We recruited male patients with AF (n=19; 46±7 years old) and male controls (n=17, 48±9 years old) with similar age. Heavy alcohol consumers (>30 g EtOH/day) and those who had consumed any prescribed drugs to treat hyperlipidemia, diabetes mellitus, or hypertension were excluded. All subjects had unremarkable medical records without histories of endocrinologic disorders. Informed consent was obtained from all patient and subjects prior to enrollment in the study and the Institutional Review Board at the Medical Center of Yeungnam University (Daegu, South Korea) approved the protocol.

Blood was drawn on overnight fasting state in AF patients and control. Blood was collected using a vacutainer (BD sciences, Franklin Lakes, NJ) containing EDTA (final 1 mM). Plasma was isolated by low speed centrifugation and stored at -80℃ until analysis. Blood parameters, lipids, and glucose concentrations were determined using an automatic blood analyzer (Chemistry analyzer AU4500 Olympus, Tokyo, Japan).

VLDL (d<1.019 g/mL), LDL (1.019<d<1.063), HDL₂ (1.063<d<1.1α25), and HDL₃ (1.125<d<1.225) were isolated from individual patient and control sera via sequential ultracentrifugation,10 with the density adjusted by the addition of NaCl and NaBr in accordance with standard protocols. Samples were centrifuged for 24 hours at 10℃ at 100,000xg 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 triglyceride (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.11 using the Bradford assay reagent (BioRad, 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 as a standard.12

In order to compare the extent of glycation between the groups, the content of AGEs in the individual lipoproteins were determined from reading the fluorometric intensities at 370 nm (excitation) and 440 nm (emission), as described previously.13

To compare the susceptibility of copper-mediated LDL oxidation, 300 µg of LDL was incubated with 5 µM CuSO₄ for up to 3 hours. During the incubation, the quantity of formed conjugated dienes was monitored by measuring the absorbance at 234 nm (Abs₂₃₄) at 37℃14 using a Beckman DU 800 spectrophotometer equipped with a MultiTemp III thermocirculator.

In order to verify the spectroscopic data, the oxLDL samples were subjected to electrophoresis on a 0.5% agarose gel for an electromobility comparison.15 The post-oxidative electrophoretic mobility of LDL was compared via electrophoresis on a 0.5% agarose gel because there is some modification of amino acids in the apo-B by the oxidation.

The ferric reducing ability of plasma (FRAP) was determined using the method described by Benzie and Strain16 with a slight modification, as described recently by our research group.17 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.

A rHDL-containing apoA-I and cholesteryl oleate was synthesized in accordance with the method described by Cho et al.1819 using trace amounts of [³H]-cholesteryl oleate (TRK886, 3.5 µCi/mg of apoA-I; GE Healthcare). 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 Colesteryl 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℃, the reaction was halted via brief centrifugation (10,000xg) for 3 minutes at 4℃. 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.20 PON-1 activity was then determined by measuring the initial velocity of p-nitrophenol production at 37℃, as determined by measuring the absorbance at 415 nm (microplate reader, Bio-Rad model 680; Bio-Rad, Hercules, CA, USA), as described previously.21

The apolipoprotein/lipoprotein compositions were compared via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with equal protein loading quantities (3 µg of total protein per lane) from individual HDL₃, and the levels of expression of apolipoprotein were analyzed via immunodetection. Anti-human apoA-I antibody (ab7613) was purchased from Abcam (Cambridge, UK). The relative band intensity (BI) was compared via band scanning with Gel Doc® XR (Bio-Rad) using 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 Student's t-test using the SPSS program (version 14.0; SPSS, Inc., Chicago, IL, USA).

As shown in Table 1, the AF group had slightly higher obese body mass index (27±4 kg/m²) than that of control group (24.7±3 kg/m²) although the blood pressures were in the normal range around 125 mmHg and 75 mmHg for systolic and diastolic blood pressure, respectively. Interestingly, the AF group showed 12% lower serum TC and 15% lower HDL-C than control group. However, AF group showed 1.5-fold elevated serum TG compared with control and TG/HDL ratio in AF group was also increased upto 1.8-fold higher than control group. TC/HDL and LDL/HDL ratios were similar between the groups (4.2-4.1 and 3.0-2.6, respectively). Thyroid stimulating hormone is elevated in the AF group, although there was no difference. The serum glucose level was elevated 19% in the AF group compared to the control group, although it was fall under the normal blood glucose range (<110 mg/dL). AF group had a normal creatinine and blood urea nitrogen levels, while the uric acid level was elevated 40% in the AF group compared to the control group. The AF group had a significantly elevated hsCRP level (2-fold higher than the control group), while hepatic inflammatory parameters (AST and ALT) were in the normal range.

The AF group had much lower FRA activity than the control group, as shown in Fig. 1A, indicating that the ferric ion reduction ability was severely impaired. However, there was no significant difference in PON activity between the groups, as shown in Fig. 1B.

Under the same protein concentration, the cholesterol content in VLDL were similar between the groups, however, TG was 1.5-fold more enriched in AF group. In LDL, cholesterol content was 13% reduced in AF group, while TG content was 2-fold elevated. In HDL₂, cholesterol content was slightly reduced and TG content was 2-fold elevated in AF group. In HDL₃, the TG content in AF group elevated 2-fold more than control, while cholesterol contents were similar between the group.

All lipoproteins from the AF group contained more oxidized species by conjugated diene determination (A₂₃₄), as shown in Fig. 2A and TBARS quantification with the MDA standard (Fig. 2B). From conjugated diene determination, VLDL in AF group showed the highest extent of oxidation, while LDL and HDL from AF group showed slightly higher than control. TBARS assay showed AF group showed higher MDA content in all lipoproteins especially in LDL, 5-fold higher than control.

As shown in Fig. 2C, without cupric ion treatment, the conjugated diene level (A₂₃₄) of AF-LDL was increased after an 80 min incubation at 37℃, while the control-LDL did not show an increase of A₂₃₄. Treatment with cupric ion accelerated more increase of A₂₃₄ in the AF-LDL group after 40 min, while the control-LDL showed an increase of A₂₃₄ after 60 min. AF-LDL was more sensitive to oxidation under with or without cupric ion. Electrophoresis revealed that AF-LDL was more sensitive to the cupric ion-mediated oxidation with time dependent manner since it migrated faster than the control-LDL as indicated by arrow (Fig. 2D).

The AF group showed weaker HDL₃-associated PON activity than the control, while HDL₂-associated PON activity was not detected in both groups (Fig. 3A). Ferric ion removal ability was also lowered in AF-HDL₃ (Fig. 3B). Electrophoretic analysis (15% SDS-PAGE) with HDL₂ revealed that no major different band patterns were found between the groups (Fig. 4A). While the level of apoA-I expression in HDL₃ was not different between the groups, the AF group had a 10% lower level of expression of apoA-I in HDL₂ from Western blot analysis (Fig. 4B). The AF group exhibited a 21% higher level of expression of the paraoxonase level in HDL3 as shown in Fig. 4B.

After a 48-hour incubation, treatment AF-VLDL (16 µg of protein) or AF-LDL (30 µg of protein) resulted in much stronger red intensity than the same amount of control VLDL and LDL, as shown in photo of Fig. 5. This result indicates that VLDL and LDL from the AF group could be more easily taken up into macrophages by phagocytosis. Determination of oxidized species with the culture media revealed that AF-VLDL- and AF-LDL-treated media contained 40% and 23% more oxidized species, respectively, as shown in graph of Fig. 5.