synthesized in accordance with the method described by Cho (20) using trace amounts of [³H]-cholesteryl oleate (TRK886, 3.5 μCi/mg of apoA-I; GE Healthcare) with a slight modification (22). The CE-transfer reaction was allowed in 300 μl reaction mixtures that contained equal amounts of the individual lipoproteins (20 μl, 10–20 μg of protein) as a cholesteryl ester transfer protein (CETP) source and rHDL-agarose (50 μl, 0.25 mg/ml) and human LDL (50 μl, 0.25 mg/ml) as a CE-donor and CE-acceptor, respectively. After incubation at 37˚C, the reaction was halted via brief centrifugation (10,000 × g) for 3 min at 4˚C. The supernatant (150 μl) was then subjected to scintillation counting, and the percentage transfer of [³H]-CE from rHDL to LDL was calculated.

Paraoxonase-1 (PON-1) activity toward paraoxon was determined by evaluating the hydrolysis of paraoxon into p-nitrophenol and diethylphosphate, which was catalyzed by the enzyme (23). PON-1 activity was then determined by measuring the initial velocity of p-nitrophenol production at 37˚C, as determined by measuring the absorbance at 405 nm (microplate reader, Bio-Rad model 680; Bio-Rad, Hercules, CA, USA), as described previously (13).

The individual lipoprotein fractions (10 μl, 20 μg) were used as an enzyme source for the PAF-AH reaction with an Lp-PLA₂ assay conducted according to the method described by Boyd et al (24). Briefly, [³H]-platelet activating factor (hexadecyl-2-acetyl sn-glyceryl-3-phosphorylcholine, NET910, 0.1 mCi/ml; Perkin-Elmer Life and Analytical Sciences, Boston, MA, USA) and 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine were used as substrates for the reaction. A substrate solution containing 10 μl of [³H]-PAF (1 μCi, 50 μM) and 12 μM of cold PAF was incubated using each HDL solution as a source for 30 min. The reaction was then stopped by vortexing the solutions with 600 μl of CHCl₃:MeOH (2:1, v/v), after which the aqueous layer (150 μl) was removed. The aqueous layer was then vortexed again with CHCl₃, after which it was centrifuged and the upper phase was used for scintillation counting.

In order to compare the electromobility of the patient and control samples, the migration of each lipoprotein (LDL, HDL₂ and HDL₃) was evaluated by agarose electrophoresis. The gels were then dried and stained with 0.125% Coomassie Brilliant Blue, after which the relative band intensities were compared via band scanning using Gel Doc^® XR (Bio-Rad) with Quantity One software (version 4.5.2).

The apolipoprotein/lipoprotein compositions were compared via sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) with identical protein loading quantities (6 μg of total protein per lane) from individual HDL₃, and the levels of expression of apolipoprotein were analyzed via immunodetection. Anti-human apoC-III antibody (AB821) was purchased from Chemicon (Temecula, CA, USA). Anti-human CETP antibody (ab19012) and LCAT antibody (ab786) were purchased from Abcam (Cambridge, UK). The relative band intensity (BI) was compared via band scanning with Gel Doc^® XR (Bio-Rad) using the Quantity One software (version 4.5.2).

All data are expressed as the mean ± SD from at least three independent experiments with duplicate samples. Data comparisons were assessed by the Student’s t-test using the SPSS program (version 14.0; SPSS, Inc., Chicago, IL, USA).

The serum TC concentrations were similar between the groups (204±57 and 200±32 mg/dl, respectively), which remained in the normal range, as suggested by the guidelines of the National Cholesterol Education Program (NCEP)-adult treatment panel (ATP)-III. The LDL-C level was similar between the groups (105±38 and 108±33 mg/dl for the AP patients and controls, respectively). However, the HDL-C level was slightly lower in the AP patients than the controls. The ratio of HDL-C-to-TC was significantly lower in the AP patients (34±2%) compared to the control group (40±4%). The serum TG level was not significantly different between the groups (136–175 mg/dl).

Properties of lipoprotein are good biomarkers which reflect the progress of cardiovascular and renal disease. As shown in Table I, the AP group had a similar composition of lipid and protein in VLDL with the control group. Although the TC and protein content in LDL was similar between the groups, the TG content in LDL was significantly higher in the AP group (38 mg of TG/mg of TP) compared to the control group (30 mg of TG/mg of TP). In the HDL₂ fraction, the AP group had a much lower TC content and a higher TG content (44% higher TC and 32% lower TG) than the control group. Immunodetection revealed that the level of expression of apoC-III was elevated in the HDL₃ fraction of the AP group (Fig. 1).

As shown in Table II, although the CE-transfer activity of the VLDL fraction was similar between the groups (~2–3% CE-transfer), the LDL fraction of the AP group had 2-fold increased CE-transfer activity. The CETP activity of the HDL fraction also increased in the AP group (a 70 and 34% increase for HDL₂ and HDL₃, respectively), compared to the control. Immunodetection revealed that CETP was highly expressed in the HDL₃ fraction of the AP group (Fig. 1).

The LCAT activity was significantly lower in the HDL₃ fraction of the AP group, while no difference existed in the HDL₂ fraction between the groups. The LCAT activity for CE-conversion from FC was lowered in the HDL₂ fraction in the AP and control groups, as shown in Table II. The level of expression of LCAT was nearly undetectable in the AP group (lane 1–5) except in one patient (Fig. 1).

The HDL₃ from the AP group had weaker antioxidant activity (172% increase from the initial level) than the control group (198% increase) when the same amount of protein in HDL (1.5 mg/ml) was used as an antioxidant source (Fig. 2A). The extent of oxidation in the native state was compared by relative electrophoretic mobility on 0.7% agarose gel electrophoresis. The HDL₂ from the AP group migrated faster than the control group without cupric ion treatment, indicating that HDL₂ of the AP group was more oxidized in the native state (Fig. 2B). More highly oxidized HDL has a faster mobility due to a smaller particle size and an increase in charge. In particular, HDL₂ from the AP group was 2-fold more susceptible to cupric ion-mediated oxidation, as shown in Fig. 3, indicating that the antioxidant potential was significantly decreased in the AP group. Specifically, electron microscopy revealed that HDL₂ from the AP group had a smaller particle size than the control; HDL₂ from the AP group was 18–20 nm in width and length, while HDL₂ from the control group was 22–25 nm in width and length. These results suggest that more highly oxidized HDL has faster electromobility and reduced particle size.

The HDL₂-associated PON activity was lower in the AP group than in the control group (112±10 vs. 164±25 μU/mg of protein) (Fig. 4A). Moreover, the AP group had a 3-fold lower HDL₃-associated PON activity than the control group (109±16 vs. 561±36 μU/mg of protein, respectively).

Although there was no significant difference in the HDL₂ fraction used as the PAF-AH source, the activity was significantly lower in the AP group when the HDL₃ fraction was used (Fig. 4). HDL₃ from the AP group showed 40% less activity than the control group (15±2 and 26±3 pmole PAF/h/mg of protein for the AP and control groups, respectively).

The LDL from the AP patient had ~1.8-fold higher levels of MDA than the control without cupric ion treatment, indicating a greater extent of oxidation of LDL in the AP group in the native state (Fig. 5). Under treatment with cupric ion (final 10 μM), the LDL from the AP and control group showed 2.3 and 1.1 nmole of MDA, respectively, suggesting that the AP group LDL was more sensitive to cupric-ion mediated oxidation.