| Background Chronic obstructive pulmonary disease (COPD) is a multiple component disorder with systemic effects. Skeletal muscle dysfunction (SMD) is one of the most important systemic manifestations of COPD. SMD has been recognized as a contributing factor related to reduced exercise capacity, impaired quality of life, and increased mortality and health care utilization. There is increasing concerns on the potential role of peripheral muscles dysfunction in COPD as it might be a potential site of intervention for improving patient's functional status. A variety of mechanisms related to SMD in COPD have been postulated, such as systemic inflammation, malnutrition, muscle disuse and atrophy, etc; while none of the above mentioned mechanisms could fully explain SMD in COPD. Quadriceps function assessment was used in majority of the studies for assessment of peripheral skeletal muscle function as it is readily accessible and is a primary locomotor muscle. Accurate assessment of skeletal muscles function is important for investigation of the mechanism of SMD in COPD. Comprehensive evaluation of muscles function should include the assessment of both of strength and of endurance capabilities. The conventional test for assessment of quadriceps strength is the maximal volitional contraction (QMVC), which is depending on subjects'volitional effort and affected by learning effects. In the past twenty years, magnetic stimulation of femoral nerve has been employed to assess quadriceps strength non-volitionally, which is not influenced by subjects'effort and co-operation. Furthermore, proper evaluation of the prevalence and severity of SMD in COPD relies also on the proper control data from the age-matched normal subject. So, adequate normal values of quadriceps function in age-matched healthy elderly were important for the study.Objective The purposes of the study are to establish normal predicted values for quadriceps muscle function tests in healthy elderly, which can provide proper control data for age-matched COPD patients, to evaluate the prevalence and severity of SMD in patients with COPD, and to explore the related risk factors. Meanwhile, the methods of quadriceps function testing were compared in order to provide evidence for selection of testing for clinical evaluation.Materials and Methods1. Study subjectsSeventy-one patients with stable COPD and 60 age-matched healthy volunteers were recruited for the study. Patients were recruited from outpatients'clinics of Guangzhou Institute of Respiratory Disease (State Key Lab of Respiratory Disease) during the period from Mar 2007 to Oct 2007. The diagnosis of COPD was according to the criteria recommended by the GOLD guideline. Significant co-morbidities were excluded by medical history, physical check-up and conventional lab investigations. Subjects exclusion criteria included history of exacerbation in the preceding month, co-morbidities of cardiac, rheumatologic or neuromuscular disorders or unwilling to participate in the study. Subjects in control group were recruited from health check-up department of First Affiliated Hospital of Guangzhou Medical College.2. Quadriceps function assessmentQuadriceps function test was performed with the technique described by Polkey et al, with a specially designed chair from which the back was removed and laid flat. The test was performed with the subject'knee flexed at 90 degree over the end of the chair. The ankle of the dominant leg was placed in an inextensible strap that was connected to a strain gauge, and the strain gauge was calibrated after each test with weights of known amounts. The force output and compound muscle action potential (CMAP) of surface electromyography (SEMG) was recorded simultaneously. SEMG was recorded for quadriceps muscles of rectus femoris, vastus lateralis and vastus medialis. Quadriceps muscle functional test included the three items: twitch tension (TwQ) evoked by magnetic stimulation of the femoral nerve, strength of maximal volitional contraction (QMVC), and endurance time.2.1 TwQTwQ was induced by magnetic stimulation of the femoral nerve. Subjects were studied supine on the apparatus, with knee joints flexed at 90 degree over the end of the apparatus. Femoral nerve stimulation was performed using a 70-mm figure-of-eight coil powered by a Magstim 200 stimulator. The coil head was positioned high in the femoral triangle just lateral to the femoral artery. Minor positional adjustments were made to the coil whilst simultaneously monitoring quadriceps force during stimulation to determine the optimum position. TwQ evoked by stimulation at 100% intensity of the stimulator power output (TwQ100%) was used to determine the best spot. The best spot that resulted in the largest TwQ100% was identified and marked in order that the same position was used throughout the range of stimulator outputs. To avoid twitch potentiation, there was a rest period of 20 minutes before the start of the magnetic stimulation. After identifying the best location, stimulations of varying intensities were administered at a stepwise increment in the stimulator's output. For each subject, stimulation was administrated at 40%, 60%, 80%, 90% and 100% intensity of the stimulator power output, respectively.Quadriceps non-volitional strength was assessed with the maximal TwQ (TwQmax). To determine whether TwQ reached to the TwQmax, a near plateau in TwQ and CMAP with increasing power output were observed, indicating maximal depolarization of the femoral nerve. In the present study, TwQmax was achieved in 121 subjects (54 normal subjects and 67 patients), and the result of TwQmax was finally determined as the mean of three reproducible TwQ100% for these subjects. TwQmax was not achievable in 10 participants (6 normal subjects and 4 patients), as a plateau in either TwQ or CMAP amplitude was not reached with the increasing intensity of the stimulator power output up to 100%. Compared with the 121 subjects mentioned above, 9 of the 10 participants had higher BMI (24.531 kg/m2) and a lower ratio of TwQ100%:QMVC (<0.16); the remain one subject had a normal BMI but presented a much higher TwQ100% (25kg), with a higher variability of TwQ100%(>20%). Therefore, the data from the 121 subjects was used for TWQmax in statistical analysis, excluding the data from the 10 subjects.2.2 QMVCQMVC was measured as the subject in an upright position, with hip and knee joints at 90 degree of flexion. Subjects were required to try to extend their dominant leg as hard as possible against the inextensible strap mentioned above. A computer screen was in front of the subjects for the force generated was visible to subject and investigator; so the computer screen was served as a positive feedback mechanism to help subjects to perform the test. Repeated efforts were made with vigorous encouragement until there was no improvement in performance. Efforts were sustained for about 35 seconds and rested for about 4565 seconds between each contraction in an effort to avoiding fatigue. If maximal values were reproducible (<10% variability) for consecutive three times, i.e., the generated strength reached a plateau, the highest value of these three contractions was considered as the QMVC.2.3 Endurance timeEndurance of the quadriceps was evaluated during an isometric contraction. After 10 minutes of rest following the QMVC maneuvers, subjects were instructed to maintain a tension corresponding to 55%65% of their own QMVC until exhaustion. The feedback mechanism served by the computer screen helped subjects to maintain the determined submaximal tension. Subjects were strongly encouraged to pursue until tension dropped to 50% QMVC or less for more than 3 seconds (S). Thus, quadriceps endurance was defined as the time to fatigue (Tf), and the time at which the isometric contraction at 60% of maximal voluntary capacity could no longer be sustained.2.4 Data acquisitionThe signal was amplified 1 KHz and passed to PowerLab 8/16 SP hardware and a computer running Chart 5.2 software. SEMG signal from quadriceps was sampled at 2 kHz when QMVC and enduring tests were performed; and it was sampled at 10 KHz when magnetic stimulation of femoral nerve was administered. The amplitude of SEMG in QMVC was determined by calculating the root mean square (RMS-100 ms time constant) over the same contraction period for each muscle. Amplitudes of CMAP were determined by calculating the difference between the peak and the minimum of M-wave amplitudes when TwQ was elicited.3. Nutritional status assessmentSubject's nutritional status was evaluated by using a multiparameter nutritional index (MNI), which consisted of anthropometric measurements and visceral proteins level. Anthropometric measurements included body weight, triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC). Albumin and transferrin plasma concentration was used as visceral proteins store. In addition, serum triglyceride and cholesterol levels were also determined.4. Quadriceps muscle mass evaluationQuadriceps muscle mass was evaluated indirectly with midthigh muscle circumference (MTMC), which calculated by mid-thigh skinfold thickness and circumference. MTMC was considered different from other anthropometric indices because midthigh muscles mainly consist of quadriceps, which was the muscle we aimed to study. 5. Spirometric functionSpirometric function was tested in all of the participants. For the patients with COPD, it was required the confirmation of irreversible airflow limitation with post-bronchodilator FEV1/FVC<70%.6. Level of daily physical activityLevel of daily life physical activity (PA) was evaluated by using a PA questionnaire adapted for the elderly in China. The original questionnaire on habitual PA consisted of 19 items, scored the past 3 year's household activities, sports activities, and other physically active leisure-time activities and gave an overall PA score. The subjects were asked to describe type of the PA, hours per week spent on it, and period of the year in which the PA was normally performed.7. CytokinesEight cytokines were screened in the present study. Antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to analyze the level of cytokines in plasma, and immunocytochemistry (ICC) was employed to detect the expression of nuclear factor KppaB (NF-κB) in peripheral blood lymphocytes and neutrocytes.The concentration of seven cytokines in plasma was determined in duplicate by using ELISA kits. These cytokines included surfactant protein D (SPD), myostatin, tumor necrosis factor (TNF-α), interlukin (IL)-6, IL-1β, TNF-like weak inducer of apoptosis (TWEAK) and C reactive protein (CRP). The expression of NF-κB in peripheral blood lymphocytes and neutrocytes was detected by using immuno-chemistic kits. Semi-quantitative methods were employed to analyze the results of the ICC.7. Study ProcedureMost of the experiments were performed in the morning. Blood sample collection and anthropometric measurements required subjects to present with an empty stomach. After breakfast, skeletal muscle performance was assessed. Quadriceps function was tested as TwQ was performed firstly, with QMVC and endurance capabilities followed in order. Before each of the items of the functional test, subjects were asked to rest quietly for 20 minutes. At the end of the morning or in the afternoon of the day, spirometric variables were measured, and finally, subjects were asked to respond to the questionnaire of physical activities.8. Statistical analyses Statistical analysis was performed using SPSS 10.0 statistical package for windows. Measurement data were summarized by mean±SD, and categorical data were summarized by number (percentage). P value <0.05 was considered statistically significant. Two independent-sample t-tests and Chi-Square test were used for univariate testing between the COPD patients and the controls. The variability for both QMVC and TwQmax were investigated by using the following two methods: (1) Intersubject variability, which was determined by calculating the coefficient of variation [CV=(SD/mean)×100%]; (2) Reproducibility of the measurements, which was determined by calculating the variation rate in the repeated measurement data from 3 times, and the variation rate=[(maximum-minimum)/(maximum+minimum)]×100%. In both controls and patients groups, multiple regression models were developed by stepwise method to determine the independent factors respectively contributing to the 3 items of quadriceps performance, i.e., TwQmax, QMVC and endurance. Pearson's correlation analysis was employed to determine the relationship between the variables of strength and endurance time, and between the volitional and non-volitional contraction.Results1. Characteristics of the subjectsThere were 60 normal subjects in control group, with 21 male and 39 female, and there were 71 patients in COPD group, with 54 male and 17 female; significant difference was observed in gender constituent ratio between the two groups, with P<0.01.The mean age was (63.98±5.77) years in controls and (65.17±6.80) years in the patients (P=2.29); and the mean height was (161.03±7.80) cm in controls and (162.22±7.14) cm in the patients, respectively, with P=0.36. The results showed that the patients and controls were matched, with respect to age and height. No subjects were receiving nutritional support therapy or had participated in any rehabilitation program previously.2. Pulmonary functionSpiro metric function test showed that, the mean forced expiratory volume for 1 S (FEV1) was (97.10±8.90) %pred in normal subjects, while it was (37.76±14.93) %pred in COPD patients, and there was significant difference between the two groups (P<0.001). The results showed that patients with COPD had, on average, severe airflow limitation as defined by the GOLD criteria.3. Nutritional statusMultiparameter nutritional index (MNI) was (7.75±3.86) score in the COPD patients and (1.13±0.96) score in normal subjects, with significant difference between the two groups (P<0.001).The results showed that malnutrition was commonly existed in the patients with COPD.4. Quadriceps muscle massMTMC was (37.77±3.73) cm in the COPD patients and (43.19±3.55) cm in normal subjects, with P<0.001, showing that the patients had decreased muscle mass compared with normal subjects.5. Level of daily physical activityPA score was (5.25±1.38) score and (7.97±1.21) score, in the COPD patients and normal subjects, respectively, with significant difference existing between the two groups (P<0.001). The PA questionnaire showed that the patients with COPD had less physical activities in their daily lives compared with the normal subjects.6 Quadriceps function assessment and methodological issues 6.1 Quadriceps function assessmentWhen analyzing the results in subgroups of men and women, QMVC was [(23.26±5.74) kg v (42.06±7.61) kg for male, and (15.55±4.25) kg v (29.24±4.58) kg for female]; endurance time was [(51.13±18.73) S v (81.09±22.58) S for male, and (56.31±15.49) S v (83.44±23.64) S for female]; TwQmax was [(7.25±2.29) kg v (13.91±3.90) kg for male, and (4.18±0.89) kg v (7.76±1.75) kg for female] in the patients and in controls, respectively, with each of P<0.001.6.1.1 Predicted equation for normal valuesFor the healthy elderly (5876 years old), the normal values of the quadriceps functional tests were defined as the dependent variant, and the candidate factors related to quadriceps function were analyzed as the independent variant by the stepwise regression correlation models. Consequently, the predicted normal values were given as the following 3 equations: QMVC(kg)=0.383+ 7.278×(sex) +1.967×PA score (score) +0.257×weight (kg) Tf(S)=2.143×MTMC(cm)+11.467×PAscore(score)+1.244×weight(kg)-34.759×(sex)-162.478 TwQmax (kg)=0.370×height (cm) + 0.898×PAscore (score)-56.7756.1.2 Prevalence of quadriceps dysfunction According to the lower limit of the 95% confidential interval of the data from the normal subjects, QMVC was impaired in 94% of the COPD patients; Tf was impaired in 78% of the COPD patients, and TwQmax was impaired in 90% of the patients. There were, 94% of the patients with quadriceps dysfunction, in terms of any item of the three indices, and 59% of them with quadriceps dysfunction, in terms of all the 3 items including QMVC, Tf and TwQmax.According to the lower limit of 95% confidential interval of the predicted normal values showed above, QMVC was impaired in 95% of the patients, Tf was decreased in 45% of them, and TwQmax was impaired in 85% of them. There were, 95% of the patients with quadriceps dysfunction, in terms of any item of the three indices, and 46% of them with all the 3 items including QMVC, Tf and TwQmax.6.1.3 Severity of quadriceps dysfunctionQMVC was decreased by 45%, Tf by 37%, and TwQmax by 47%, respectively, in male patients, compared with normal male subjects; QMVC, Tf and TwQmax were decreased by 48%, 33% and 44%, respectively, in the female patients, compared with the female controls.According to the prediction equation for normal values showed above, QMVC was only 65%, Tf was 73%, and TwQmax was 67% of predicted values, respectively, in the COPD patients.6.1.4 Characteristic of quadriceps dysfunctionWith respect to extent of the quadriceps dysfunction, QMVC, Tf and TwQmax were decreased by 46%, 35% and 45%, respectively, compared with normal subjects. Overall, strength showed a larger extent than endurance time in the impairment of the quadriceps functional tests.In total, quadriceps function was commonly and substantially impaired in the COPD patients, compared with the age-matched normal subjects.6.2 Variability of volitional and non-volitional techniques6.2.1 Reproducibility of repeated measurementsReproducibility of the measurements was assessed by calculating the variation rate of the repeated measurement data from 3 testings. The variation rates of TwQmax and QMVC were [2.3% v 3.3% for male, 3.4% v 3.7% for female] in normal subjects, and [3.9% v 8.5% for male; 3.3% v 5.1% for female] in COPD patients, respectively. According to the results, TwQmax presented a higher reproducibility than QMVC in all of the subjects. However, the variation rates for both TwQmax and QMVC were less than 10% in all of the groups.6.2.2 Inter-subject variabilityInter-subject variability was assessed with the coefficient of variation (CV). When analyzing subgroups of women or men, CV of TwQmax and QMVC was [28% v 18% for male; 23% v15% for female] in normal subjects, and [32% v 24% for male; 21% v 27% for female] in COPD patients, respectively. The result showed that TwQmax demonstrated greater variability than QMVC in all of the groups except of the female patients group, in which variability was lower for TwQmax than it for OMVC, according to the CV.7. CytokinesConcentrations of the 4 cytokines in plasma were elevated in COPD patients as compared with control subjects. The results were showed as following: SPD (ng/ml): (31.97±5.54) vs (47.52±17.26); Tweak (pg/ml): (447.57±84.25) vs (583.39±241.50); myostatin (ug/ml): (31.76±9.39) vs (48.71±21.01); TNF-α(pg/ml): (6.23±4.49) vs (13.91±2.86) (all p<0.05). Another 4 cytokines including IL-6, IL-1β, CRP, and NF-κB were also showed the tendency of elevation in COPD patients comparing with control subjects, but no statistical significance was observed (all p<0.05).7. Correlations7.1 Multivariate stepwise regression analysisIn normal control subjects, stepwise regression analysis showed that QMVC could be predicted by sex, PA scores and weight, with R2 of 0.61 (p<0.001); for endurance time, PA scores, MTMC, weight and sex were the predictors to Tf variance, with R2 of 0.58 (p<0.001); and for TwQmax, height and PA scores were the predictors, with R2 of 0.67 (p<0.001).In the COPD patients, the results showed that FEV1%pred had the biggest effect on QMVC, except of gender difference, according to the values of standardized coefficients (β); R2 suggested that 6 independent variables including sex, FEV1%pred, MNI, SPD, TWEAK and myostatin were contributors of QMVC, together explaining 89% of QMVC variance. For endurance time, FEV1%pred and TNF-αwere contributors to Tf, explaining 25% of Tf variance. While for TwQmax, there was not any parameter correlated with it.7.2 Pearson's correlation analysis for different methods of quadriceps functional testsThere was a significant correlation between TwQmax and QMVC in normal subjects (r=0.793, p=0.000), and a mild correlation between them in the COPD patients (r=0.396, p=0.001). The ratio of TwQmax/QMVC was 0.29 in normal subjects, and it was 0.32 in the COPD patients.Quadriceps strength didn't correlate with endurance time, in terms of either QMVC or TwQmax, in any of the groups.Conclusions1. For the healthy elderly age between 58-76yrs, the predicted normal values of the quadriceps functional tests should be as follows: QMVC(kg)=0.383+ 7.278×(sex) +1.967×PA score (score) +0.257×weight (kg) Tf(S)=2.143×MTMC(cm)+11.467×PAscore(score)+1.244×weight(kg)-34.759×(sex)-162.478 TwQmax (kg)=0.370×height (cm) + 0.898×PA score (score)-56.7752. Quadriceps function commonly and substantially impaired in COPD patients compared with the healthy elderly, in terms of both strength and endurance capability, with strength impaired to a larger extent.3. Airflow limitation, systemic inflammation, malnutrition and muscle atrophy are the main risk factors of SMD in COPD, with the former is the most significant one. Our study results support the notions that SMD is a complex related to multiple risk factors, with several mechanisms involved in it, and none of any one mechanism can fully explain SMD in COPD.4. Systemic inflammatory cytokines including SPD, myostatin, TWEAK and TNF-αmight be potential candidate biomarkers for SMD in COPD.5. As for the methodology, we conclude that each of the techniques has its own advantages and limitations. TwQmax has higher reproducibility, which is appropriate for repeated measurements in dynamic observation; while it presents with a higher inter-subject variability. In contrast, QMVC was shown to be lower in inter-subject variability, which offers a better measurement for subjects with full consciousness; whereas magnetic stimulation might be an ideal alternative technique for those who can not fully perform QMVC. With respect to endurance test, it was subject to the effort and cooperation of the subject and should not be used as choice of evaluation of quadriceps function in routine clinical practice. |
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