2. Materials and Methods
musculoskeletal pain, T2DM, pain
DISEASE, ADVERSE EVENT, INSULIN RESISTANCE, BLIND, BLOOD, COLD, RENAL DISEASE, CHRONIC DISEASE
Study design and population: This is a randomized, triple-blinded clinical trial (Registered in ClinicalTrials.gov, identifier: NCT03792646, developed at Universidade São Judas Tadeu in partnership with the Laboratory of the Study of Movement of the Institute of Orthopedics and Traumatology (IOT) of Hospital das Clínicas (HC) of the Faculty of Medicine of the University of São Paulo (FMUSP). Approved by the Ethics Committee of the FMUSP n° CAAE: 39202214.8.0000.0065.Participants: The population of the present study consisted of 28 men with T2DM, aged 68.2 (±4.0) years. Participants were recruited from senior groups at the Laboratory for the Study of Movement (LEM) with the first contact made by telephone call. After verifying potential eligibility, they were invited to come to LEM for the interview and first set of assessments.For inclusion, the study had the following criteria: living with T2DM on a stable dose of medication (oral antidiabetic agents or insulin) for over a year, availability to participate in the RT program for three months at the University Hospital, Glycated hemoglobin (HbA1c) between 6.0 and 9.0%, and renal function assessed by the Modification of Diet in Renal Disease (MDRD) method above 60 mL/h. Similarly, liver function was assessed by the blood levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT), which was expected to be up to 2.5 times higher than the limit of the method, and no impairment of the musculoskeletal system, such as pain or any condition that could cause inability to sustain a whole training session without restrictions, according to the protocol. They also could not have any decompensated or untreated chronic disease. As exclusion criteria, the study established the inability to carry out assessments, and during the training period, the participants could not have more than three absences or any related adverse event, such as an outcome of musculoskeletal pain or worsening of any prior disease symptoms or signs. After this approach, all the participants signed the Informed Consent (IC) form and were invited for a second set of evaluations.The selected participants had to undergo an evaluation by a physician to check clinical conditions and safety to engage in RT.Laboratory analysis: Blood samples were also requested for laboratory analyses from the participants who were clinically approved. Peripheral blood samples (20 mL) were collected in tubes, containing EDTA anticoagulant at the baseline and at the end of 12 weeks of protocol. Whole blood with EDTA was centrifuged to obtain plasma, which was used to evaluate the circulating concentration of glucose, insulin, fructosamine, hepatic enzymes, and glycated hemoglobin (HbA1c), using commercial kits, according to the manufacturer’s instructions (Labtest, Minas Gerais, Brazil).Renal function was obtained by the Modification of Diet in Renal Disease (MDRD) method, using the formula: glomerular filtration rate GFR = 175 × Serum (Cr) (−1.154) × age (−0.203) × 1.212 (if black) × 0.742 (if female). Moreover, insulin resistance was calculated by the HOMA-IR formula: (Homa-IR: [(Glucose (mg/dL) × (0.0555) × Insulin (UI/mL)] ÷ 22.5) [Clinical assessment: Socio-demographic characterization was performed using a questionnaire, regarding age, education, profession, marital status, physical activity, and current health conditions.Assessment of muscle strength (1RM): The 1RM test was defined as the maximum amount of weight lifted in a simple maximum effort, in which each participant completed an entire movement that could not be repeated a second time. The maximum load was the last one, in which each participant performed a movement with adequate performance standards. All of the participants in the study participated in two sessions of the 1RM test. The first one was for the participants to become familiar with the test, and the second one was for consideration of valid results.Evaluation of Handgrip Strength (HGS): For this evaluation, a JamarAssessment of functional tasks: To assess dynamic balance NeuroCom Balance MasterIn this study, the following protocols were used: sit and stand (Sit-to-Stand), walk and return (Step/Quick Turn), and go up and down steps (Step Up/Over). Before starting the evaluation, personal data of the evaluated participant was filled with full name, age, gender, and body mass. The tests were repeated three times with an interval of 30 s between them.Sit-to-Stand: Each participant was instructed to sit on a backless bench, feet apart, knees flexed at 90 degrees, and to stand up quickly and safely, remaining standing for a few seconds.Step/Quick Turn: Each participant was previously instructed to walk on the platform, turn 180 degrees, and return to the starting point. The test started from the left side and then the right side.Step Up/Over: To perform the test, a 20 cm step was placed in front of each participant, which they could see in front of them. The participants were instructed by the evaluator to step onto the step with their left leg and with their body in an upright position, stepping over it with their right leg, and stepping down with their left leg on the platform (floor) [Randomization, allocation, blinding, and loss to follow-up. Group allocation was made, according to a randomized and stratified numerical sequence, generated before the first allocation to the groups. The study consisted of two parallel arms with a CG and a PG. Both groups did the same physical exercises. However, one group was supplemented with WP and the other with Control (Maltodextrin). An independent researcher, who did not know the numerical codes, prepared a random numerical sequence. The numerical sequence was kept in opaque envelopes and numbered sequentially, following the order generated by the software. The allocation to the groups was made by another independent researcher, who also did not know the numerical code that identified the groups.Triple blind: Equally, the researcher who carried out the assessments, the one who carried out the interventions and the participants were blinded to the applied intervention.The tabulation of data from all evaluation forms and the mathematical and statistical data treatment were also carried out by an independent researcher and blinded to the groups.Losses to follow-up were treated by intention-to-treat, which was considered the appropriate analysis for testing “superiority” in randomized clinical trials [Exercise training and supplementation: Nutritionists administered supplementation after training in both groups. Only they knew the allocation of participants, but they were not present during the RT session. Both PG and CG were supplemented immediately after each session, by ingesting 20 g of the respective supplement diluted in cold water.Nutritional Guidance: All participants received nutritional guidance to standardize daily WP intake over the experimental protocol period. As recommended by the PROT-AG Study Group for older adults individuals (>65 years) [Resistance training (RT): The training sessions had a frequency of twice a week for 12 weeks. The RT protocol targeted the major muscle groups: pectoral press, leg press, leg extension, leg curl, plantar flexion, and abdominal crunch. In each exercise, three sets of 8 to 12 repetitions were performed. The intensity was first determined in 70% of the values obtained in the one-repetition maximum (1RM) test [Statistical analysis: Data were stored and analyzed using the Statistical Package for the Social Sciences (SPSS) 24.0 program. The normality and homogeneity of the variances and the adherence to the Gauss curve were obtained, using the Shapiro-Wilk and Levene tests, respectively. The descriptive analysis of the sample was done, using the mean and standard deviation. The Student’s T-test was used to compare the quantitative sample’s baseline, and categorical variables were presented in terms of their absolute and relative values, and the chi-square test and/or Fisher’s exact test were used to verify the association. Parametric tests (two-way ANOVA) were used for the variables that satisfied assumptions (muscle strength, functional tasks, glycemic control, body composition, and handgrip strength). The accepted significance level was 5% (The calculation of the effect size was performed by calculating the square partial eta (ƞ2). The results were analyzed, according to the criteria: small (0.02), medium (0.13), and large (0.26).The size of the sample was calculated through a pilot study with five T2DM older adults. The chosen variable was the primary outcome of muscle strength analyzed by the isokinetic peak torque (PT) assessment of the dominant limb quadriceps, with which we assumed the following two-tailed hypothesis: alpha value (error probability 1) of 5%; beta value, and test power of 80%. For these values, 13 patients were considered in each group.