Blue-200501-114OC-R3 - online data supplement clean version Title Hypogonadism, quadriceps weakness and exercise intolerance in chronic obstructive pulmonary disease

Authors Monique Van Vliet, Martijn A. Spruit *, Geert Verleden, Ahmad Kasran, Erik Van Herck, Fabio Pitta, Roger Bouillon, and Marc Decramer

ONLINE-ONLY REPOSITORY

* MVV and MAS contributed equally to this study.

Blue-200501-114OC-R3 - online supplement clean version Methods Patients 259 consecutive patients who attended the outpatient clinic of chronic obstructive pulmonary disease were asked to participate in the present study. Inclusion criteria were primary diagnosis of chronic airway obstruction (ratio of forced expiratory volume in one second to forced vital capacity of 70% or less), and stable clinical condition without recent exacerbation. Exclusion criteria included current androgen and anti-androgen therapy, partaking in pharmacological trials, primary diagnosis of obstructive sleep apnea syndrome, comorbidities which may affect androgen status (for example: prostate carcinoma), and active waiting on the list for lung transplantation. 157 patients were reluctant to participate or were excluded (figure E1). The consenting COPD patients had similar characteristics as those who were not included, except for their age (table E1). The remaining 102 patients (78 men) voluntarily consented to partake in the present study. For obvious reasons, only the results of the male patients were taken into account to answer the aforementioned questions. The results of the 78 men with COPD were compared with those of 21 men with similar age distribution, but with a normal pulmonary function. The circulating levels of the inflammatory markers were only determined in the male patients with COPD to study the possible relationship between low androgen status and systemic inflammation. Control subjects were recruited from relatives and friends by students of the Department of Rehabilitation Sciences of the Faculty of Kinesiology and Rehabilitation Sciences of the Katholieke Universiteit Leuven. Exclusion

E1

Blue-200501-114OC-R3 - online supplement clean version criteria included female sex, an obstructive or restrictive pulmonary function, active participation in sporting activities, a current androgen and antiandrogen therapy, partaking in pharmacological trials and comorbidities which may affect androgen status. All participants had to come for 1 morning to the outpatient clinic to perform the tests as described below. The Medical Ethical Board of the University Hospitals Leuven approved this cross-sectional comparative study. All participants were Caucasian and gave oral and written informed consent.

Blood analyzes To minimize possible ‘noise’ of circadian rhythm

E1

and/or of exercise

E2

on

circulating levels of various cytokines, venous blood was drawn from supine subjects between 08.30 and 09.00 hours before functional testing. Samples were analyzed for C-reactive protein, soluble TNF receptors p55 and p75

E3

,

interleukin-8 E4, follicle stimulating hormone, luteinizing hormone, testosterone and sex hormone-binding globulin. Moreover, free testosterone was calculated. Circulating levels of C-reactive protein (CRP) were determined using an immunoturbidimetric assay (Roche Diagnostics Corporation, Indianapolis, Indiana, USA), according to the manufacturers’ specifications (detection limit: 1.0 mg/l). The systemic levels of CXCL8 was determined using Human Inflammation Cytometric Bead Array (Becton Dickinson Biosciences, San Diego, CA; range: 1.0 to 625 pg/ml)

E5

). Data were obtained and analysed by

using FACScanTM flow cytometer and Cell Quest software.

E2

Blue-200501-114OC-R3 - online supplement clean version Soluble tumor necrosis factor (TNF) receptors p55 and p75 were measured with enzyme-linked immuno assays (EASIA KAC 1762 (range: 0.5 to 47.0 ng/ml) and 1772 (range: 1.7 to 120 ng/ml), respectively, Biosource Europe S.A., Nivelles, Belgium). Immunoradiometric assay kits were used for the in vitro quantitative measurement of human testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) (KIP1709, FSH-IRMA KIP0841 and LHsp-IRMA KIP1311, respectively, Biosource Europe S.A., Nivelles, Belgium). The detection limits were 5 ng/dl, 0.1 mIU/ml and 0.2 mIU/ml, respectively. Sex hormone binding-globulin (SHBG) was determined by using a radio immuno assay (Orion Diagnostica Oy, Espoo, Finland). The detection limit was 0.02 µg/dl. Free testosterone has been calculated using the formula of Vermeulen and co-workers, which has shown to result in comparable values as obtained with an equilibrium dialysis E6. Upper and lower limits of normal of the various hormones were those determined in healthy control subjects and provided in the manual by the manufacturers of the commercially available kits. Of interest, the prevalence of hypogonadism in the present control group is close to values reported in an unrelated Northern-American study E7. The thresholds, therefore, appear to be valid.

E3

Blue-200501-114OC-R3 - online supplement clean version

Functional measurements Pulmonary function Forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) were measured according to the European Respiratory Society guidelines for pulmonary function testing

E8

. In addition, the diffusing capacity

for carbon monoxide (TL,CO) was measured by the single breath method (Sensor Medics 6200, Bilthoven, the Netherlands). Results were expressed as a percentage of the predicted normal values of Quanjer and co-workers E8.

Quadriceps muscle force Isometric quadriceps peak torque was measured using a Cybex Norm (EnrafNonius, Delft, the Netherlands). Peak extension torque was evaluated in sitting position at 60 degrees of knee flexion and 90 degrees of hip flexion. The test was performed at least three times, and the best of two reproducible tests was used for analysis. Values were related to reference values of Decramer and co-workers E9. The noise of this measurement was determined in a separate group of outpatients with clinically stable COPD (n=8, 6 men; median age: 64 years (IQR: 61 to 67); median body mass index: 27 kg/m2 (IQR: 21 to 30); median FEV1/FVC: 43% predicted (IQR: 35 to 63)). Patients’ quadriceps peak torque was assessed on two separate occassions with a 3 days median interval (IQR: 1.5 to 5). The median difference between the two measurements was 1.5% of the predicted value (IQR: 0.6 to 3.8).

E4

Blue-200501-114OC-R3 - online supplement clean version Functional exercise capacity Functional exercise performance was measured by a 6-minute walking test. This test was performed in a 53-meter hospital corridor. Encouragement was standardized

E10

. Transcuteneous oxygen saturation and heart rate were

measured during this test for safety reasons. The best of two tests was used and expressed as percent of the predicted value E11.

Comorbidities

Comorbid conditions have been classified using the Charlson index, which is a simple weighted index that takes into account the number and the seriousness of comorbid disease

E12

. A score of 1 is allocated to myocardial

infarct, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, COPD, connective tissue disease, ulcer disease, mild liver disease, and diabetes. A score of 2 is allocated to hemiplegia, moderate to severe renal disease, diabetes with end-organ damage, and malignancies, including leukemia and lymphoma. A score of 3 is allocated to moderate or severe liver disease, while acquired immune deficiency syndrome and metastatic malignancies are attributed a score of 6. In addition, the scores on the age-comorbidity index (Charlson index II) were calculated

E13

. Each

decade of age over 40 years adds 1 point to risk (e.g 50 to 59 years, 1 points; 60 to 69 years, 2 points; etc.) and these points for age have been added to the score from the Charlson comorbidity index, as calculated above.

E5

Blue-200501-114OC-R3 - online supplement clean version Statistical analyses

Data are presented as mean ± standard deviation (SD) or as median (interquartile range, IQR) as appropriate. A two-tailed unpaired t-test or a Wilcoxon test was used to determine differences between male patients with COPD and healthy control subjects. Patients who were stratified according to possible causes of low androgen status were compared using a KruskalWallis test or a

2

test. Relationships were studied using Spearman

correlations coefficient (rs). A priori, a two-sided level of significance was set at p 0.05 E14.

Online-only results Correlations between hormones of the pituitary-gonadotrophic axis in COPD Circulating levels of FSH and LH were positively related to the circulating levels of SHBG [rs=0.28, p=0.0155; and rs=0.37, p=0.0009, respectively] in the patients with COPD. Moreover, FSH tended to be significantly related to the circulating levels of free testosterone only [rs=-0.20, p=0.0822]. LH was not related to the circulating levels of testosterone [p=0.19] or free testosterone [p=0.98]. Circulating levels of testosterone were positively related to the circulating levels of free testosterone [rs=0.86, p=0.0001] and SHBG [rs=0.57, p=0.0001]. Eugonadal healthy control subjects had a mean quadriceps peak torque of 100±15% of the predicted values, while hypogonadal healthy control subjects had a mean quadriceps peak torque of 84±19% of the predicted values (p=0.03).

E6

Blue-200501-114OC-R3 - online supplement clean version Reference List

E1. Vgontzas AN, Papanicolaou DA, Bixler EO, Lotsikas A, Zachman K, Kales A, Prolo P, Wong ML, Licinio J, Gold PW et al. Circadian interleukin-6

secretion

and

quantity

and

depth

of

sleep.

J.Clin.Endocrinol.Metab 1999;84:2603-7. E2. Pedersen BK, Steensberg A, Fischer C, Keller C, Ostrowski K, Schjerling P. Exercise and cytokines with particular focus on muscle-derived IL-6. Exerc.Immunol.Rev. 2001;7:18-31. E3. Spruit MA, Thomeer MJ, Gosselink R, Troosters T, Kasran A, Debrock AJ, Demedts MG, Decramer M. Skeletal muscle weakness in patients with sarcoidosis and its relationship with exercise intolerance and reduced health status. Thorax 2005;60:32-8. E4. Spruit MA, Gosselink R, Troosters T, Kasran A, Gayan-Ramirez G, Bogaerts P, Bouillon R, Decramer M. Muscle force during an acute exacerbation in hospitalised patients with COPD and its relationship with CXCL8 and IGF-I. Thorax 2003;58:752-6. E5. Cook EB, Stahl JL, Lowe L, Chen R, Morgan E, Wilson J, Varro R, Chan A, Graziano FM, Barney NP. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow

cytometry:

allergics

vs.

non-

allergics.

J.Immunol.Methods

2001;254:109-18.

E7

Blue-200501-114OC-R3 - online supplement clean version E6. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation

of

free

testosterone

in

serum.

J

Clin.Endocrinol.Metab 1999;84:3666-72. E7. Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR. Longitudinal effects of aging on serum total and free testosterone levels in

healthy

men.

Baltimore

Longitudinal

Study

of

Aging.

J

Clin.Endocrinol.Metab 2001;86:724-31. E8. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur.Respir.J.Suppl 1993;16:5-40. E9. Decramer M, Lacquet LM, Fagard R, Rogiers P. Corticosteroids contribute

to

muscle

weakness

in

chronic

airflow

obstruction.

Am.J.Respir.Crit Care Med. 1994;150:11-6. E10. Guyatt GH, Pugsley SO, Sullivan MJ, Thompson PJ, Berman L, Jones NL, Fallen EL, Taylor DW. Effect of encouragement on walking test performance. Thorax 1984;39:818-22. E11. Troosters T, Gosselink R, Decramer M. Six minute walking distance in healthy elderly subjects. Eur.Respir.J. 1999;14:270-4. E12. Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic.Dis. 1987;40:373-83.

E8

Blue-200501-114OC-R3 - online supplement clean version E13. Charlson M, Szatrowski TP, Peterson J, Gold J. Validation of a combined comorbidity index. J Clin.Epidemiol. 1994;47:1245-51. E14. Altman DG, Gore SM, Gardner MJ, Pocock SJ. Statistical guidelines for

contributors

to

medical

journals.

Br.Med.J.(Clin.Res.Ed)

1983;286:1489-93.

E9

Blue-200501-114OC-R3 - online supplement clean version Table E1. Characteristics of Consenting and Not-included Patients with COPD Consenting COPD patients

Non-included patients

p-value

Gender, M / F

78 / 24

105 / 52

-

Age, years

65 (9)

68 (10)

0.02

BMI, kg/m2

26 (5)

25 (6)

0.27

FEV1, % pred

45 (17)

43 (16)

0.34

FEV1/FVC, %

41 (11)

43 (12)

0.18

TL,CO, % pred

50 (19)

47 (19)

0.17

GOLD, I to IV

3 / 25 / 44 / 30

-

3 / 40 / 57 / 57

Values are expressed as mean (standard deviation), except for gender and disease severity based on Global Initiative for Chronic Obstructive Lung Disease guidelines (GOLD, absolute numbers). BMI=body mass index; kg·m-2=body weight per square height; FVC=forced vital capacity; l=liters; % predicted=percent of the predicted value; FEV1=forced expiratory volume in the first second; %=percent; TL,CO=transfer factor for carbon monoxide of the lungs.

E10

Blue-200501-114OC-R3 - online supplement clean version Table E2. Prevalence of comorbidities in patients with COPD and healthy control subjects COPD patients

Healthy control subjects

Diabetes

13%

5%

Peripheral vascular problems

4%

-

History of myocardial infarction

12%

-

PTCA

8%

-

Ulcera

5%

-

Moderate renal failure

1%

-

33% of the outpatients with COPD had one to three comorbidities. PTCA=percutaneous transluminal coronary angioplasty

E11

Blue-200501-114OC-R3 - online supplement clean version Legends Figure E1. Enrolment scheme of the patients with COPD from the outpatient clinic until the tests. OSAS=obstructive sleep apnoe syndrome. LTX=lung transplantation. WPW=Wolf Parkinson White syndrome.

Figure E2. Distribution of 78 male patients with COPD stratified according to circulating levels of testosterone (ng/dl).

Figure E3. Circulating levels of testosterone (ng/dl) versus circulating levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in 45 hypogonadal male patients with COPD (circulating levels of testosterone < 300 ng/dl).

Figure E4. Male patients with COPD stratified according to the current use of oral corticosteroids (‘yes’ (=open) or ‘no’ (=closed) and for the partial arterial oxygen tension (