Exclusion criteria. The exclusion criteria were as follows: (1) diabetes mellitus; (2) any acute or chronic inflammatory processes; (3) symptomatic congestive ...
SUPPLEMENTAL RESEARCH DESIGN AND METHODS Exclusion criteria The exclusion criteria were as follows: (1) diabetes mellitus; (2) any acute or chronic inflammatory processes; (3) symptomatic congestive heart failure; (4) unstable coronary artery disease, myocardial infarction or stroke within 6 months preceding the study; (5) impaired renal function or nephrotic syndrome; (6) liver or biliary tract diseases; (7) malabsorption syndromes; (8) thyroid disorders; (9) malignancy within 5 years preceding the study; (10) treatment with other hypolipemic drugs within 3 months prior to the study; (11) concomitant treatment with other drugs known either to affect plasma glucose or lipid levels or to interact with statins and fibrates; (12) concomitant treatment with glucocorticosteroids and non steroid anti inflammatory drugs; (13) ongoing hormonal replacement therapy or oral contraception, and (14) patients with poor compliance. Study design MS was diagnosed on the basis of the criteria of the ATP III Expert Panel of the U.S. National Cholesterol Education Program as the concomitant presence of at least three of the following five clinical features: waist circumference (central obesity) more than 102 cm in men and more than 88 cm in women, fasting blood glucose 100 mg/dl or more, triglycerides 150 mg/dl or more, HDL-cholesterol less than 40 mg/dl in men and less than 50 mg/dl in women, and arterial systolic/diastolic blood pressure 130/85 mm Hg or more. The exclusion criteria are presented above. The study was performed according to the Declaration of Helsinki and approved by the local ethics committee. All participants provided their written informed consent. All enrolled MS patients were given detailed advice about how to achieve the goals of lifestyle modification, which were a reduction in weight of 7% or more if necessary, total fat intake less than 30% of total energy intake, saturated fat intake less than 7% of energy consumed, cholesterol intake less than 200 mg per day, an increase in fiber intake to 15 g per 1,000 kcal, and moderate to vigorous exercise for at least 30 min per day. On the basis of fasting plasma glucose, MS patients were allocated into one of the two groups: patients with pre-diabetes (n=183) (plasma glucose at least 100 mg/dl but less than 126 mg/dl and/or a 2-h post-challenge glucose level at least 140 mg/d but less than 200 mg/dl) and patients with normal glucose tolerance (NGT, n=59) (fasting plasma glucose level less than 100 mg/dl and a 2-h post-challenge glucose level less than 140 mg/dl). The former group was additionally divided into three subgroups: (a) patients with isolated IFG (n=61) (fasting plasma glucose at least 100 mg/dl but less than 126 mg/dl, and a 2-h post-challenge glucose level less than 140 mg/dl); (b) patients with isolated IGT (n=62) (fasting plasma glucose less than 100 mg/dl, and plasma glucose concentration 2 hours after a 75-g oral glucose load at least 140 mg/dl but less than 200 mg/dl); and (c) patients with concomitant IFG and IGT (IFG+IGT, n=60) (fasting plasma glucose at least 100 mg/dl, and a 2-h post-challenge glucose level between 140 and 200 mg/dl). Using a computer-generated randomization schedule the patients belonging to each group were randomized in a double-blind fashion to fenofibrate (200 mg), atorvastatin (40 mg) or placebo, which were administered once daily for 90 days. Patients were randomized. For fenofibrate, a micronized form was used, which is more effective and convenient than its immediate-acting form. Patients with MS were compared with the control group including 48 age- and sex-matched healthy subjects without lipid- and glucose metabolism abnormalities. Treatment safety was monitored at entry and then fortnightly throughout the study. Overt myopathy, elevated levels of aminotransferases (>3 times above the normal limit) and of creatine kinase (>10 times above the
normal limit) were considered an indication for withdrawal of treatment. Compliance was monitored during each visit by tablet count and was considered satisfactory when the number of tablets taken by a patient ranged from 90 to 110%. Laboratory assays Lipid/lipoprotein profile, glycated hemoglobin (HbA1c), plasma insulin, fasting and 2-h post-challenge glucose levels, high sensitivity C-reactive protein (hsCRP), hemostatic markers and monocyte production of tumor necrosis factor-α (TNF-α), interleukin-1β, interleukin-6 and monocyte chemoattractant protein-1 (MCP-1) were determined before and after 30 and 90 days of therapy. To avoid any diurnal variations in the studied markers, all blood samples were taken between 8.00 and 9.00 a.m. after a 12-h overnight fasting in a quiet, temperature-controlled room (24-250C). The samples were immediately coded so that the person performing laboratory assay was blind to subject identity and study sequence. To minimize analytical errors, all samples were tested in duplicate and results were calculated as the average value of these two measurements. Plasma lipids (total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides) were determined by a colorimetric method using commercial kits (bioMerieux, France) 12 hours after the last meal. LDL levels were measured directly. Total free fatty acids were measured by an enzymatic assay (Alpha Laboratories, Eastleigh, Hants, UK). Levels of apoprotein A-I and apoprotein B were assessed by immunoturbidimetry using reagents from Incstar Corp. (Stillwater, MN). Oxidized LDL-cholesterol levels were measured using an enzyme-linked immunosorbent assay (ELISA) method (Mercodia, Uppsala, Sweden). Plasma levels of CRP were assessed using a high-sensitivity monoclonal antibody assay (MP Biomedicals, Orangeburg, NY). The lower limit of sensitivity of this method was 0.1 mg/l. Plasma insulin was determined with a commercial radioimmunoassay kit (Linco Research Inc, St Charles, MO). This assay does not cross-react with human pro-insulin. The homeostatic model assessment (HOMA) index was calculated from the following equation: fasting serum glucose (mmol/l) x fasting insulin level (µU/ml)/22.5. Because of pulsatile secretion, insulin concentration used in this equation constituted the mean value of three consecutive measurements (blood samples were collected in 5-minute intervals). HbA1c was measured using a commercially available kit purchased from Sigma (St. Louis, MO, USA). Both fibrinogen and factor VII were assessed by an automated HEMOLAB coagulometer. Fibrinogen levels were determined by the Clauss method, while factor VII activity was assessed by an one-step method using factor VII deficient plasma. Plasminogen activator inhibitor-1 (PAI-1) antigen levels were assessed by a commercially available ELISA method (Asserachrom, Diagnostica Stago, France). Peripheral blood mononuclear cells were separated by histopaque (Sigma, St. Louise, MO) density gradient centrifugation. Then monocytes were isolated from the peripheral blood mononuclear cells by negative immunomagnetic separation using Pan-T and Pan-B Dynabeads (Dynal, Oslo, Norway). This procedure enabled us to isolate inactive monocytes without artificial uncontrolled stimulation. The isolated cells were labeled with monoclonal antibody (Daco, Glostrup, Denmark) against the monocyte specific positive antigen CD14. The procedure gave 90% of CD14 positive cells in the isolated fraction. The viability of immunomagnetically isolated monocytes were >98% as assessed by trypan blue exclusion. Monocytes were suspended in RPMI 1640 medium supplemented with 10% FCS (low in endotoxin) (Gibco, Grand Island, NY), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10 µg/ml fungizone (Gibco, Grand Island, NY). The cells were counted on a Coulter counter (Coulter Electronics, Mijdrecht, The Netherlands) and the number of monocytes was adjusted to 1x106 /ml. A constant number of cells 2 ©2010 American Diabetes Association. Published online at http://care.diabetesjournals.org/cgi/content/full/dc10-0272/DC1.
(1x106 monocytes per well) was placed in a plastic 24-well microtiter plate (Becton-Dickinson, Franklin Lakes, NJ) and left intact for 2 h to make them adhere to the bottom. Then the medium was changed and cultures were incubated for 24 h. Incubations were performed in triplicate at 370C in a humidified atmosphere containing 5% CO2 in the air. After a 24-h incubation, the supernatant was carefully aspirated and frozen at a temperature of – 800C until assayed for basal cytokine release. The removed medium was then replaced with medium supplemented with a submaximal dose of lipopolysaccharide (Sigma, St. Louise, MO) (1 µg/ml) and the cells were incubated in the mentioned conditions for an additional 24 h. At the end of the incubation, the supernatant was again collected and stored until determination of cytokine release. TNF-α, interleukin-1β, interleukin-6 and MCP-1 levels were determined using commercial ELISA kits (R&D Systems, McKinley Place N.E. Minneapolis, MN) according to the manufacturer’s instructions. The minimum detectable levels for the assessed cytokines were: 4.4 pg/ml, 1.0 pg/ml, 3.9 pg/ml, and 5.0 pg/ml, respectively, for TNF-α, interleukin-1β, interleukin-6, and MCP-1. The intra- and inter-assay coefficients of variation in our laboratory were as follows: fibrinogen – 3.6% and 2.3%, factor VII - 4.3% and 3.2%, PAI-1 – 8.7% and 5.0%, hsCRP - 4.3% and 5.9%, oxidized LDLs - 4.0% and 7.4%, insulin – 4.0% and 6.0%, HbA1c – 1.1% and 2.3%, TNF-α - 4.4% and 8.7%, interleukin-1β- 3.4% and 4.1%, interleukin-6 - 2.5% and 5.9%, and MCP-1 - 4.0% and 4.8%. Power calculations The sample size needed for the study was calculated using Sample Power software (SPSS, Chicago, IL) based on data from our previous studies. A power calculation using 80% power and type I error of 0.05 indicated that at least 200 patients would need to be randomized to detect a difference in the effects of treatment between the groups. Taking into account possible drop-outs and possible estimation and measurement inaccuracies, the number of participants (not counting the control group) was increased to 242 patients. Statistical analysis First, the distribution of the variables was analyzed. Outcomes for hsCRP, HOMA, fibrinogen, factor VII, PAI-1, TNF-α, interleukin-1β, interleukin-6 and MCP-1 were natural-log transformed to satisfy assumptions of normality and equal variance. Because lipid, lipoprotein, free fatty acid, glucose, HbA1c and after logarithmic transformation, also the other values were normally distributed, parametric tests were used for statistical analysis. Comparisons between the groups were performed using 1-way ANOVA followed by the post-hoc Bonferroni test. Student's paired t test was applied to compare pre-, inter- and post-therapy data within the same treatment group. For categorical variables χ2 test was used. Values were considered statistically significant at p
