EXERCISE-INDUCED QUADRICEPS OXIDATIVE STRESS AND PERIPHERAL MUSCLE DYSFUNCTION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE

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1 Online Supplement for: EXERCISE-INDUCED QUADRICEPS OXIDATIVE STRESS AND PERIPHERAL MUSCLE DYSFUNCTION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE Physical Activity Levels of physical activity were assessed through a physical activity questionnaire adapted for older retired adults (E1). The questionnaire scores the past year s household activities, sports activities, and other physically active leisure time activities and gives an overall physical activity score. Subjects were asked to describe the type of activity, hours per week spent on it, and the period of the year during which the activity was normally performed. All activities were classified according to the posture and movement. This questionnaire provides a reliable and valid method for classifying the activity level of older subjects as high, medium, or low, a score 9 meaning low physical activity level thus classifying the subjects as being sedentary. Mid-Thigh Muscle Cross-sectional Area Measurement A computed tomography of the dominant thigh halfway between the pubic symphisis and the inferior condyle of the femur was performed using a fourth-generation Toshiba Scanner 900S (Toshiba INC., Tokyo, Japan) (E2). Each image was 10-mm thick and was taken at 120kV and 200mA with a scanning time of 1 second while the subject was lying in the supine position. The mid-thigh muscle cross-sectional area was obtained by measuring the surface area of the tissue with a density of 40 to 100 Houndsfield Units (HU) (E3). All computed tomography images were analyzed blindly by one investigator. Quadriceps Strength Measurement E1

2 The quadriceps maximal voluntary strength was measured while subjects performed dynamic knee extensions against an hydraulic resistance (HF STAR, Hydrafitness Total Power, Henley Health Care, Belton, TX). The device consists of a chair equipped with one lever for each lower limb than can be pulled or pushed against a piston with six different levels of resistance. With this system, the generated force increases with the effort while the velocity of contraction remains almost constant (E4). To ensure that the best possible effort was made, subjects were carefully instructed to perform maximal effort at high velocity for each of the six resistance levels, and they were closely supervised during the procedure. Starting with the lowest level of resistance, the subjects were asked to perform three sets of movement at 5-minute intervals until they reached a level of resistance at which they could no longer complete the full range of movement. Under these conditions, the generated strength reached a plateau, defined as maximal voluntary contraction. Twitch Quadriceps Force Assessment To determine whether local exercise induced contractile muscle fatigue, we quantified the force of the dominant quadriceps during magnetic stimulation of the femoral nerve before and after exercise. This was done using a commercially available magnetic stimulator (Magstim 200; Magstim Co Ltd., Wales, UK) equipped with a figure-of-eight 42-mm coil, according to the method of Mador and colleagues (E5). Briefly, after resting for 15 minutes, the subject was asked to lie on a table and the leg was stabilized in a standard position on a wood frame with the knee and the hip flexed at 45º. The ankle was attached to a force gauge through a nonelastic ankle strap and the force generated by the quadriceps contraction was recorded and stored on computer. The coil was positioned on the femoral nerve just lateral to the femoral artery. The optimal position of the coil was located and used for subsequent twitches. A minimum of 10 E2

3 unpotentiated twitches were obtained before and 10 minutes after exercise. All twitches were obtained at 100% stimulator output. Twitch quadriceps force values are expressed as the means of the three best twitches. To determine whether femoral nerve stimulation was supramaximal, a single unpotentiated twitch was systematically obtained in each subject at 30, 50, 70, and 90% of power output. A plateau in twitch quadriceps force with increasing power output indicated that the femoral nerve was maximally stimulated. Skeletal Muscle Data Analysis Determination of markers of oxidative stress. Muscle thiobarbituric acid reactive substances (TBARs) were used as markers of muscle lipid peroxidation and were determined fluorimetrically using the method described by Ohkawa and colleagues (E6). The reproducibility of TBARs, calculated as coefficients of variation, was < 10%. In subjects in whom sufficient amount of muscle tissue was still available (COPD, n = 8; controls, n = 7), we also measured protein oxidation by evaluating the levels of protein carbonyls using immunoblotting (Oxyblot kit; Serologicals Corporation, Norcross, VA). The conventional assay for protein carbonyls is a colorimetric procedure that measures the binding of dinitrophenylhydrazine (DNP). However, western blotting using anti-dnp antibody provides a much more sensitive method than colorimetric procedures. Frozen muscle samples were homogenized in 6 vol/wt homogenization buffer (10 mm Tris, 3 mm EGTA, 275 mm sucrose, 50 mm DTT, 1 ml/100 ml protease inhibitor cocktail [Sigma, Oakville, Ontario], ph 7.2). After centrifugation at 1,000 g for 10 minutes, the pellet was discarded. Fifteen µg of protein were used per derivatization reaction. Total muscle protein in each sample was determined with the Bradford technique (Bio-Rad, Hercules, CA). Proteins were denatured by the addition of 12% SDS and subsequently derivatized by adding 10 µl of 1 DNP solution and incubated for 15 minutes. Finally, 7.5 µl of neutralization solution was E3

4 added to the sample mixture. Both derivatized and underivatized (negative control) muscles samples were then separated on Tris-glycine sodium dodecyl-sulfate polyacrylamide gels (SDS- PAGE) and transferred onto nitrocellulose membranes. Membranes were probed with polyclonal anti-dnp moiety antibody. Positive proteins were detected with horseradish peroxidase conjugated anti-rabbit secondary antibody and an enhanced chemiluminescence kit (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK ). Total levels of carbonyls were calculated by adding the integrated density of individual protein bands (Alpha Innotech Corporation, San Leandro, CA). References E1. Voorips LE, Ravelli ACJ, Dongelmans PCA. A physical activity questionnaire for the elderly. Med Sci Sports Exerc 1991;8: E2. Bernard S, Leblanc P, Whittom F, Carrier G, Jobin J, Belleau R, Maltais F. Peripheral muscle weakness in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;158: E3. Kelley DE, Slasky BS, Janosky J. Skeletal muscle density: effects of obesity and noninsulin-dependant diabetes mellitus. Am J Clin Nutr 1991;54: E4. Sale DG. Testing strength and power. In: MacDougall JD, Wonge HA, Green HJ, editors Physiological testing of the high-performance athlete. Human Champaign, Illinois: Kinetics Publishers; p E4

5 E5. Mador MJ, Kufel TJ, Pineda L. Quadriceps fatigue following cycle exercise in patients with COPD. Am J Respir Crit Care Med 2000;161: E6. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animals tissues by thiobarbituric acid reaction. Anal Biochem 1979;95: E5