METHODS
Participants and preliminary testing.A group of eight subjects (one woman and 7 men) with an average age of 61±7 years, BMI of 30±4 kg·m-2 and hypercholesterolemia treatment with statins was recruited. These subjects were diagnosed with the metabolic syndrome based on the criteria previously defined [10], as showed in Table 1 (Met Synd group). Subjects were physically active and medicated with statins (“ator”, “pita”, “sim”, “rosu”-vastatin) during at least 6 months before the onset of the study. The diary dosage for each drug was prescribed by participant’s primary care physicians which followed the Spanish National Institute of Health guidelines for management of lipids as a cardiovascular risk factor. Eight subjects of 30±10 years-old subjects (1 woman and seven men) were recruited as the control metabolically healthy group (i.e., Met Healthy) with the characteristics shown in Table 1. All the individuals signed a witnessed, informed consent of the protocol approved by the local Hospital’s Ethics Committee following the declaration of Helsinki (revised October 2013). Subjects underwent a medical physical examination and completed a maximal cardiopulmonary graded exercise test (GXT) to exhaustion on an electronically braked cycle ergometer (Ergoselect 200, Ergoline, Germany) with ECG monitoring (Quark T12, Cosmed, Italy) to screen for myocardial diseases and determine their maximal oxygen consumption (VO2MAX). After a cool down and 15 min of passive rest including rehydration, a short (2 - 3 min) confirmatory test was performed at 110% of the maximum load reached during the previous ramp test [11]. The VO2MAXobtained was used to set exercise intensity during the exercise bout. This is a sub-study part of a larger clinical trial evaluating the effects of 4-month exercise training and habitual medication in individuals with metabolic syndrome (ClinicalTrials.gov Identifier: NCT03019796).
Experimental design. Using repeated-measures crossover trial design subjects completed two trials. Met Synd group performed these trials by duplicate, taking either a placebo or their habitual statin medication using a randomized control trial (RCT) design. Upon study enrollment, Met Synd participants turned in their statin prescription drugs to the team physician for masking into larger capsules. Identical tablets were used for placebo but filled with dextrose. On the morning of every fifth day for the duration of the experiment (4 weeks), participants turned in their empty research pill bottle to receive a new prescription bottle. In that way, we altered participants’ drug intake between placebo and statins in a double-blinded fashion. Placebo was taken for 4 days (i.e., 96-hr) prior to the trials because this time exceeds by 5-fold the longer-lasting statin half-life prescribed to our subjects (i.e., Rosuvastatin, 19 h). During the first trial, subjects filled out an activity/diet diary and were instructed to replicate those for the 48-h before every trial. Met Synd subjects underwent 4 trails to measure postprandial lipoproteins, a) substituting their habitual statin medication by placebo medicine (REST+PLAC trial), b) taking their habitual statin medicine (REST+STA trial), c) placebo medicine combined with a bout of intense aerobic exercise (EXER+PLAC trial), d) combining exercise and statin medicine (EXER+STA trial). Met Healthy group did not take statins and thus underwent only the REST and EXER trials. Trials were separated by at least a week among them.
Experimental trials. Subjects arrived at the laboratory between 7 and 8 AM after an 10-12 hours overnight fast preceded by a standardized 322 kcals dinner (325 gr of pre-cooked pork tenderloin in mushroom sauce with 4 gr of fat, 9.5 gr of carbohydrate, 6 gr of protein per 100 g, 500 mL of water and a medium-sized apple). Upon arrival, subjects’ body weight (Hawk, Mettler Toledo, USA), body composition (BIA using Tanita BC-418-MA, Japan) were assessed. Subjects lie in a gurney while a catheter (20G, BD Insyte, Becton and Dickinson, Spain) was inserted in an antecubital vein and a 3-way stopcock attached (Luer-lock, CPK IV, Farmaban, Spain). After 20 min of lying in a quiet, (22±1 ºC and 25±6% humidity). Then, a blood sample was withdrawn (i.e., -60 min blood sample) and blood pressures (ECG gated electro-sphygmomanometer; Tango, Suntec Medical; NC; USA) measured in triplicate. This blood sample was used as a baseline for the calculations of the incremental area under the curve for blood triglycerides. Following, in trials with exercise (EXER+PLAC and EXER+STA trial) subjects pedaled continuously during 41 min alternating intensities (i.e., 40%-70%-85% VO2MAX) interspersed with 5 min of low-intensity pedaling to end with 5 min warm-down (40% VO2MAX). We chose this interval aerobic exercise scheme because a recent meta-analysis review deems that high-intensity interval training is more effective in reducing PPTG [12]. The exercise bout entailed 41 min of moderately-high average intensity cycling (i.e., 72% of HRMAX) which represented a tolerable exercise bout that could be implemented on health promotion for the Met Synd population. After exercise subjects returned to the gurney and after 20 min of supine rest a blood sample was drawn (i.e., 0 min, blood sample).
High-Fat meal. Following, subjects sat and ingested within 10 min a high-fat meal, 244 g of Oreo® frozen cheesecake (Granderroble Deserts, Spain) containing every 100 grams, 24 gr of dairy-derived fat (0.71±0.03 g kg-1 BW), 34 gr of simple sugar carbohydrate (1.06±0.04 g·kg-1 BW) and 5 gr of milk-derived protein (0.20±0.01 g· kg-1 BW). The cheesecake was blended with 150 mL of skimmed milk (Asturiana®, Spain) for a total volume of 394±21 mL. The meal amounted to 11.6 kcal·kg-1 body weight for a group average of 862±109 kcals. This meal was chosen because it was commercially available, the source of fat (i.e., mostly dairy) is what is commonly used in fat meal experiments and we could deliver in a sizable volume (394 mL) more than 0.7 g of fat·kg-1 BW which is the threshold for high-fat meal consideration [13]. During the 5 hours that followed the high-fat test meal ingestion, subjects remained seated in the lab and blood samples were collected hourly (i.e., samples 60, 120, 180, 240 and 300 min) while the catheter was maintained patent by flushing with 5 cc of 0.9% saline (Grifols, Spain) after each blood collection.
Data from blood triglyceride concentration was modeled by the trapezoidal rule to calculate the iAUC as follows [14]:
iAUC (mg·dL-1* 5 h) = (2 * (S60 min + S120 min + S180 min + S240 min) + S300 min) - (9 * S0 min)
Blood analysis. 5-cc blood samples were collected in tubes with a clot activator (Vacutainer®; USA) to upon centrifugation, obtain serum. Another 3 cc were mixed with 3K EDTA and then centrifuged to obtain plasma. Serum was analyzed for triglyceride, total cholesterol, and HDL-c. Then, LDL-c was calculated using the Friedewald formula [15]. Blood triglycerides with glycerol-3-phosphate oxidize method (interassay CV; 0.8-1.7%). Total serum cholesterol by an enzymatic method with a single aqueous reagent (iCV; 1.1-1.4%). HDL-c using accelerator selective detergent method (iCV; 1.7-2.9%). Glucose was analyzed using glucose oxidase peroxidase method with the intra-inter assay coefficient of variation (iCV; 0.9-1.2%). These analyses were run in an automated analyzer (Mindray BS 400 Medical Instrumentation, China). Apolipoproteins B levels were quantified through enzyme-link immunosorbent assay (ELISA), according to the manufacturer instructions, Apo B100 (iCV; 2.6-10.4%; Mabtech AB, Nacka, Sweden) and Apo B48 (iCV; 2.8 – 8.6%; Shibayagi, Shibukawa, Japan).
Statistical analysis. Shapiro-Wilk test confirmed that the main dependent variable, basal TG, was normally distributed. Power analysis suggested that 8 participants would be required as determined by using the variance in postprandial TG iAUC at an effect size of β = 0.80 and an α = 0.05. Data collected on arrival at the laboratory (anthropometrics, blood lipid, heart rate, blood pressure) were analyzed using 1-way (treatment) repeated-measures ANOVA. Data collected overtime was summarize as AUC and analyzed using 2-factor (exercise × statin) ANOVA in the MetSynd group. To analyze differences between Met Synd and Met Healthy groups we used a mixed-design analysis of variance (ANOVA) in all reported variables. After a significant F test, pairwise differences were identified using posthoc Tukey’s HSD. Data are presented as means ± standard deviation unless otherwise noted. All analyses were performed with SPSS version 21 (Chicago, IL). Statistical significance level was set at P≤0.05, if not indicated.