Scientific Papers

Effects of a physical exercise program on HIF-1α in people with Chronic Obstructive Pulmonary Disease living at high altitude: study protocol for a clinical trial | Trials

Intervention description {11a}

The program will consist of 8 weeks with 3 sessions per week of physical exercise, for a total of 24 sessions. Each week will include sessions for training the conditional physical capabilities and sessions for health education. The training loads will be adjusted from the increased volume of work, the intensity of the exercise, and the training method. The program presents the following specifications (Fig. 2).

Fig. 2
figure 2

Physical exercise program

RR: respiratory reeducation, EE: endurance exercise, RE: resistance exercise, FE: flexibility exercises, HE: health education, HRmax: maximum heart rate, OMNI-RES Scale: perceived effort scale in strength exercises, RM: maximum repetition, PERFLEX Scale: scale of perceived exertion in flexibility exercises, COPD: chronic obstructive pulmonary disease

Explanation for the choice of comparators {6b}

The CON group will perform the same 8-WVP exercise program as the EXP group, since the purposes of the investigation are to determine the differential molecular and physiological responses between people with COPD and people of CON.

Criteria for discontinuing or modifying the interventions {11b}

Study participants may withdraw from the study at any time during the protocol. If adverse events associated with the intervention or the measurements occur concurrently, the study will end and the data obtained up to that moment will be analyzed. The following symptoms will be considered as discontinuation criteria: coughing, bronchospasm, chest pain, dizziness, headache, and exacerbation signs of respiratory distress after finishing the physical effort.

If an uncontrolled exacerbation of respiratory symptoms occurs after the physical exercise program, non-pharmacological management will be given as follows: (i) start oxygen therapy until a SpO2 level ≥ 90%, (ii) maneuvers of feedback of the respiratory cycle, (iii) constant monitoring of respiratory pattern and vital signs, (iv) accompaniment to the nearest health center. If it would be necessary to start respiratory pharmacological treatment, the specific medication to each patient according to their personal medical formulation will be administered.

Strategies to improve adherence to interventions {11c}

As part of the intervention for the 8-WVP program, health education training and workshops will be developed regarding respiratory and musculoskeletal care topics during and after the practice of physical exercise, as well as topics associated with the benefits of physical exercise. This education training is aimed to promote the understanding of the participants in the 8-WVP program to improve their adherence to the study.

Patient participation in study design

The participants in this research will decide the specific concepts to be developed in the education sessions, considering the particular needs of each subject, the tastes and preferences of knowledge, and the thematic areas proposed in Fig. 2. The identification of the concepts to be developed will be done through open questions during the development of the physical exercise sessions.

Relevant concomitant care permitted or prohibited during the trial {11d}

Participation in alternate physical activity or physical exercise programs implemented by other professionals. Pharmacological use of erythropoietin (EPO), corticosteroids, nonsteroidal anti-inflammatory, and antibiotics.

Provisions for post-trial care {30}

A telephone follow-up for 3 weeks after participation in the study will be tracked to assess complications after the study and to continue the promotion of healthy lifestyle habits. Once the study will be over, the results will be communicated to all the participants in a joint session.

Outcomes {12}

The primary outcomes of this investigation will be related to the expression of the HIF-1α protein and its incidence on the response of VEGF-mediated angiogenesis and EPO-regulated erythropoiesis. Secondary outcomes will be associated with the performance of lung function (measured by spirometry), physical performance (measured by ergospirometry and dynamometry), and hematological changes after physical exercise (measured by systematized complete blood count).

Primary outcomes

  • –Protein expression by enzyme-linked immunosorbent assay (ELISA). To analyze of the primary variables HIF-1α, VEGF, and EPO, 8 mL of venous blood will be taken. Blood will be centrifuged at 2400 g for 10 min at 4 °C to isolate the plasma, which will be separated into aliquots of 200 μL in Eppendorf microtubes and subsequently stored at − 80 °C in the Exercise Physiology Laboratory of the Universidad Nacional de Colombia until analysis. Protein quantification HIF-1 [22], VEGF [23], and EPO [24] in plasma will be carried out using the sandwich-type ELISA molecular technique using specific antibodies to immobilize the study proteins on the plate for their subsequent detection. Finally, a chemiluminescence detection will be performed using a spectrophotometer SpectraMax ®M5 (San José, USA). The following antibodies will be used: (i) human/mouse total HIF-1α DuoSet – R&D Systems, (ii) human VEGF quantikine ELISA Kit – R&D Systems, (iii) human erythropoietin quantikine—R&D Systems (Minneapolis, USA).

Secondary outcomes

  • Pulmonary function. It will be evaluated by means of spirometry, employing spirometer COSMED microQuark PC (Rome, Italy) and using the Omnia 2.0 COSMED operating system (Rome, Italy). The execution procedure and analysis of the results will be carried out following the recommendations of the American Thoracic Society (ATS) and European Respiratory Society (ERS) [25]. The valid measures will be considered after the highest value of three satisfactory measurements. The parameters to analyze will be Forced Expired Volume in the first second (FEV1), Forced Vital Capacity (FVC), FEV1/FVC Ratio, and changes in FEV1 (ΔFEV1) and FVC (ΔFVC).

  • Ergospirometry. Cardiopulmonary exercise testing (CPET) will be performed using a Velotron DynaFit Pro cycle ergometer—RacerMate (Seattle, USA) adjusted for the comfort of the participants. The Cosmed Quark-B2 gas analysis system (Rome, Italy) will be used to measure the variables resulting from the cardiopulmonary test. An incremental protocol will be followed by all participants, respecting the physical tolerance of each participant [26]. The protocol will consist of (1) 1 min at rest on the cycle ergometer, (2) 2 min of warm-up at 30 rpm without load, (3) beginning of the test with a pedaling at 60 rpm for 2 min with a load of 30 W and increasing 15 W the load every 2 min until fatigue, (4) 2 min of active recovery, pedaling without load at 40 rpm, (5) 4 min of passive recovery in a sitting position. The following parameters will be analyzed: maximum oxygen consumption (V̇O2max), peak oxygen consumption (V̇O2peak), carbon dioxide production (V̇CO2), pulse-oximetry (SpO2), maximum voluntary ventilation (MVV), ventilatory reserve (VR) (calculated after 1 min of maximal exercise ventilation: V̇Epeak/MVV), maximum heart rate (HR max), heart rate reserve (HRR) (calculated as heart rate at maximum exercise: HR peak/HRmax) [27].

  • Muscular strength. The measurement of peripheral muscle strength will be carried out using a digital dynamometer (Saehan—model DHD-1, Seoul, Korea) and performing the test on the dominant limb. Three repetitions with 1 min of rest between them will be recorded and the highest value will be considered [28].

  • Automated blood count. Venous blood collection will be carried out from a peripheral blood sample by venous puncture in the antecubital vein. Before the procedure, a tourniquet will be made and a 21-gauge needle in a 10-cc hypodermic syringe will be used. Blood will be collected with the Vacutainer collection system and the sample placed in tubes with ethylenediaminetetraacetic acid (EDTA). For each participant, 4 mL of venous blood will be collected for an automated blood count in Mindray CAL 6000 Blood Cell Analysis System (Shenzhen, China). The following parameters will be obtained: reticulocyte count, red blood cell (RBC) count, hemoglobin concentration ([Hb]), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCH).

  • Physical activity level. The level of physical activity will be measured using an ActiGraph GT3X + triaxial accelerometer (Pensacola, USA) which registers acceleration with variations of magnitudes from 0.05 to 2.5 g (g = 9.8 m/s2) [29] with a frequency range of 0.25 to 2.5 Hz for 60 s. The accelerometer will be attached to the participant’s dominant waist or hip using a belt. Recordings will be obtained during 7 days, taking as an efficient measurement of 5 valid days for which the first and last day of measurement will be excluded [30]. A period of continuous use of the accelerometer with time from 8 to 10 h will be taken as a valid day. For the analysis of the data, the times of non-registration will be excluded, defined as the period of 60 min of counting 0, which is the evidence of non-use of the device. To define the level of physical activity, the records are as follows: (i) METs/h, METs/day and average METs, (ii) energy expenditure in kcal/h, kcal/day and average kcal, (iii) number of steps per hour, number of steps per day, and average number of steps.

  • Quality of life associated with health. To evaluate quality of life, the COPD Assessment Test (CAT) will be applied, which is a survey that determines the person’s quality of life in relation to the impact of the disease on the patient’s well-being. Comparisons of health-related quality of life (CAT) will be performed exclusively in the COPD group to determine the effects of the physical exercise program in subjects with the same health condition.

Participant timeline {13}

The enrollment procedure, interventions, and evaluations of the participants are presented below (Fig. 3):

  • Day 0: Selection and enrollment. Snowball recruiting.

  • Day 1. Determination of health condition: (1) cardiovascular health condition with electrocardiogram, (2) identification of neurological or musculoskeletal comorbidities, (3) filling in the clinical history of the investigation, and (4) signature of informed consent.

  • Day 2. Evaluation of physical performance: (1) lung function by spirometry, (2) measurement of cardiopulmonary resistance by ergospirometry, (3) assessment of muscle strength in extremities using dynamometry, (4) systematized blood count, (5) ELISA determination, and (6) quality of life assessment with CAT questionnaire in people with COPD.

  • Day 3 to day 27. Each participant will follow the 8-WVP program at home.

  • Days 28 and 29. Reassessment of physical performance through the same tests and protocols developed on day 02 and discharge evaluation.

  • Day 30. Socialization of the results to participants and interested parties.

Fig. 3
figure 3

Chronology of enrollment, interventions, and evaluations

Sample size {14}

The sample size for the longitudinal studies was calculated using the Diggle formula [31] for both differences between individuals and between groups, taking as reference an expected effect size of 7.10 (δ) for the expression of EPO, VEGF, and HIF-1α. Variances (δ2) of 6.76 are considered according to each effect measure reported in the literature [14, 32, 33]. Two measurements will be considered (pre and post) for the two comparison groups, with a type I error of α = 0.05, power P = 0.80 and exploring correlations (ρ) of 0.47 according to each parameter. In this way, a total of 10 people are projected and an attrition of 20% is calculated. Therefore, 6 people with COPD are projected for the experimental group and 6 people without COPD for the control group. Being a study of repeated samples, a total of 6 measurements per participant will be analyzed for a total of 72 samples for the analysis of the results.

Recruitment {15}

The recruitment process will be carried out by snowball involving people attending the National University hospital – UN and the Universidad Nacional de Colombia. The principal investigator will contact the interested participants by a telephone call in which the nature of the study will be explained. If people express interest in participating, a home visit will be scheduled to explain the details of the study, resolve concerns, and provide any necessary clarifications. After confirmation of compliance with the selection criteria, the informed consent will be signed, and then the evaluation process will start. Any participant who wishes to withdraw voluntarily during the study may do so and this will not affect the quality of care for the other participants.

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