Mg loading and exercise on COPD Amaral AF et al. parameters of stable COPD patients (17). The treatment was associated with a reduction in lung hyperinflation and an improvement in respiratory muscle strength. The objective of the present paper is to report the effects of acute Mg IV loading on the exercise performance of stable COPD subjects. We hypothesized that these patients would have a high frequency of hypomagnesemia and that Mg treat- ment would lead to improvements in their maximal exercise performance, primarily in the most stable subjects with low Mg levels. MATERIALS AND METHODS Patients All patients had a diagnosis of COPD according to the Global Initiative for Chronic Obstructive Lung Disease criteria (18). All individuals were former smokers who had quit smoking for at least 12 months. They also had been in a stable clinical condition for at least two months before admission to the study. Patients with a history of asthma or allergy, renal failure, heart failure, arrhythmias or cardiac electrical disturbances, or a significant disease other than COPD, were excluded. Individuals on chronic oral steroids, diuretics, or multimineral supplements were also excluded. The study was approved by the institutional medical ethics committee, and all of the volunteers gave their written informed consent at the initial visit. The protocol has also been registered at ClinicalTrials.gov (NCT00500864). The pulmonary function data from 18 of the present patients have been included in a previously published paper related exclusively to the evaluation of Mg effects on spirometric parameters and respiratory muscle strength (17). Study design This was a randomized, double-blinded, placebo-con- trolled crossover study. The patients attended an initial visit during which a clinical history was provided, pulmonary function tests were performed, basal arterial blood gases were determined, and blood for the Mg measurements was obtained. The volunteers were then randomized by drawing lots to receive one of two sequences of treatment: a placebo on day 1 and Mg on day 2, or Mg on day 1 and a placebo on day 2. Although we attempted to keep a fixed interval of two days between day 1 and day 2, the personal issues of some subjects led to the extension of this interval up to five days. The placebo or the Mg was administered slowly by an IV infusion through venous access in the arm. A total of 33 ml of a 20% Mg sulfate aqueous solution (Aster, Sorocaba, Brazil) was diluted in 67 ml of saline, correspond- ing to 2 g of the salt. This dose was given to the patients based on the recommendations in previously published papers (19). The placebo was similarly administered as 100 ml of saline. All researchers and technicians were blinded to the treatment administered, except for the pharmacist who prepared the solution immediately before the tests. On the study days, the patients were subjected to the following procedures: (i) pulmonary function tests; (ii) an IV infusion of Mg or the placebo; (iii) blood venous sampling for plasma Mg measurements 30 minutes after the end of the infusion; (iv) a new set of pulmonary function tests performed 45 minutes after the end of the infusion; and v) a symptom-limited maximal cardiopulmonary exercise test performed within 90 and 120 minutes after the end of the IV 616 CLINICS 2012;67(6):615-621 infusion. All physiological and biochemical measurements were performed in the morning, with the patients abstaining from the use of bronchodilators and anti-inflammatory or any other type of medication for at least 18 hours prior to the experimental procedures. Pulmonary function tests Spirometry data were obtained using a water-sealed Godard spirometer (SensorMedics, Bilthoven, The Netherlands). The forced vital capacity (FVC), forced expiratory volume in the first second (FEV1) and the forced expiratory flow at 25–75% of the expiratory curve (FEF25–75%) were calculated employing representative curves. The total lung capacity (TLC) and residual volume (RV) were measured using the helium dilution technique. The procedures were performed according to the American Thoracic Society’s recommendations, and the predicted normal values were based on Crapo et al. (20-22). Exercise test Symptom-limited incremental maximal cardiopulmonary exercise tests were performed in a cycle ergometer (Corival 400, Lode, Holland) according to the Brazilian Cardiology Society guidelines (23). All patients were submitted to a ramp protocol comprising one resting minute, four minutes biking at no load, and computer-driven automatic load increments every minute after that. The ramp increments were set in an individualized way, according to clinical estimates of the subject’s exercise capacity. The exercise protocols were planned for test durations between 8 and 12 minutes. The patients were encouraged to maintain a velocity of 60 rpm, and the same protocol was applied on both occasions. The heart rate and rhythm were continuously monitored by an electrocardiogram and an analogic polygraph (HP 7754A, Palo Alto, CA, USA). Breath-by-breath respiratory measurements were registered using an ergospirometer (CPX/D MedGraphics, St. Paul, Minnesota). The arterial oxygen saturation was continuously monitored by a pulse oximeter (Moriya 1001, Japan). The arterial blood pressure was non-invasively measured at two- minute intervals. The perceptions of dyspnea and leg effort were measured at the end of the tests, using the modified Borg scale. Magnesium levels Basal plasma magnesium levels were measured in the venous blood samples collected into lithium-heparin tubes during an initial visit. Blood samples were also obtained from the arm that had not received the IV infusions on the two protocol days. The samples were stored at –80˚C after processing, and all analyses were performed on the same occasion using a Perkin Elmer model 3110 atomic absorp- tion spectrometer (Waltham, MA, USA). Statistical analysis The data are reported as the means and the standard deviations. The data obtained on days 1 and 2 were compared according to the recommended methodology for investigations with a crossover design, including an assess- ment for period effects and treatment/period interactions (24,25). Paired t-tests were employed in this setting. Post hoc comparisons between the variables with the patients classi- fied according to the degree of their response to the Mg infusion were performed employing non-paired t-tests. A p- value #0.05 was considered to be statistically significant.