Methods

The ethics committee of the University of São Paulo Medical School approved the present investigation, and all participants provided written informed consent. The study cohort included 30 subjects (10 healthy controls and 20 with the MS) selected from the outpatient clinic of the Heart Institute, Hospital das Clínicas of the University of São Paulo Medical School. Their age range was 21 to 70 years, and they were nonsmokers and abstainers or only moderate alcohol consumers (<25 g/day). None had cardiovascular, renal, liver, gastrointestinal, pulmonary, endocrine, or oncologic diseases. None was receiving antioxidant vitamin supplementation or drugs known to affect lipoprotein metabolism.

The presence of the MS was defined according to the American Heart Association/National Heart, Lung, and Blood Institute scientific statement. The criteria for the clinical diagnosis of the MS was the presence of ≥3 of the following characteristics: elevated waist circumference (≥102 cm in men and ≥88 cm in women), elevated triglycerides (TG) (≥150 mg/dl) or receiving drug treatment for elevated TG, reduced HDL cholesterol (≤40 mg/dl in men, ≤50 mg/dl in women) or receiving medication for reduced HDL cholesterol, elevated blood pressure (≥130 mm Hg systolic blood pressure or ≥85 mm Hg diastolic blood pressure) or receiving antihypertensive drug treatment with a history of hypertension, elevated fasting glucose (≥100 mg/dl) or receiving medication for elevated glucose.

Patients with the MS underwent exercise training for 3 months. A bicycle cardiopulmonary exercise test was performed by healthy controls at baseline and by the MS group at baseline and after the 3-month exercise period. The peak oxygen uptake was considered at the end of the test (ramp protocol with 10- to 15-W increments every minute until exhaustion) when the subject no longer maintained the bicycle velocity at 60 rpm. The heart rates corresponding to the anaerobic threshold and the respiratory compensation point were determined. The training program consisted of a 3-times/week supervised and controlled training load (45 min/day) on a bicycle ergometer, as previously described. The exercise was considered of moderate intensity and compared to brisk walking. The participants began their training at the heart rate corresponding to the anaerobic threshold. The exercise was then progressively intensified, according to each patient’s subjective effort perception, up to the heart rate corresponding to 10% less than the respiratory compensation point.

Blood samples were obtained after the patient had fasted for ≥12 hours, always at the same point of the menstrual cycle in the women, and ≥16 hours after the last exercise session. The total cholesterol (TC), HDL cholesterol, TG, and plasma glucose levels were measured using commercially available kits and standardized techniques. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula.

Apolipoprotein A-I was determined using a commercial kit (Wako Pure Chemical, Osaka, Japan), and the serum samples were assayed for paraoxonase-1 activity using paraoxon as a substrate, as previously described.

The preparation of a lipidic nanoemulsion labeled with ¹⁴ C-phospholipid, ³ H-triglycerides, ¹⁴ C-cholesterol, or ³ H-cholesteryl ester (all from Amersham International, Amersham, United Kingdom) was performed, as previously described. The incubation of the radioactive nanoemulsion with the patient’s whole plasma, the chemical precipitation of the apolipoprotein B-containing lipoproteins and the nanoemulsion, and the measurements of the radioactivity transferred from the nanoemulsion to the supernatant containing the HDL fraction were performed as previously described. Venous blood collected in tubes containing ethylenediaminetetraacetic acid was kept on ice and immediately centrifuged at 4°C. The plasma was then mixed with sucrose (final concentration 0.6% wt/vol) as a cryoprotectant for lipoproteins and frozen at −80°C in plastic cryovials.

LDL and HDL were fractionated using 2 different methods. LDL was isolated using an adaptation of the single vertical spin discontinuous density gradient ultracentrifugation method. In brief, the plasma density was adjusted to 1.400 kg/L by the addition of solid potassium bromide. This solution was then overlaid with saline and ultracentrifuged for 120 minutes at 70,000 rpm at 4°C. The LDL was then removed by aspiration of the orange band from the top of the tube. HDL was fractionated using single spin isopycnic density gradient ultracentrifugation. Five major HDL subfractions were obtained: large, light HDL2b and HDL2a and small, dense HDL3a, HDL3b, and HDL3c. The lipoproteins were stored at 4°C and used within 10 days. Before being used in the experiments, ethylenediaminetetraacetic acid and potassium bromide were removed from the LDL and HDL solutions by exhaustive dialysis with phosphate-buffered saline and Chelex 100 (Bio-Rad Laboratories, California) for 24 hours at 4°C, with the buffer changed 3 times.

The content of protein, TC, and TG in the HDL subfractions was analyzed using commercially available kits and standardized techniques. Free cholesterol and phospholipids were determined using commercial kits (Wako Pure Chemical, Osaka, Japan), and the cholesterol ester content was calculated by the difference between the TC and free cholesterol.

The oxidizability of a reference LDL, retrieved from a control subject and used throughout the study, was analyzed by the in vitro resistance to oxidation with copper sulfate, using standardized methods. The antioxidative activities of the HDL subfractions were evaluated by the influence of the HDL subfractions on the resistance of the reference LDL to oxidation with copper sulfate, slightly modified, as described previously. In brief, the reference LDL (100 μg of protein/ml) was incubated at 37°C in phosphate-buffered saline in the presence of copper sulfate (5 μm) for 4 hours. The accumulation of conjugated dienes was continuously measured as the increment in absorbance at 234 nm. Two characteristic phases were identified: the lag phase and the propagation phase. Individual HDL subfractions (80 μg of protein/ml) were added to the LDL directly before oxidation. We used the HDL2a and HDL3b subfractions as representatives of HDL2 and HDL3. HDL2a was obtained from the patients with the MS before and after the exercise training, was co-incubated with the reference LDL, and the resistance to oxidation with copper sulfate was evaluated. The percentage of variation in the lag phase time for oxidation (after vs before training) was determined. The same procedure was performed with HDL3b.

Statistical analyses were performed using the Statistical Package for Social Sciences for Windows, version 10 (SPSS, Chicago, Illinois). All values are expressed as the mean ± SD, unless otherwise indicated. An α probability of p <0.05 was required for statistical significance. Between-group differences in the continuous variables were analyzed using the Student t test or Wilcoxon nonparametric test for dependent samples, as applicable. The chi-square test was used to compare the proportions between the controls and the MS group.

Results

Compared to the controls, the MS group had a greater body mass index (32.4 ± 3.3 vs 24.3 ± 2.6 kg/m ² , p <0.05), waist circumference (110 ± 10 vs 88 ± 7 cm, p <0.05), blood pressure (141 ± 10/92 ± 4 vs 120 ± 6/75 ± 5 mm Hg, p <0.05), TC (203 ± 20 vs 192 ± 25 mg/dl, p <0.05), LDL cholesterol (123 ± 21 vs 116 ± 22 mg/dl, p <0.05), and plasma glucose (112 ± 19 vs 81 ± 6 mg/dl, p <0.05).

The effectiveness of exercise training in the MS group was demonstrated by an approximately 20% increase in the peak oxygen uptake on the cardiopulmonary test (from 19.9 ± 4.3 to 23.7 ± 4.6 ml/kg min, p <0.05). Exercise did not affect the body mass index (32.4 ± 3.3 vs 32.4 ± 3.2 kg/m ² before and after training, respectively, p >0.05) but did reduce the waist circumference (from 110 ± 10 to 107 ± 9 cm, p <0.05) and blood pressure (from 141 ± 10/92 ± 4 to 135 ± 7/89 ± 5 mm Hg, p <0.05).

Exercise training did not significantly change the HDL cholesterol ( Table 1 ), TC (203 ± 20 vs 196 ± 27 mg/dl before and after training, respectively, p >0.05), LDL cholesterol (123 ± 21 vs 122 ± 27 mg/dl before and after training, respectively, p >0.05), or apolipoprotein A-I (1.43 ± 0.25 vs 1.45 ± 0.21 g/L before and after training, respectively, p >0.05) plasma levels. However, a significant increase occurred in the LDL resistance to oxidation with the addition of HDL2a or HDL3b subfraction from the MS group after training compared to the addition of the respective HDL2a or HDL3b subfraction before training ( Figure 1 ).

Table 1

Individual data on plasma lipids and transfer of labeled lipids to HDL of study subjects, before and after training

Pt. No.	Gender	Age (years)	Lipid Concentration (mg/dl)				Transfer of Labeled Lipids to HDL (%)
			HDL Cholesterol		TG		14 C-PL		3 H-TG		14 C-C		3 H-CE
			Before	After	Before	After	Before	After	Before	After	Before	After	Before	After
MS
1	Female	45	40	39	157	154	21.4	27.2	4.3	3	10	9.3	4.1	3.9
2	Female	58	41	41	193	208	21.5	25.9	3.4	3.1	8.1	8.6	3.4	4
3	Female	29	34	36	230	210	22.8	26.7	4.4	3.4	8.8	8.4	4.1	5.4
4	Female	49	41	44	177	179	28.2	25.3	3.7	2.8	7	7.7	2.4	3.6
5	Female	61	40	39	282	234	30.3	26.1	3.4	3.3	8.1	8.6	3.3	4.1
6	Female	54	40	43	326	111	27.9	27.1	4.8	2.7	8.1	7.8	2.5	4
7	Female	49	49	53	162	91	25.2	26.4	3.1	2.7	8	8.5	2.4	4.2
8	Female	42	39	34	230	213	25.4	25.7	3.2	4.3	7.8	8.3	2.3	3.3
9	Female	57	34	30	370	330	29.4	26.2	3	5.4	7.5	7.6	2.1	3.4
10	Female	56	40	36	276	191	27.4	25.6	3.2	4.5	8.7	8.9	2.9	2.8
11	Male	69	34	37	155	95	25	25.3	3.8	6.1	8.6	10.1	6.9	3.1
12	Male	47	31	34	159	156	32.2	24.7	4.6	5.5	8.5	8.9	3.3	3.7
13	Male	57	26	27	273	270	26	23.2	4.4	5.1	6.4	7.1	4.1	4.7
14	Male	40	35	27	166	104	30.3	24.9	3.4	3.3	7	8.3	3.3	4.1
15	Male	56	32	43	164	98	28.2	24.8	3.7	2.8	7.4	7.7	2.4	3.5
16	Male	50	36	37	265	200	26	23.2	4.4	5.1	6.1	7.1	4.1	4.2
17	Male	52	31	31	233	301	25.4	25.7	3.2	4.3	7.8	8.3	2.3	3.3
18	Male	47	36	44	195	206	32.2	25.8	4.6	5.5	7.7	8.6	3.3	3.7
19	Male	31	33	34	139	129	23.7	23.7	3.3	5.2	8.6	7.5	2.1	3.4
20	Male	55	37	40	195	192	30.2	26.5	4.4	2.9	6.8	9.1	4.4	4.5
Mean ± SD		50 ± 10 ⁎	36 ± 5 †	37 ± 6 †	217 ± 64 †	184 ± 68 † ‡	26.9 ± 3.2 ⁎	25.5 ± 1.2 ⁎	3.8 ± 0.6 †	4.1 ± 1.2 †	7.9 ± 0.9 †	8.3 ± 0.8 ⁎ ‡	3.3 ± 1.1 †	3.8 ± 0.6 ⁎ ‡
Controls
1	Female	41	51		73		31.1		6.4		7		4.8
2	Female	43	60		114		27		6.6		9.3		4.1
3	Female	37	56		75		24.6		4.8		7		3.6
4	Female	43	47		146		28		6.1		9.8		5
5	Male	46	57		150		27.4		8.3		10.3		3.4
6	Male	46	48		148		28.6		6.4		9.4		3.2
7	Male	63	43		146		23.9		6.1		9.3		4.6
8	Male	42	44		142		24.2		7.1		10.5		5.3
9	Male	44	69		128		27.1		8.5		9.7		5.5
10	Male	45	47		76		25.4		6.9		6.9		4.1
Mean ± SD		45 ± 7	52 ± 8		120 ± 33		26.7 ± 2.3		6.7 ± 1.1		8.9 ± 1.4		4.4 ± 0.8

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