MD Giovanna Castoldi
email: giovanna.castoldi@unimib.it

Stefania Galimberti

Chiara Riva

Ruggero Papagna

Federico Querci

Marco Casati

Gianpaolo Zerbini

Gianluigi L Caccianiga

Carlo Ferrarese

Marco Baldoni

M G Valsecchi

A Stella

Association between serum values of C-reactive protein and cytokine production in whole blood of patients with Type 2 diabetes

Keywords: type 2 diabetes, C-reactive protein, inflammation, interleukins, lipopolysaccharide

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Epidemiological studies indicate that diabetes mellitus can accelerate atherosclerotic processes and increase the incidence of cardiovascular events and strokes [START_REF] Grundy | Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association[END_REF]. It has been demonstrated that inflammation plays an important role in atherosclerosis [START_REF] Reiss | Atherosclerosis: immune and inflammatory aspects[END_REF] and in the pathogenetic mechanisms of some cardiovascular events [START_REF] Gough | Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice[END_REF].

Increasing evidence indicate that the same 'traditional' cardiovascular risk factors, such as hypertension, diabetes, obesity, smoking, and dyslipidemia, might induce a 'low-grade' inflammation state through the stimulation of different cytokines [START_REF] Engstrom | Fatality of acute coronary events in relation to hypertension and low-grade inflammation: a population-based cohort study[END_REF][START_REF] Pirro | Low-grade systemic inflammation impairs arterial stiffness in newly diagnosed hypercholesterolaemia[END_REF][START_REF] Basu | Type 1 diabetes is associated with increased cyclooxygenase-and cytokine-mediated inflammation[END_REF].

Many 'pro-inflammatory' molecules have been proposed as potential markers for development of cardiovascular events [START_REF] Pearson | Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[END_REF]. Among these, the acute phase protein, C-reactive protein (CRP), has been considered as a sensitive marker to predict future cardiovascular events in patients with stable and unstable angina [START_REF] Haverkate | Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group[END_REF][START_REF] Liuzzo | The prognostic value of C-reactive protein and serum amyloid A protein in severe unstable angina[END_REF], and in primary cardiovascular prevention [START_REF] Libby | Inflammation and atherosclerosis: role of C-reactive protein in risk assessment[END_REF], even if recent evidence indicates that CRP does not add substantial predictive value for cardiovascular events beyond established risk factors [START_REF] Wilson | C-reactive protein and risk of cardiovascular disease in men and women from the Framingham study[END_REF][START_REF] Sattar | C-reactive protein and prediction of coronary heart disease and global vascular events in the prospective study of pravastatin in the elderly at risk (PROSPER)[END_REF][START_REF] Danesh | C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease[END_REF][START_REF] Sattar | High sensitivity C-reactive protein and cardiovascular disease: an association built on unstable foundation?[END_REF]. CRP has been also considered a cardiovascular risk marker in diabetes [START_REF] Pfutzner | High-sensitivity C-reactive protein as cardiovascular risk marker in patients with diabetes mellitus[END_REF] and an useful marker to predict the risk of developing type 2 diabetes [START_REF] Freeman | C-reactive protein is an independent predictor of risk for the development of diabetes in the West of Scotland Coronary Prevention Study[END_REF][START_REF] Duncan | Low-grade systemic inflammation and the development of type 2 diabetes. The Atherosclerotic Risk in Communities Study[END_REF],

indicating that the link between cardiovascular diseases and diabetes might be in part explained by the presence of inflammation.

In the last years it has been shown that in vascular cell cultures CRP stimulates the production of pro-inflammatory molecules, suggesting a direct effect of CRP in atherosclerotic processes. However, its role as a direct etiological agent in atherosclerosis and inflammatory processes remains unclear and under active investigations [START_REF] Verma | Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein[END_REF][START_REF] Pasceri | Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-20[END_REF][20][START_REF] Pepys | Proinflammatory effects of bacterial recombinant human C-reactive protein are caused by contamination with bacterial products, not by C-reactive protein itself[END_REF][START_REF] Liu | Proapoptotic, antimigratory, antiproliferative, and antiangiogenic effects of commercial C-reactive protein on various human endothelial cell types in vitro: implications of contaminating presence of sodium azide in commercial preparation[END_REF][START_REF] Swafford | C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations[END_REF][START_REF] Van Den Berg | C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations[END_REF][START_REF] Singh | C-reactive protein decreases tissue plasminogen activator activity in human aortic endothelial cells. Evidence that C-reactive protein is a procoagulant[END_REF].

Recently, it has been demonstrated in healthy subjects that the serum levels of CRP are associated to an increase in proinflammatory molecules and to an increase of IL-1β and TNF-α in LPSstimulated whole blood [START_REF] Devaraj | Defining the proinflammatory phenotype using high sensitive C-reactive protein levels as the biomarker[END_REF]. Using an ex-vivo approach, we have investigated whether serum CRP values might influence cytokine production in peripheral blood of type 2 diabetic patients in response to LPS stimulation.

METHODS

Eighty nine type 2 diabetic outpatients admitted to the Diabetes Unit of Alzano Lombardo, Bergamo, Italy, from January to July 2003 were enrolled. Patients with known malignancy, immunological or inflammatory diseases and recent (< 6 months) myocardial infarction were excluded from the study. All patients gave written informed consent to partecipate in the study. The study was approved by the local ethic committee.

Patients underwent clinical examination and blood pressure was measured using a mercury sphygmomanometer, after a 5 minute rest. Blood pressure was measured 3 times at 2 minute intervals and expressed as the mean of the 3 readings. Systolic blood pressure was indicated by the first Korotkoff sound and diastolic blood pressure was indicated by the disappearance of Korotkoff sound.

In all patients peripheral whole blood samples were collected from an antecubital vein. Fasting blood glucose, HbA1c, total cholesterol and triglycerides was measured using routinary laboratory tests. CRP was measured using a high sensitivity method (hs-CRP Test, Roche). The lower limit of detection for hs-CRP was 0.03 mg/L. The patients were divided in three groups with respect to the hs-CRP levels, according to the statement of the American Heart Association [START_REF] Pearson | Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[END_REF].

Basal values of IL-6, IL-1β, and TNF-α were measured in heparinized whole-blood samples using high sensitivity ELISA (hs-hIL-6, hs-hIL-1β, and hs-hTNF-α; GE Healthcare-Amersham). The sensitivity of these assays for the three cytokines was 0.1 pg/ml.

In these patients the pro-inflammatory responsiveness was evaluated by measuring the levels of IL-6, IL-1β, and TNF-α in whole blood samples after the stimulation of LPS [START_REF] Bour | Interaction of indomethacin with cytokine production in whole blood. Potential mechanism for brain-protective effect[END_REF][START_REF] Remarque | Patients with Alzheimer's disease display a pro-inflammatory phenotype[END_REF].

LPS stimulated IL-6, IL-1β, and TNF-α were measured by immunometric assays (Immulite IL-6, IL-1β, TNF-α), as previously described [START_REF] Sala | Peripheral cytokine release in Alzheimer patients: correlation with disease severity[END_REF]. The lower limit of detection of these assays were 2 pg/ml for IL-6, 1.5 pg/ml for IL-1β, and 1.7 pg/ml for TNF-α. Aliquots of 3 ml of the same samples were incubated for 24 hours in sterile conditions at 37°C with saline or LPS (Sigma), at the dose of 100 ng/ml. At the end of this time IL-6, IL-1β, and TNF-α were measured.

Statistical Analysis

Median and interquartile range (1 st -3 th quartile) were used for descriptive purposes due to the skewed nature of the variables considered. A generalized linear model (GLM) was applied to investigate the relationship of LPS-induced IL-1β, IL-6 and TNF-α versus hs-CRP after both log transformation and categorization in the three groups defined above. A multivariate analysis was also performed in order to adjust for the following variables: basal cytokine levels, sex, age at visit, duration of diabetes, BMI, HbA1c, LDL cholesterol, antidiabetic treatment (yes-no), antihypertensive treatment (yes-no), anticholesterol treatment (yes-no) and smoking habits (yes-no).

The influence of LPS-induced IL-6, IL-1β, and TNF-α and hs-CRP on the occurrence of cardiovascular events was evaluated by means of a logistic regression model. All tests were done at a significance level of 0.05 and were two-sided.

Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338

RESULTS

Table 1 summarizes the clinical characteristics of the patients. In this sample, in spite of insulin or oral hypoglycemic therapy, blood glucose was not completely normalized. In our population, 43 patients were treated with the association of sulphonylureas and biguanides, 7 with sulphonylureas alone, 7 with biguanides alone, 3 with acarbose, 5 with insulin, and 24 only with non pharmacological treatment. Recommended values of LDL cholesterol for diabetic patients [START_REF] Diabetes | Standards of medical care in diabetes[END_REF] were not obtained. Most of the patients were overweight and 47 (53%) of the patients show LDL cholesterol greater than 100mg/dl. Twenty patients (23% of the sample) had hs-CRP < 1mg/L (Low hs-CRP group; median 0.6 mg/l, 1 st -3 th quartile: 0.5-0.7 mg/l); 44 patients (49%) had hs-CRP between 1mg/L and 3mg/L (Medium hs-CRP group; median 2.0 mg/l, 1 st -3 th quartile: 1.5-2.3 mg/l), and 25 patients (28%) had hs-CRP > 3mg/L (High hs-CRP group; median 7.7 mg/l, 1 st -3 th quartile: 5.1-11.7 mg/l).

In 66 of the 89 patients (79% of the sample) for whom cytokines have been measured, independently on hs-CRP levels, the basal values of IL-1β were very low, near the sensitivity of the 'high-sensitivity' array (1pg/ml). Basal levels of TNF-α were undetectable, while IL-6 showed a progressive, but not significant, increase according with the increasing levels of hs-CRP (Table 1).

After LPS stimulation, we found that the level of IL-1β and IL-6 significantly increased with increasing levels of hs-CRP, while this was not true for the TNF-α production (Table 1). No differences in samples incubated with saline were observed (data not shown). Figure 1 shows the relationship of cytokines with hs-CRP in continous on the log-scale (A) and the categories of hs-CRP defined above (B). In Fig. 1B we reported the p-values obtained for the comparisons between groups, showing that the increase in IL-1β and IL-6 levels was significant in group 3 with respect to group 1. When a multivariate analysis was performed, adjusting for potential confounders, the contrast between group 1 and 3 was borderline significant for IL-1β (p=0.048) and became non significant for IL-6 (p=0.117).

During the 3 years follow-up period we have observed the development of 12 cardiovascular events: 5 (33%) in patient with low hs-CRP, 4 (11%) in patient with medium hs-CRP, and 3 (19%) in patient with high hs-CRP. No relation was found between the development of cardiovascular events and the basal hs-CRP levels or LPS-stimulated cytokine levels.

DISCUSSION

The results of the present study, carried out in type 2 diabetic patients, show an association between the serum levels of hs-CRP and the LPS-stimulated production of IL-1β and IL-6 in whole blood.

The basal values of IL-1β, IL-6 and TNF-α were low and independent of the hs-CRP measuraments. On the contrary, the cytokine production in the whole blood stimulated by LPS tended to be higher for higher values of hs-CRP, with a significant association for the IL-1β synthesis under a multivariate analysis. A weaker association was also shown between LPSstimulated IL-6 and serum hs-CRP values, while no association was found with TNF-α. These data, obtained by an ex-vivo study in a small population, suggest that type 2 diabetic patients with higher hs-CRP do not show an increase in the basal level of the cytokines considered in this study. These same patients could nonetheless have a predisposition to over-produce the different cytokines in response to a specific stimulus. The evidence that the association between LPS-stimulated IL-6 and serum hs-CRP is lost after multivariate analysis might be explained by a possible interfering action of environmental factors such as lipid composition or glucose control. How CRP and LPS-reactivity may interact remains to be clarified. Infact, it is well known that the production of cytokines induced by LPS depends on the activity of specific Toll-like receptors, such as TLR 4 [START_REF] Dauphinee | Lipopolysaccharide signaling in endothelial cells[END_REF], whereas CRP has to be considered an unspecific marker of inflammation related to IL-6.

In healthy subjects Devaraj et al. demonstrated that basal values of CRP is associated to an increase in some plasma inflammatory mediators and to an increase in LPS-stimulated whole blood TNF-α and IL-1β levels [START_REF] Devaraj | Defining the proinflammatory phenotype using high sensitive C-reactive protein levels as the biomarker[END_REF]. According to these data, we have found a strong association between serum CRP levels and LPS-stimulated production of IL-1β in whole blood, whereas a weaker association was evident for IL-6. At difference with the data obtained in healthy subjects [START_REF] Devaraj | Defining the proinflammatory phenotype using high sensitive C-reactive protein levels as the biomarker[END_REF], we could not find any association between serum CRP values and LPS-stimulated production of TNF-α in our patients. We have however to consider that in the study of Devaraj et al. [START_REF] Devaraj | Defining the proinflammatory phenotype using high sensitive C-reactive protein levels as the biomarker[END_REF] a very stringent exclusion criteria were applied to healthy subjects, including pharmacological treatments. Conversely, the exclusion criteria in our, most observational study, were limited to the major diseases that may modify the inflammatory parameters. In particular, the 38% of our patients were treated with antihypertensive drugs, mainly angiotensin converting enzyme inhibitors and angiotensin II type 1 receptor antagonists, and the 25% with 3-hydroxy-3-methylglutaryl coenzyme A inhibitors, which are known to interfere with cytokine production [START_REF] Fliser | Antiinflammatory effects of angiotensin II subtype 1 receptor blockade in hypertensive patients with microinflammation[END_REF][START_REF] Devaraj | Direct demonstration of an antiinflammatory effect of simvastatin in subjects with the metabolic syndrome[END_REF].

In vitro studies have suggested that the effects of CRP on inflammatory molecules [START_REF] Pasceri | Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-20[END_REF]20] might be dependent on the bacterial contamination or by the presence of sodium azide in the commercial CRP preparation. [START_REF] Pepys | Proinflammatory effects of bacterial recombinant human C-reactive protein are caused by contamination with bacterial products, not by C-reactive protein itself[END_REF][START_REF] Liu | Proapoptotic, antimigratory, antiproliferative, and antiangiogenic effects of commercial C-reactive protein on various human endothelial cell types in vitro: implications of contaminating presence of sodium azide in commercial preparation[END_REF][START_REF] Swafford | C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations[END_REF][START_REF] Van Den Berg | C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations[END_REF]. However, other studies have demonstrated that also sodium azide-and LPS-free CRP promotes endothelial activation and atherothrombosis [START_REF] Singh | C-reactive protein decreases tissue plasminogen activator activity in human aortic endothelial cells. Evidence that C-reactive protein is a procoagulant[END_REF]. In addition, using recombinant human CRP free of sodium azide and bacterial contamination products, it has been demonstrated that CRP, at a concentration known to predict cardiovascular events in stable and unstable angina [START_REF] Haverkate | Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group[END_REF], is also able to increase the expression of the receptor for advanced glycation end products in human endothelial cell cultures, suggesting a link among inflammation, endothelial dysfunction and atherosclerosis in diabetic patients [START_REF] Zhong | C-reactive protein upregulates receptor for advanced glycation end products expression in human endothelial cells[END_REF].

In rat model of myocardial and cerebral infarction it has been demonstrated that injections of human CRP increases myocardial and cerebral infarct size, through a complement dependent mechanism [START_REF] Gill | Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats[END_REF][START_REF] Griselli | C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction[END_REF]. Recently, the same Authors have demonstrated in rats that the administration of a specific CRP inhibitor, blunted the increase in infarct size produced by human CRP injection, indicating that CRP not only might contribute, throught immuno-inflammatory actions, to increase myocardial damage, but also that in this experimental model, the inhibition of CRP might have therapeutical effects [START_REF] Pepys | Targeting C-reactive protein for the treatment of cardiovascular disease[END_REF].

Taken togheter, these data indicate that the role of CRP as etiological agent in inflammatory processes remains to be proven.

Clinical studies have suggested that the measurements of hs-CRP might be an useful marker for predicting cardiovascular events in different pathological conditions [START_REF] Haverkate | Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group[END_REF][START_REF] Liuzzo | The prognostic value of C-reactive protein and serum amyloid A protein in severe unstable angina[END_REF][START_REF] Libby | Inflammation and atherosclerosis: role of C-reactive protein in risk assessment[END_REF][START_REF] Wilson | C-reactive protein and risk of cardiovascular disease in men and women from the Framingham study[END_REF][START_REF] Sattar | C-reactive protein and prediction of coronary heart disease and global vascular events in the prospective study of pravastatin in the elderly at risk (PROSPER)[END_REF], and in apparently healthy individuals [START_REF] Ridker | Comparison of C-reactive protein and lowdensity lipoprotein cholesterol levels in the prediction of first cardiovascular events[END_REF]. Recently, increasing evidence from large epidemiological studies did not confirm the predictive role of CRP for cardiovascular events, but indicated that CRP measuraments add only a little additional predictive value in risk stratification [START_REF] Wilson | C-reactive protein and risk of cardiovascular disease in men and women from the Framingham study[END_REF][START_REF] Sattar | C-reactive protein and prediction of coronary heart disease and global vascular events in the prospective study of pravastatin in the elderly at risk (PROSPER)[END_REF][START_REF] Danesh | C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease[END_REF] .

In our population of type 2 diabetic patients we have observed that serum values of CRP might influence cytokine production in response to LPS, suggesting a predisposition to an overproductions of different cytokines in response to a specific stimilus, with possible implication in inflammatory-atherosclerotic processes.

Figure 1 .

 1 Figure 1.

Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338 © 2007 The Biochemical Society

Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338

Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338 © 2007 The Biochemical Society

© 2007 The Biochemical Society

atherosclerosis drugs. Circulation 103, 2531-2534. Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338 © 2007 The Biochemical Society

Acknowledgments

This study was supported by the COFIN 2001. The authors acknowledge Dr. L. Perego and Dr. C. Bombardi for their technical assistance. Clinical Science Immediate Publication. Published on 16 Mar 2007 as manuscript CS20060338 © 2007 The Biochemical Society


Renate T De Jongh
email: rt.dejongh@vumc.nl

Richard G Ijzerman

Erik H Serné

Mirjam M Van Weissenbruch

Jasper J Voordouw

Henriette A Delemarre-Van De Waal

Da Coen

Stehouwer

; Coen

D A Stehouwer

Urinary cortisol is inversely associated with capillary recruitment in women: a

Keywords: glucocorticoids, microcirculation, capillary, blood pressure, hypertension, insulin resistance

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cortisol excess is characterized by a cluster of cardiovascular risk factors consisting of hypertension, insulin resistance and obesity [1;2]. The aetiology of cortisol-induced hypertension is complex and multiple mechanisms have been proposed to play a role, such as a cortisol-induced increase in peripheral vascular resistance possibly in part by direct inhibition of endothelial NO synthesis [START_REF] Whitworth | Mechanisms of cortisol-induced hypertension in humans[END_REF][START_REF] Kelly | The nitric oxide system and cortisol-induced hypertension in humans[END_REF][START_REF] Yang | Glucocorticoids and vascular reactivity[END_REF]. In turn, cortisol-induced insulin resistance may result of altered insulin-binding and intracellular signalling, reduced translocation of glucose transporters or increased lipolysis in adipose tissue [START_REF] Andrews | Glucocorticoids and insulin resistance: old hormones, new targets[END_REF].

Alternatively, cortisol may decrease the availability of glucose and insulin to muscle cells by effects on total blood flow or intramuscular blood flow patterns. Indeed, administration of corticosteroids inhibits insulin-induced increases in total skeletal muscle blood flow [START_REF] Scherrer | Suppression of insulin-induced sympathetic activation and vasodilation by dexamethasone in humans[END_REF]. Thus, associations of cortisol with elevated blood pressure and insulin resistance are likely to be the result of a complex interplay of different mechanisms. However, cortisol-induced effects on muscle blood flow may explain part of both associations.c Numerous studies have shown associations between defects in skin microvascular function and cardiovascular risk factors, such as hypertension, insulin resistance and obesity [START_REF] Shore | Microvascular function in human essential hypertension[END_REF][START_REF] Antonios | Structural skin capillary rarefaction in essential hypertension[END_REF][START_REF] Serné | Capillary recruitment is impaired in essential hypertension and relates to insulin's metabolic and vascular actions[END_REF][START_REF] Serné | Microvascular function relates to insulin sensitivity and blood pressure in normal subjects[END_REF][START_REF] Agapitov | Impaired skeletal muscle and skin microcirculatory function in human obesity[END_REF][START_REF] De Jongh | Impaired microvascular function in obesity: implications for obesityassociated microangiopathy, hypertension, and insulin resistance[END_REF]. In addition, impairment of skin microvascular function is related to increased coronary heart disease risk as estimated by the Framingham risk score [START_REF] Ijzerman | Individuals at increased coronary heart disease risk are characterized by an impaired microvascular function in skin[END_REF]. Furthermore, recent studies have proposed that insulin-mediated effects on microvascular blood flow distribution regulate peripheral muscle glucose uptake [14;15]. Also, defects in skin microvascular function are not only associated with, but also may precede, elevation of blood pressure [START_REF] Noon | Impaired microvascular dilatation and capillary rarefaction in young adults with a predisposition to high blood pressure[END_REF][START_REF] Antonios | Rarefaction of skin capillaries in borderline essential hypertension suggests an early structural abnormality[END_REF][START_REF] Antonios | Rarefaction of skin capillaries in normotensive offspring of individuals with essential hypertension[END_REF]. Moreover, large epidemiological studies have shown that retinal microvascular defects are predictive for the development of hypertension and diabetes mellitus [START_REF] Wong | Retinal arteriolar narrowing and risk of diabetes mellitus in middle-aged persons[END_REF][START_REF] Smith | Retinal arteriolar narrowing is associated with 5-year incident severe hypertension: the Blue Mountains Eye Study[END_REF][START_REF] Ikram | Retinal vessel diameters and risk of hypertension: the Rotterdam Study[END_REF]. Therefore, alterations in microvascular function seem to be particularly important in the development of insulin resistance and hypertension and it's associated cardiovascular risk.

To our knowledge, data on associations of cortisol with microvascular function have hitherto been limited. Previous studies in isolated cells and vessels have shown that corticosteroids impair angiogenesis [START_REF] Cariou | Inhibition of human endothelial cell proliferation by heparin and steroids[END_REF][START_REF] Jung | Inhibition of human angiogenesis with heparin and hydrocortisone[END_REF][START_REF] Jaggers | Potent inhibitory effects of steroids in an in vitro model of angiogenesis[END_REF] and that corticosteroids are able to disrupt microvascular architecture, leading to loss of functional capillaries [START_REF] Jung | Inhibition of human angiogenesis with heparin and hydrocortisone[END_REF]. Also, corticosteroids inhibit the insulin-induced proliferation of capillary endothelium in animal muscle, indicating an opposite effect of insulin and corticosteroids on capillary networks [START_REF] Holmang | Rapid formation of capillary endothelial cells in rat skeletal muscle after exposure to insulin[END_REF]. In addition, Walker and colleagues have previously demonstrated that locally applied corticosteroids induce blanching of human skin, which has been suggested to be due to a vasoconstrictive response in the skin microvasculature [START_REF] Walker | Increased glucocorticoid activity in men with cardiovascular risk factors[END_REF].

Thus, previous studies suggest that corticosteroids may influence both structure and function of the microvasculature.

On the basis of these data, we hypothesized that higher cortisol levels are associated with defects in microvascular function. Furthermore, we hypothesized that these defects may link increased cortisol levels with elevated blood pressure and insulin resistance.

Methods

Subjects

This study is part of a larger project in which vascular and metabolic variables were studied in prepubertal children and their parents [27;28]. In the present study only data of the parents are reported. Couples were contacted by letter and telephone. All

Microvascular measurements

The microvascular measurements were conducted in the morning after 30 minutes of acclimatisation in a quiet, temperature-controlled room (T=23.4±0.4ºC), with individuals in the sitting position and the investigated, non-dominant hand at heart level.

All individuals had abstained from caffeine-containing drinks overnight. Nailfold and iontophoresis studies were performed on the same day and by a single experienced investigator (RGIJ). Perfused nailfold capillaries in the dorsal skin of the third finger were visualised by a capillary microscope, as described previously [10;27;28]. Briefly, nailfold capillaries of the non-dominant hand were recorded before and after 4 minutes of arterial occlusion with a digital cuff. This procedure was performed twice and the mean of both measurements was used for analyses. The number of capillaries at baseline and directly after release of the cuff were counted off-line for respectively 15 and 30 seconds from a freeze-framed reproduction of the videotape and from the running videotape when it was uncertain whether a capillary was present or not. Postocclusive capillary recruitment was calculated as the absolute and relative increase in number of capillaries. The intrasubject coefficient of variation of relative post-occlusive capillary recruitment was 17.2±12.1%, as determined in 7 individuals on two occasions.

Endothelium-dependent and -independent vasodilatation of skin microcirculation was evaluated by iontophoresis of acetylcholine and sodium nitroprusside in combination with laser Doppler fluxmetry as previously described in more detail Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357 [10;27]. Acetylcholine dissolved in mannitol 3% in water (1% acetylcholine; Miochol, Bournonville Pharma, The Netherlands) was delivered using an anodal current (7 doses of 0.1 milliamps (mA) for 20 s with a 60-s interval). Nitroprusside dissolved in water for injection (0.1% sodium nitroprusside; Nipride, Roche, The Netherlands) was delivered using a cathodal current (9 doses of 0.2 mA for 20 s with a 90-s interval).

Acetylcholine-dependent and nitroprusside-dependent vasodilation were measured on the middle phalanx of the third and fourth finger, respectively. Approximately 15 minutes elapsed between these two measurements. The responses to acetylcholine and sodium nitroprusside were calculated as the absolute and relative increase from baseline to the plateau phase (during the final two iontophoretic deliveries). Intrasubject coefficients of variation of the relative increase were 13.5±7.7% for acetylcholine and 18.7±23.4% for sodium nitroprusside, as determined in 7 individuals on two occasions.

Anthropometrical, blood pressure and insulin sensitivity measurements

Anthropometrical measurements were performed twice and the mean of two measurements was used for analyses. Weight and height were determined barefoot and wearing light clothing. Waist and hip circumference were measured as the horizontal circumference at the level of the umbilicus and both great trochanters, respectively. Ambulatory monitoring (Spacelabs 90207, Redmond, Washington, USA) was used to obtain 24-hour recordings of blood pressure and heart rate. The non-dominant arm was used with an appropriately sized cuff. The monitors were programmed to take blood pressure and heart rate readings every 20 minutes from 7.00 -22.00 and every 30 minutes from 22.00 -7.00.

Insulin-mediated glucose disposal was assessed by the hyperinsulinaemic, euglycaemic clamp technique, as previously described [START_REF] Serné | Microvascular function relates to insulin sensitivity and blood pressure in normal subjects[END_REF]. Briefly, insulin (Velosulin; Novo Nordisk, Bagsvaerd, Denmark) was infused in a primed continuous manner at a rate of 60 mU.kg -1 .hour -1 for 2 h. Normoglycaemia was maintained by adjusting the rate of a 20% D-glucose infusion based on plasma glucose measurements performed at 5minute intervals. Whole body glucose uptake (M) was calculated from the glucose infusion rate during the last 60 minutes and expressed per unit of plasma insulin concentration (M/I). For convenience the M/I ratio was multiplied by 100.

Biochemical methods

Fasting blood samples were drawn and samples were stored frozen (-20˚C) until analyses. Plasma glucose, serum total cholesterol, HDL cholesterol and triglyceride concentrations were measured by enzymatic techniques (Hitachi 747 model 100, Roche diagnostics, Mannheim, Germany). LDL cholesterol was calculated by the Friedewald formula. Plasma insulin concentrations were measured by radioimmunoassay techniques (Immunoradiometric Assay, Medgenix Diagnostics, Fleurus, Belgium).

Urinary free cortisol was measured by radioimmunoassay (Coat-A-Count, DPC, Los

Angeles, USA) with inter-assay and intra-assay coefficients of variation of 6% and 9% in 100 and 190 samples, respectively.

Statistical methods

Variables are presented as mean ± standard deviation (SD), or, in case of a nonparametric distribution, as median and interquartile range. Triglycerides, urinary cortisol showed a skewed distribution and the log transformed variables were used in statistical analyses. The independent samples t-test was used to study differences between men and women. Analysis of covariance was used to examine gender differences after adjustment for other variables. Partial correlation analyses were used to investigate relationships among urinary cortisol, capillary recruitment, blood pressure, insulin sensitivity and measures of adiposity. Multiple regression analysis was used to analyse whether the cortisol-blood pressure and the cortisol-insulin sensitivity associations could statistically be explained by differences in skin capillary recruitment or measures of adiposity. Correlation and multiple regression analysis require that, for each variable, measurements are independent. Because our study of 56 adults includes 26 couples and cortisol levels may correlate positively among spouses, we examined the correlation of this variable between men and women. Partial correlation analysis demonstrated plasma cortisol and urinary cortisol were not positively associated between men and women within the couples (r=0.10, p=0.6 and r=-0.06, p=0.8). A twotailed P-value of <0.05 was considered significant. All analyses were adjusted for age.

All analyses were performed on a personal computer using the statistical software package SPSS version 11.0 (SPSS, Chicago, IL, USA).

Results

Characteristics of the study population

Characteristics are presented for men and women separately (Table 1). Men were older and characterized by higher body mass index (BMI), waist circumference, waist-to-hipratio (WHR), systolic blood pressure (SBP), total cholesterol and triglycerides, and by lower acetylcholine-mediated vasodilation and nitroprusside-mediated vasodilation. In addition, men were characterized by higher levels of urinary cortisol excretion and creatinine excretion. Adjustment for age or BMI did not change these findings (data not shown).

Relationships among cortisol, microvascular function, blood pressure, insulin sensitivity and measures of adiposity

Partial correlation analyses demonstrated that greater urinary cortisol excretion was associated with less capillary recruitment in women, but not in men. In addition, in women, urinary cortisol was positively associated with SBP and measures of adiposity, and negatively associated with insulin sensitivity (Table 2 and Figure 1). Urinary cortisol was not associated with acetylcholine-mediated vasodilation in women or men (r=0.17, P=0.4 and r=0.12, P=0.6, respectively) or nitroprusside-mediated vasodilation ( r=-0.25, P=0.2 and r=0.21, P=0.3, respectively). Adjustment for BMI or WHR did not materially affect these associations, except that the association of urinary cortisol with insulin sensitivity in women was no longer significant after adjustment for BMI or WHR (r=-0.19, p=0.4 and r=-0.24, P=0.3, respectively).

Interaction analyses demonstrated significant gender differences in the associations of urinary cortisol with capillary recruitment (P=0.01 for the interaction variable), SBP (P=0.007), BMI (P=0.02) and WHR (P=0.03). Interaction with gender for the association of urinary cortisol with insulin sensitivity tended to be significant (P=0.1). Correction of urinary cortisol for creatinine levels did not significantly change these findings.

Similar conclusions were reached if absolute increases in capillary recruitment, acetylcholine-mediated vasodilation and nitroprusside-mediated vasodilation were used instead of relative increases.

The contribution of capillary recruitment and anthropometrical measures to the cortisol-blood pressure and the cortisol-insulin sensitivity relationship in women

We used multiple regression analyses to investigate whether impaired capillary recruitment could statistically explain (part of) the association of urinary cortisol levels with SBP in women (Table 3). Model 1 demonstrates that cortisol (independent variable) was positively associated with SBP (dependent variable). The regression coefficient of the association between urinary cortisol and SBP decreased by approximately 37% and was no longer significant when capillary recruitment was entered into the regression analysis (model 2). The regression coefficient decreased by 24% but remained significant after introduction of BMI into the regression model (model 3). This suggests that, at least statistically, capillary recruitment and to a lesser extent BMI partly explain the relationship between urinary cortisol and SBP. If capillary recruitment was entered to the model in addition to BMI, the regression coefficient further decreased by 31% indicating that capillary recruitment, at least statistically, explained part of the relationship independently of BMI (model 4). If WHR instead of BMI was added to model 1, the regression coefficient decreased by 12% (β 19.0, 95%-CI 5.3 to 32.7) and remained significant (P=0.009), suggesting that WHR plays a less important role in the relationship between urinary cortisol and blood pressure than BMI.

In order to examine the contribution of capillary recruitment and measures of adiposity to the relationship between urinary cortisol and insulin sensitivity in women, we used insulin sensitivity as dependent variable. Indeed, urinary cortisol was inversely associated with insulin sensitivity in women (β -0.90, 95%-CI -1.78 to -0.02, P<0.05).

Addition of capillary recruitment did not decrease the regression coefficient (β -1.09, 95%-CI -2.24 to 0.07, P=0.06) indicating that capillary recruitment cannot explain the association between urinary cortisol and insulin sensitivity. Addition of BMI or WHR did reduce the regression coefficient with 68% (β -0.29, 95%-CI -1.12 to 0.55, P=0.5) and 47% (β -0.47, 95%-CI -1.39 to 0.44, P=0.3), respectively, indicating a potential role for BMI and WHR in the association between urinary cortisol and insulin sensitivity in women.

Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357

Discussion

The main finding of the present study is an association between urinary free cortisol excretion and capillary recruitment in healthy women and the absence of this relationship in healthy men. Subsequent regression analysis demonstrated that part of the relationship between urinary cortisol excretion and blood pressure in women can, at least statistically, be explained by impaired capillary recruitment.

In the present study we found striking gender differences. Higher urinary cortisol excretion rates in men than in women are in agreement with most [START_REF] Litchfield | Increased urinary free cortisol: a potential intermediate phenotype of essential hypertension[END_REF][START_REF] Lamb | Urine-free cortisol excretion: evidence of sex-dependence[END_REF][START_REF] Dacou-Voutetakis | Urinary free cortisol and its nyctohemeral variation in adolescents and young adults with IDDM: relation to endothelin 1 and indices of diabetic angiopathy[END_REF][START_REF] Masi | Gender and ethnic differences in urinary stress hormones: the population-based Chicago Health, Aging, and Social Relations Study[END_REF][START_REF] Barton | Cortisol production rate and the urinary excretion of 17-hydroxycorticosteroids, free cortisol, and 6 beta-hydroxycortisol in healthy elderly men and women[END_REF], but not all previous studies [START_REF] Finken | Cortisol metabolism in healthy young adults: sexual dimorphism in activities of A-ring reductases, but not 11beta-hydroxysteroid dehydrogenases[END_REF][START_REF] Weaver | Sexual dimorphism in 11 beta hydroxysteroid dehydrogenase activity and its relation to fat distribution and insulin sensitivity; a study in hypopituitary subjects[END_REF][START_REF] Yehuda | Relationship between 24hour urinary-free cortisol excretion and salivary cortisol levels sampled from awakening to bedtime in healthy subjects[END_REF]. These gender differences may, at least partly, be explained by differences in cortisol binding. Earlier studies have reported higher concentrations of cortisol binding globulin in women compared to men [START_REF] Fernandez-Real | Serum corticosteroidbinding globulin concentration and insulin resistance syndrome: a population study[END_REF][START_REF] Stolk | Gender differences in the associations between cortisol and insulin in healthy subjects[END_REF][START_REF] Purnell | Association of 24-hour cortisol production rates, cortisol-binding globulin, and plasma-free cortisol levels with body composition, leptin levels, and aging in adult men and women[END_REF]. To our knowledge, gender differences in relationships between urinary free cortisol and blood pressure or insulin sensitivity have not previously been reported. However, in line with our findings, Stolk and colleagues have previously shown a relationship between plasma free cortisol, assessed by the ratio of fasting cortisol over cortisol binding globulin, and fasting insulin levels, as a measure of insulin resistance, in women, but not in men [START_REF] Stolk | Gender differences in the associations between cortisol and insulin in healthy subjects[END_REF]. There are several explanations for our finding of gender differences in the association of cortisol with capillary recruitment. Firstly, in our study, men were more, in particular centrally, obese than women. Associations among cortisol and other variables may differ within different strata of obesity. Unfortunately, our study population was not large enough to perform analyses stratified for measures of adiposity. Secondly, a sexual dimorphism in activity of 11 β-hydroxysteroid dehydrogenase (11β-HSD), which regulates the interconversion of inactive cortisone to active cortisol in several tissues, has been shown in humans [40;41]. 11β-HSD is also present in vascular endothelial and smooth muscle cells [42;43], which suggests that local 11β-HSD expression may play a role in regulation of local glucocorticoid activity and its effect on vascular function. Gender differences in local vascular 11β-HSD activity might explain differences in associations between urinary cortisol and variables related to vascular function such as capillary recruitment, blood pressure and insulin sensitivity.

Cortisol inhibits endothelium-dependent vasodilation in the human forearm [START_REF] Mangos | Cortisol inhibits cholinergic vasodilation in the human forearm[END_REF] and inhibition of cortisol production prevents stress-related endothelial dysfunction in human resistance vessels [START_REF] Broadley | Inhibition of cortisol production with metyrapone prevents mental stress-induced endothelial dysfunction and baroreflex impairment[END_REF]. We did not find an association between cortisol and impaired endothelium-dependent vasodilation in the skin microvasculature of the hand. This discrepancy may be due to different study designs. We studied cross-sectional data in healthy humans, whereas studies on macrovascular function used interventions to induce cortisol elevation. Another explanation is that hand and forearm (micro)vascular beds may differ in their responses to pharmacological stimuli [START_REF] Benjamin | Measuring forearm blood flow and interpreting the responses to drugs and mediators[END_REF].

Several earlier findings suggest that impairment of tissue capillary perfusion contributes to and precedes the development of hypertension. Hypertension is characterized by increased capillary pressure [47;48], while venous pressure is normal [START_REF] Widgren | Reduced venous compliance in normotensive men with positive family histories of hypertension[END_REF]. This implicates that capillary or postcapillary vascular resistance is increased and explains part of the increase in total peripheral vascular resistance in hypertension. Also, mathematical modeling of in vivo capillary networks shows an exponential relationship between capillary number and network resistance [START_REF] Hudetz | Percolation phenomenon: the effect of capillary network rarefaction[END_REF]. Furthermore, capillary rarefaction has been observed before the development of overt hypertension [START_REF] Noon | Impaired microvascular dilatation and capillary rarefaction in young adults with a predisposition to high blood pressure[END_REF][START_REF] Antonios | Rarefaction of skin capillaries in borderline essential hypertension suggests an early structural abnormality[END_REF][START_REF] Antonios | Rarefaction of skin capillaries in normotensive offspring of individuals with essential hypertension[END_REF] and, moreover, muscle capillary rarefaction predicts the increase in mean arterial pressure in two decades [START_REF] Hedman | Alterations in skeletal muscle morphology in glucose-tolerant elderly hypertensive men: relationship to development of hypertension and heart rate[END_REF], suggesting a pathogenic role. Thus, our findings of an inverse association between capillary recruitment and cortisol in women are in line with a role of impaired capillary perfusion in the development of corticosteroid-induced hypertension in women. However, at this point, it should be emphasized that cause and effect cannot be distinguished in our study because of its cross-sectional design.

Therefore, it cannot be excluded that (part of the) impaired capillary recruitment is a result of the cortisol-induced increase in blood pressure or related mechanisms instead of a pathophysiological link. To further elucidate these issues, intervention studies and studies on the temporal relationships of cortisol-induced hypertension and microvascular defects are warranted.

Previous studies have shown that impairment of capillary recruitment is associated with a reduction of muscle glucose uptake and that physiological coupling between both is plausible [14;15]. However, in the present study, capillary recruitment did not mediate the association between cortisol and insulin sensitivity. A possible explanation for this phenomenon may be that effects of cortisol on nutritive capillary blood flow and concomitant insulin and glucose delivery are overruled by effects on other mechanisms that determine peripheral glucose uptake. Corticosteroids inhibit insulin-induced translocation of glucose transporters [52;53] and influence insulin receptor binding and intracellular second messenger signaling [START_REF] Andrews | Glucocorticoids and insulin resistance: old hormones, new targets[END_REF]. Thus, our finding that capillary recruitment does not mediate the relationship between cortisol and insulin sensitivity may indicate that effects of cortisol on other determinants of peripheral glucose uptake than amount of nutritive flow may be rate-limiting.

A limitation of the present study was that a single 24-hour urine collection was used instead of an average of several collections. 24-Hour urinary sampling may not be practical and may be characterized by low compliance [32;36]. In order to minimize sampling errors in the present study all participants were provided with collection bottles and clear instructions and were questioned afterwards about their compliance. Also, there is uncertainty whether urinary cortisol can best be expressed in term of cortisol/creatinine ratio or total cortisol excretion [START_REF] Greenberg | Urinary creatinine excretion is not stable: a new method for assessing urinary toxic substance concentrations[END_REF]. Correction for the amount of creatinine excreted corrects for urine collection errors. However, it is dependent on diet and muscle mass and therefore is likely to underestimate true urinary cortisol excretion in men compared to women [START_REF] Greenberg | Urinary creatinine excretion is not stable: a new method for assessing urinary toxic substance concentrations[END_REF]. In the present study, correction for urinary creatinine excretion did not change the main findings.

In the present study we examined the contribution of different measures of adiposity to relationships among cortisol, insulin sensitivity and blood pressure.

However, it may be argued whether it is legitimate to consider measures of adiposity as confounder, because different scenarios for the role of adiposity in these relationships can be depicted. There is considerable evidence that elevated cortisol causes fat accumulation at the level of the adipocyte by enhancing lipoprotein lipase activity [START_REF] Bjorntorp | The regulation of adipose tissue distribution in humans[END_REF].

In such a scenario, adiposity is not a confounder of the cortisol -insulin resistance relationship, but rather an intermediary variable. Secondly, adiposity may lead to inappropriately elevated cortisol levels through enhanced responsiveness of the hypothalamic-pituitary-adrenal axis to various stimuli and through peripheral alterations of cortisol production and metabolism [START_REF] Pasquali | Activity of the hypothalamic-pituitaryadrenal axis in different obesity phenotypes[END_REF]. In this scenario adiposity is the pathophysiological first step leading to increased cortisol levels and therefore again should not be considered a confounder. Finally, adiposity may lead to hypercortisolism on the one hand and both insulin resistance and hypertension on the other through nonrelated pathophysiological mechanisms. Only in this scenario adiposity is a true Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357 confounder. These issues are controversial and more work is obviously needed to disentangle these complex interrelationships.

In conclusion, our present data provide the first evidence of an inverse association between urinary cortisol and skin capillary recruitment in healthy women, but not in men. Furthermore, in women, capillary recruitment explains part of the cortisol-blood pressure relationship. However, additional studies are necessary to examine whether capillary recruitment is a true pathophysiological link in this relationship in women. Also, the gender differences remain unexplained in the present study and studies on gender differences in particularly local vascular 11 βhydroxysteroid dehydrogenase activity would of considerable interest. Nevertheless, our findings in healthy women suggest that, on the one hand, any impairment of capillary recruitment may predispose women to develop high blood pressure when exposed to excess cortisol concentrations. On the other hand, improvement of capillary function may partly protect women from the development of cortisol-associated high blood pressure. Therefore, interventions directed at preservation or improvement of microvascular function may be worthwhile in women in whom cortisol excess is to be expected.

Legends to Figures

  participants were of Caucasian origin. After exclusion of individuals with vasoactive medication, diabetes mellitus type 2, rheumatoid arthritis and incomplete data for cortisol measurements (2 individuals), 26 women and 30 men, containing 26 couples, participated in this study. The study was undertaken with approval of the local Ethics Committee and performed in accordance with the Declaration of Helsinki. Informed consent was obtained from each individual before start of the study.

  Correlations among capillary recruitment, urinary cortisol and systolic blood pressure in

Figure 1 .

 1 Figure 1.

Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357

Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357

Table 1 .

 1 Baseline characteristics of the study population Data are presented as mean±SD, or as median (interquartile range). PRH = peak reactive hyperaemia, Ach = acetylcholine, SNP = sodium nitroprusside, PU = perfusion units,*P<0.05, † P<0.01, ‡ P<0.001 men vs. women

	Men	Women

Table 3 .

 3 Regression analysis with 24h systolic blood pressure as dependent variable in

	26 women
		β	95%-CI	P	R square P model
	1. Log urinary cortisol (nmol/24h) 21.5	9.2 to 33.6 0.001	0.35	0.001
	2. Log urinary cortisol (nmol/24h) 13.5	-1.7 to 28.7	0.08	0.43	0.002
	Capillary recruitment (%)	-0.18 -0.40 to 0.03	0.1
	3. Log urinary cortisol (nmol/24h) 16.4	3.2 to 29.5	0.03	0.43	0.001
	BMI (kg/cm 2 )	0.9	-0.1 to 2.0	0.09
	4.Log urinary cortisol (nmol/24h)	11.3	-4.0 to 26.6	0.1	0.47	0.002
	Capillary recruitment (%)	-0.14 -0.36 to 0.08	0.2
	BMI (kg/cm 2 )	0.7	-0.4 to 1.8	0.2
	All models are adjusted for age

Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357

Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

women (left panels) and 30 men (right panels). Clinical Science Immediate Publication. Published on 13 Feb 2007 as manuscript CS20060357 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

R.T. de Jongh is supported by a grant from The Netherlands Organization for Health

Research and Development (ZonMw 940-37-025).


Hans-Joachim Kabitz

Stephan Walterspacher

David Walker

M.D. Wolfram Windisch
email: wolfram.windisch@uniklinik-freiburg.de

Inspiratory muscle strength in chronic obstructive pulmonary disease depending on disease severity

Keywords:

Staging criteria for COPD include symptoms and lung function parameters, but the role of reduced inspiratory muscle strength related to disease severity remains unclear. Therefore, the present study tested if inspiratory muscle strength is reduced in COPD and related to disease severity according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria and to assess its clinical impact.

Maximal inspiratory mouth pressure (PImax), sniff nasal pressure (SnPna) and twitch mouth pressure (TwPmo) following bilateral anterior magnetic phrenic nerve stimulation were assessed in 33 COPD patients (8 GOLD 0 , 6 GOLD I , 6 GOLD II , 7 GOLD III and 6 GOLD IV ) and in 28 matched controls. Furthermore, all participants performed a standardized six minute walking test. PImax (11.6±2.5 vs. 7.3±3.0 kPa; p<0.001), SnPna (9.7±2.5 vs. 6.9±3.3 kPa; p<0.001) and TwPmo (1.6±0.6 vs. 0.8±0.4 kPa; p<0.001) were markedly lower in COPD patients. TwPmo was decreasing with increasing COPD stage. TwPmo was correlated to walking distance (r=0.75; p<0.001), dyspnea (r=-0.61; p<0.001) and blood gas values after exercise (r>0.57; p<0.001 in all instances). Inspiratory muscle strength as reliably assessed by TwPmo decreases with increasing severity of COPD and should be considered as an important factor to rate disease severity and to reflect burden in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is predicted to become the third most frequent cause of death in the world by 2020 [START_REF] Murray | Mortality by cause for eight regions of the world: Global Burden of Disease Study[END_REF]. Treatment guidance adapted to disease severity is rated following staging criteria according to several important guidelines, namely, the American Thoracic, the European Respiratory and the British Thoracic Society as well as the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [START_REF] Sutherland | Management of chronic obstructive pulmonary disease[END_REF]. Within these guidelines, disease severity is rated according to symptoms and forced expiratory volume in one second (FEV 1 ) only.

However, it has been clearly pointed out that FEV 1 , although essential for diagnosis and disease progression, does not adequately reflect all symptomatic manifestations of COPD [START_REF] Celli | The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease[END_REF].

Furthermore, other factors such as body mass index (BMI), dyspnea and exercise capacity have been indicated to be predictive for death, and this has been summarized to the Body-Mass Index, Airflow Obstruction, Dyspnea, and Exercise Capacity (BODE) Index [START_REF] Celli | The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease[END_REF].

In addition to these clinical parameters, inspiratory muscle strength -as assessed by the maximal inspiratory mouth occlusion pressure (PImax) -has been shown to be an independent determinant of survival in COPD patients [START_REF] Gray-Donald | Nutritional status and mortality in chronic obstructive pulmonary disease[END_REF]. Moreover, hypercapnic respiratory failure following inspiratory muscle weakness [START_REF] Begin | Inspiratory muscle dysfunction and chronic hypercapnia in chronic obstructive pulmonary disease[END_REF] is suggested to be the leading cause of death in COPD patients [START_REF] Zielinski | Causes of death in patients with COPD and chronic respiratory failure[END_REF], thus underlining the importance of reduced inspiratory muscle strength in the course of COPD.

However, studies on respiratory muscle function have produced contradicting results [START_REF] Similowski | Assessment of diaphragm function using mouth pressure twitches in chronic obstructive pulmonary disease patients[END_REF], [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], [START_REF] Topeli | Can diaphragmatic contractility be assessed by twitch airway pressures in patients with chronic obstructive pulmonary disease?[END_REF], [START_REF] Levine | Human diaphragm remodeling associated with chronic obstructive pulmonary disease: clinical implications[END_REF], [START_REF] Ottenheijm | Diaphragm dysfunction in chronic obstructive pulmonary disease[END_REF], [START_REF] Ottenheijm | Titin and diaphragm dysfunction in chronic obstructive pulmonary disease[END_REF], and impairments of inspiratory muscle strength have not been related to disease severity in any study. Therefore, the present prospective study was aimed at testing if inspiratory muscle strength is reduced in COPD, and if it is related to disease severity according to GOLD criteria [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF], [START_REF] Who | GOLD[END_REF]. Finally, the clinical impact of reduced inspiratory muscle strength in COPD has been addressed.

Materials and Methods

The protocol was approved by the institutional review board for human studies of the Albert-Ludwig University Freiburg, Germany, and was performed in accordance with the ethical standards stated in the Declaration of Helsinki. Informed written consent was obtained from all participants.

Participants

Thirty-three male outpatients suffering from COPD according to GOLD guidelines [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF], [START_REF] Who | GOLD[END_REF] were studied. All patients included in the study were clinically stable with no episode of exacerbation within two months preceding the study. Medication was provided according to GOLD guidelines [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF], [START_REF] Who | GOLD[END_REF]. None of the patients included in the study received oral steroids or suffered from malnutrition, but further selection was not performed to provide data on a representative collective of COPD patients. In addition, 28 healthy, non-smoking men matched for age and body mass index served as controls. All participants were naive to magnetic stimulation.

Lung function, blood gas analysis, and exercise testing

Lung function parameters were measured immediately prior to tests on inspiratory muscle strength using body-plethysmography (Masterlab-Compact ® , Jaeger, Hochberg, Germany) according to the ERS statement [START_REF] Quanjer | 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[END_REF]. Intrathoracic gas volume (ITGV) assessed during bodyplethysmography was used as the best available measure of functional residual capacity (FRC) in COPD patients [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF]. A standardized six minute walking test was performed with measurement of the six minute walking distance (6MWD), dyspnea assessed by the Borg Dyspnea Scale (BDS) and blood gas analysis [16].

Pressure and airflow recordings

All airflow and pressure recordings were measured using the pneumotachograph ZAN 100 Flowhandy II ® and the pressure transducer ZAN 400 ® (ZAN ® GmbH, Oberthulba, Germany).

PImax sustained for 1 second (PImax 1.0 ) was assessed at residual volume (RV) as previously reported [START_REF] Windisch | Peak or plateau maximal inspiratory mouth pressure: which is best?[END_REF]. Sniff nasal pressure (SnPna) was obtained at FRC based on previous recommendations [START_REF] Heritier | Sniff nasal inspiratory pressure. A noninvasive assessment of inspiratory muscle strength[END_REF]. Twitch mouth pressure (TwPmo) was recorded during bilateral anterior magnetic phrenic nerve stimulation (BAMPS) (Magstim ® 200 2 , Magstim ® Inc., Wales, United Kingdom) [START_REF] Mills | Bilateral magnetic stimulation of the phrenic nerves from an anterolateral approach[END_REF], [START_REF] Polkey | Clinical aspects of respiratory muscle dysfunction in the critically ill[END_REF]. Supramaximality of BAMPS has been reliably demonstrated in several previous studies and was therefore not re-tested in the current protocol [START_REF] Mills | Bilateral magnetic stimulation of the phrenic nerves from an anterolateral approach[END_REF], [START_REF] Man | Postprandial effects on twitch transdiaphragmatic pressure[END_REF], [START_REF] Mador | Bilateral anterolateral magnetic stimulation of the phrenic nerves can detect diaphragmatic fatigue[END_REF]. A fully automated and controlled inspiratory pressure trigger was used at 0.5 kPa, as has been described previously [START_REF] Windisch | Influence of different trigger techniques on twitch mouth pressure during bilateral anterior magnetic phrenic nerve stimulation[END_REF], [START_REF] Kabitz | Controlled twitch mouth pressure reliably predicts twitch esophageal pressure[END_REF]. Furthermore, TwPmo was corrected for lung volume (TwPmo-c), as TwPdi decreases by 0.34 kPa/l above normal ITGV [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF]. The ratio between TwPes and TwPdi, and hence between TwPmo and TwPdi, is about 0.4 in COPD patients [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], given that TwPmo is virtually analogous to TwPes. However, since lung volume correction has been established for twitch pressures only, neither PImax nor SnPna pressures could be corrected for lung volume. To confirm adequacy of pressure transduction as demonstrated previously [START_REF] Kabitz | Controlled twitch mouth pressure reliably predicts twitch esophageal pressure[END_REF], in three COPD patients TwPes, twitch gastric pressure (TwPga) and TwPdi were measured using a thin conventional double-balloon catheter (ZAN ® GmbH, Oberthulba, Germany) according to previous recommendations [25], [START_REF] Anonymous | ATS/ERS Statement on respiratory muscle testing[END_REF]. Breathing frequency (f b ), FRC and the time between triggerimpulse and pressure maximum of TwPmo (t trig-max ) were automatically calculated. The pressure (P trig ) and the inspiratory flow (F trig ) during triggerimpulse were measured to control adherence to trigger criteria [START_REF] Windisch | Influence of different trigger techniques on twitch mouth pressure during bilateral anterior magnetic phrenic nerve stimulation[END_REF].

Statistical analysis

Statistical analysis was performed using Sigma-Stat ® (Version 3.1, Systat Software, Inc., Point Richmond, California, USA). All normally distributed data are presented as mean and standard deviation (SD). The unpaired t-test was used comparing two groups. When comparing more than two groups, one way analysis of variance (ANOVA) was performed using the F-Test. Pearson Product Moment Correlation was used for metric data. Linear regression analysis was performed when appropriate [START_REF] Bland | Regression analysis[END_REF]. Bland and Altman analysis was used for agreement in method comparison [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. A p-value <0.05 was considered statistically significant.

Results

To provide detailed information on disease severity depending data all single stages of COPD according to COLD criteria are presented in Table 1 andTable 2. However, these findings were purely descriptive. Further statistical analysis was performed summarizing early and advanced stages of COPD to assure reasonable number of cases. Therefore, group comparison was performed between control subjects, COPD 0-II and COPD III-IV only.

Anthropometric data and lung function parameters are presented in Table 1. Results of the six minute walking test and blood gas values are given in Table 2. All COPD patients clearly stated that their exercise capacity was limited by dyspnea and not by skeletal muscle exhaustion or weakness of the legs. Volitional (PImax 1.0 , SnPna) and non-volitional (TwPmo) tests on inspiratory muscle strength and corresponding data are shown in Table 3. TwPmo was sufficiently assessed in all patients and in 21 controls. Six controls declined magnetic stimulation and two controls declined sniff maneuvers. In one control subject the generated pressure-time curves did not meet the requirements of previously published criteria [START_REF] Windisch | Influence of different trigger techniques on twitch mouth pressure during bilateral anterior magnetic phrenic nerve stimulation[END_REF]; [START_REF] Kabitz | Controlled twitch mouth pressure reliably predicts twitch esophageal pressure[END_REF]. Values for TwPmo in COPD patients and controls are illustrated in Figure 1. TwPmo-c values are shown in

Discussion

The present study is the first to investigate inspiratory muscle strength in a representative collective of male COPD patients depending on disease severity according to GOLD criteria [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF], [START_REF] Who | GOLD[END_REF]. The major finding is that inspiratory muscle strength decreases with increasing disease severity. Even in patients with mild to moderate COPD, inspiratory muscle strength was markedly reduced compared to healthy matched controls. Interestingly, laboratory studies have recently shown that reduced muscle fiber strength compromising diaphragmatic contractility and reduced passive tension generation are present even in patients with mild to moderate COPD [START_REF] Levine | Human diaphragm remodeling associated with chronic obstructive pulmonary disease: clinical implications[END_REF], [START_REF] Ottenheijm | Diaphragm dysfunction in chronic obstructive pulmonary disease[END_REF], [START_REF] Ottenheijm | Titin and diaphragm dysfunction in chronic obstructive pulmonary disease[END_REF]. Therefore, the present clinical study confirms these laboratory investigations.

However, this finding is in contrast to previous clinical studies where reduced inspiratory muscle strength in COPD was solely attributed to reduced diaphragmatic force generation induced by hyperinflation, rather than compromised diaphragmatic contractility. This was based on the observation that the measured inspiratory muscle strength was suggested not to be reduced when retrospectively corrected for lung volume [START_REF] Similowski | Contractile properties of the human diaphragm during chronic hyperinflation[END_REF], [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], [START_REF] Man | Non-volitional assessment of skeletal muscle strength in patients with chronic obstructive pulmonary disease[END_REF], [START_REF] Laghi | Disorders of the respiratory muscles[END_REF], [START_REF] Man | Magnetic stimulation for the measurement of respiratory and skeletal muscle function[END_REF]. However, only COPD patients with severe to very severe disease were included in these studies, and the stages of disease were not addressed. Furthermore, the present study indicates that inspiratory muscle strength is still reduced even after correction for lung volume. This is supported by the observation that hyperinflation is insufficient in predicting inspiratory muscle strength [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF].

Moreover, inspiratory muscle strength was substantially lost, even early in the course of the disease when hyperinflation was not significantly detectable. Therefore, mechanisms other than hyperinflation must be responsible for reduced inspiratory muscle strength in mild to moderate COPD. In the present study, further reduction in inspiratory muscle strength in severe to very severe stages was observed if TwPmo was not corrected for lung volume. In contrast, when TwPmo was corrected for lung volume there was still a markedly reduction in TwPmo in all COPD patients compared to controls but no significant difference could be detected between early and advanced COPD stages. Therefore, two mechanisms are proposed to be responsible for reduced inspiratory muscle strength in COPD: Firstly, reduced diaphragmatic contractility beginning in early stages of the disease that is independent of hyperinflation; and secondly, reduced diaphragmatic force generation due to hyperinflation in severe to very severe disease stages only.

There is one major reason suggested to be responsible for the discrepancies discussed above: inspiratory muscle strength was assessed by using TwPmo in the present study but TwPdi was used in previous studies [START_REF] Polkey | Exhaustive treadmill exercise does not reduce twitch transdiaphragmatic pressure in patients with COPD[END_REF], [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], [START_REF] Man | Non-volitional assessment of skeletal muscle strength in patients with chronic obstructive pulmonary disease[END_REF]. However, TwPdi is likely to overestimate intrathoracic pressure generation due to an increasing gastric pressure component in patients with increasing hyperinflation [START_REF] Polkey | Exhaustive treadmill exercise does not reduce twitch transdiaphragmatic pressure in patients with COPD[END_REF], [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF]. Since intrathoracic (and not gastric) pressure generation is the clinically relevant part of diaphragm contractility [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], methods that exclusively assess intrathoracic pressure are suggested to be more suitable in rating inspiratory muscle strength when hyperinflation is present.

A point of criticism might be that group comparison is hindered by the fact that there is a statistical difference in age regarding controls and patients with COPD III-IV . Diaphragm strength is known to be slightly reduced in the elderly [34]. However, this reduction occurred when comparing a difference in mean age of 44 years and was considered not to be of functional importance [34]. In addition, a markedly reduction in inspiratory muscle strength was observed in patients with COPD 0-II as outlined above. As this group showed no difference in age compared to controls, findings on reduced inspiratory muscle strength are suggested not to be confused by poorly matched groups. It might be argued that TwPmo, as used in the present study, does not reliably assess intrathoracic pressure generation due to an elevated airway time constant in COPD [START_REF] Marazzini | Diffference between mouth and esophageal occlusion pressure during CO2 rebreathing in chronic obstructive pulmonary disease[END_REF], [START_REF] Murciano | Comparison of esophageal, tracheal, and mouth occlusion pressure in patients with chronic obstructive pulmonary disease during acute respiratory failure[END_REF]. This could lead to a dampened and delayed TwPmo compared to TwPes when measured at relaxed FRC [START_REF] Similowski | Assessment of diaphragm function using mouth pressure twitches in chronic obstructive pulmonary disease patients[END_REF]. Nonetheless, it has been clearly shown that gentle inspiratory efforts can markedly reduce the time constant in healthy subjects [START_REF] Jaeger | Effect of the cheeks and the compliance of alveolar gas on the measurement of respiratory variables[END_REF] and in COPD patients, thus enabling the adequate measurement of TwPmo in COPD [START_REF] Similowski | Assessment of diaphragm function using mouth pressure twitches in chronic obstructive pulmonary disease patients[END_REF]. Moreover, a fully automated and controlled inspiratory pressure trigger was used in the present study. Therefore, a time-delayed pressure transduction could be avoided as indicated by a t trig-max that was non-delayed in COPD patients compared to controls. Additionally, the relationship between TwPmo and TwPes was very close with acceptable limits of agreement in the three COPD patients measured with enteral balloon catheters. Here, it has to be clearly stated that these findings can be used as an indicator only since sample size was too small to generalize these results. Future studies on a representative collective of COPD patients are needed to confirm these findings. Nevertheless, the current authors would suggest that intrathoracic pressure generation was reliably assessed by the measurement of TwPmo. This suggestion is strongly supported by the fact that important clinical parameters such as 6MWD, dyspnea and blood gas values were significantly correlated to inspiratory muscle strength. Therefore, decreased inspiratory muscle strength is suggested to provide a substantial burden for COPD patients. This has only been demonstrated when using TwPmo, thus underlining the importance of non-volitional test for the assessment of inspiratory muscle strength [START_REF] Man | Magnetic stimulation for the measurement of respiratory and skeletal muscle function[END_REF]. However, 6MWD could not be predicted when considering both lung function parameters and TwPmo. This indicates that there is a complex interaction between these important variables with regard to exercise limitation in COPD. Moreover, the relationship between inspiratory muscle strength and endurance in COPD patients was not assessed in the present study, and this should be investigated in future studies. In addition, it has been shown that hypercapnic respiratory failure due to inspiratory muscle weakness is the leading cause of death in patients suffering from COPD [START_REF] Begin | Inspiratory muscle dysfunction and chronic hypercapnia in chronic obstructive pulmonary disease[END_REF], [START_REF] Zielinski | Causes of death in patients with COPD and chronic respiratory failure[END_REF]. In accordance with previous observations [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF], TwPmo was significantly lower in patients with a PaCO 2 2 <43 mmHg. This again underlines the important role of reduction in respiratory muscle strength.

In conclusion, inspiratory muscle strength decreases in COPD patients with increasing disease severity, as rated by GOLD. Two mechanisms are proposed to be responsible for this: compromised diaphragmatic contractility beginning in early disease stages and further reduction in inspiratory muscle strength following hyperinflation in advanced COPD. Reduced inspiratory muscle strength is related to several important clinical parameters such as exercise capacity, dyspnea and gas exchange. However, the importance of reduced respiratory muscle strength compared to impaired lung function with regard to exercise limitation needs further investigation.

Nevertheless, impaired inspiratory muscle function is suggested to provide substantial burden for COPD patients. Therefore, inspiratory muscle strength as assessed by PImax, SnPna and TwPmo should be considered as an important factor to rate disease severity and to reflect disability in COPD.

Tables

Figure 2 .

 2 Figure 2. Correlation analysis between tests on inspiratory muscle strength, lung function parameters and the six-minute walking test with correlation coefficients exceeding 0.55 are presented in Table4. TwPmo-c did not correlate with any of these parameters. In addition, multiple linear regression analysis revealed that neither FEV 1 (p=0.15) and FEV 1 /FVC (p=0.25) nor TwPmo (p=0.07) were significant predictors for 6MWD, although TwPmo almost reached statistical significance.Mean PImax 1.0 (4.8±1.7 kPa), mean SnPna (3.6±1.2 kPa) and mean TwPmo (0.3±0.2 kPa) were significantly lower in COPD patients with a PaCO 2 43 mmHg (n=7), when compared to mean PImax 1.0 (8.0±2.9 kPa), mean SnPna (7.8±3.1 kPa) and mean TwPmo (1.0±0.4 kPa) in those with a PaCO 2 <43 mmHg (n=26) (p<0.01 in all instances). Considering all single twitch replies (n=9) in three COPD patients (GOLD stage I, III, III), mean TwPes was 1.0±0.4 kPa, mean TwPga was -0.9±0.1 kPa and mean TwPdi was 1.7±0.7 kPa.Regression analysis and Bland and Altman analysis was performed to evaluate correct pressure transduction for TwPmo in COPD patients only. No further conclusions were drawn out of these results, since the sample size was too small. Regression analysis revealed a very close relationship between TwPmo and TwPes (r=0.98; p<0.0001). The mean of the difference

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Box plots for lung volume corrected twitch mouth pressure (TwPmo-c) in patients with COPD 0-II , COPD III- IV and controls. TwPmo in controls was not corrected because lung volumes did not exceed normal intrathoracic gas volume.

Clinical Science Immediate Publication. Published on 29 Mar 2007 as manuscript CS20060362 © 2007 The Biochemical Society

Clinical Science Immediate Publication. Published on 29 Mar 2007 as manuscript CS20060362 © 2007 The Biochemical Society

Clinical Science Immediate Publication. Published on 29 Mar 2007 as manuscript CS20060362 © 2007 The Biochemical Society

Table 1 .

 1 Anthropometric data and lung function parameters in patients with COPD 0-IV and controls.

	n	Age	BMI	Smoking	FEV 1	FVC	FEV 1 /FVC	TLC	RV	ITGV
		[years]	[kg/m 2 ]	[pack years]	[%pred]	[%pred]	[%]	[%pred]	[%pred]	[%pred]

Table 2 .

 2 Blood gas analysis and data from six minute walking test in patients with COPD 0-IV and controls.

	n	6MWD	BDS	PaO 2		PaCO 2	pH		SaO 2
		[m]		[mmHg]		[mmHg]			[%]
			rest	exercise	rest	exercise	rest	exercise	rest	exercise

Table 3 .

 3 Tests on inspiratory muscle strength and corresponding data in patients with COPD 0-IV and controls.

		n	PImax 1.0	SnPna	TwPmo	TwPmo-c	t trig-max	F trig	P trig
			[kPa]	[kPa]	[kPa]	[kPa]	[ms]	[ml/s]	[kPa]
	Controls	28	11.6 (2.5)	9.7 (2.5) *	1.6 (0.6) †	1.6 (0.6) †	183 (30) †	34 (20) †	0.5 (0.0) †
	COPD 0-IV	33	7.3 (3.0)	6.9 (3.3)	0.8 (0.4)	1.0 (0.3)	183 (53)	43 (20)	0.5 (0.1)
	p-value		<0.001	<0.001	<0.001	<0.001	NS	0.03	NS
	COPD 0-II	20	8.1 (3.0)	8.2 (3.3)	1.0 (0.3)	1.1 (0.3)	195 (55)	45 (23)	0.5 (0.1)
	COPD III-IV	13	6.2 (2.7)	4.9 (1.9)	0.5 (0.4)	0.9 (0.3)	165 (48)	41 (16)	0.5 (0.0)
	p-value		NS	0.003	<0.001	NS	NS	NS	NS
	COPD 0	8	10.0 (2.8)	10.5 (3.8)	1.2 (0.2)	1.2 (0.2)	178 (34)	48 (22)	0.5 (0.1)
	COPD I	6	7.0 (2.0)	6.6 (2.1)	1.1 (0.2)	1.2 (0.3)	190 (72)	38 (16)	0.5 (0.0)
	COPD II	6	6.6 (2.9)	6.6 (1.2)	0.7 (0.3)	0.8 (0.4)	224 (55)	48 (31)	0.5 (0.0)
	COPD III	7	7.6 (2.6)	6.2 (1.5)	0.7 (0.4)	0.9 (0.4)	185 (44)	43 (17)	0.5 (0.0)
	COPD IV

Clinical Science Immediate Publication. Published on 29 Mar 2007 as manuscript CS20060362 © 2007 The Biochemical Society

Acknowledgements

We would like to thank Roland Merklein (ZAN ® GmbH, Oberthulba, Germany) for software assistance and technical advice.

Controls

28 52 ( 11) [START_REF] Anonymous | ATS/ERS Statement on respiratory muscle testing[END_REF] (3) 0 (0) 105 [START_REF] Quanjer | 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[END_REF] 105 ( 16) 81 ( 5) 97 [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF] 106 [START_REF] Anonymous | ATS/ERS Statement on respiratory muscle testing[END_REF] 86 [START_REF] Quanjer | 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[END_REF] COPD 0-IV 33 57 ( 13) 26 ( 4) 37 [START_REF] Anonymous | ATS/ERS Statement on respiratory muscle testing[END_REF] 63 [START_REF] Laghi | Disorders of the respiratory muscles[END_REF] 83 ( 29) 59 ( 14 4) 83 [START_REF] Similowski | Assessment of diaphragm function using mouth pressure twitches in chronic obstructive pulmonary disease patients[END_REF] Values are expressed as mean (SD). BDS = Borg Dyspnea Scale; 6MWD = distance walked in 6 minutes; NS = not