Detection of Erythropoietin in Exhaled Breath Condensate of Nonhypoxic Subjects Using a Multiplex Bead Array

As a noninvasive method, exhaled breath condensate (EBC) has gained importance to improve monitoring of lung diseases and to detect biomarkers. The aim of the study was to investigate, whether erythropoietin (EPO) is detectable in EBC. EBC was collected from 22 consecutive patients as well as from healthy individuals. Using a multiplex fluorescent bead immunoassay, we detected EPO in EBC, as well as tumour necrosis factor-α (TNF-α) in 13 out of 22 patients simultaneously (EPO 0.21 ± 0.03 in U/mL and TNF-α 34.6 ± 4.2 in pg/mL, mean ± SEM). No significant differences for EPO levels or correlation between EPO and TNF-α were found but TNF-α was significantly higher in patients with chronic obstructive pulmonary disease (COPD) than in non-COPD (obstructive sleep apnoea, OSA, and lung healthy patients). This is the first report of detection of EPO in EBC. Due to the small study size more data is needed to clarify the role of EPO in EBC

Signatures of three-nucleon interactions in few-nucleon systems

Recent experimental results in three-body systems have unambiguously shown that calculations based only on nucleon-nucleon forces fail to accurately describe many experimental observables and one needs to include effects which are beyond the realm of the two-body potentials. This conclusion owes its significance to the fact that experiments and calculations can both be performed with a high accuracy. In this review, both theoretical and experimental achievements of the past decade will be underlined. Selected results will be presented. The discussion on the effects of the three-nucleon forces is, however, limited to the hadronic sector. It will be shown that despite the major successes in describing these seemingly simple systems, there are still clear discrepancies between data and the state-of-the-art calculations.Comment: accepted for publication in Rep. Prog. Phy

Short Communication Detection of Erythropoietin in Exhaled Breath Condensate of Nonhypoxic Subjects Using a Multiplex Bead Array

As a noninvasive method, exhaled breath condensate (EBC) has gained importance to improve monitoring of lung diseases and to detect biomarkers. The aim of the study was to investigate, whether erythropoietin (EPO) is detectable in EBC. EBC was collected from 22 consecutive patients as well as from healthy individuals. Using a multiplex fluorescent bead immunoassay, we detected EPO in EBC, as well as tumour necrosis factor-α (TNF-α) in 13 out of 22 patients simultaneously (EPO 0.21 ± 0.03 in U/mL and TNF-α 34.6 ± 4.2 in pg/mL, mean ± SEM). No significant differences for EPO levels or correlation between EPO and TNF-α were found but TNF-α was significantly higher in patients with chronic obstructive pulmonary disease (COPD) than in non-COPD (obstructive sleep apnoea, OSA, and lung healthy patients). This is the first report of detection of EPO in EBC. Due to the small study size more data is needed to clarify the role of EPO in EBC. Copyright © 2006 Christian Schumann et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION Exhaled breath condensate (EBC) has gained increasing importance in noninvasive monitoring of airway inflammation EPO is a 30.4 kd glycoprotein, which has a span of 165 amino acids. It has glycosylated chains, which are necessary for the biological function. Additional organs, apart from the kidney, liver, and uterus, have been found to secrete EPO, including peripheral vascular smooth muscle cells, endothelial cells, and insulin-producing cells Previously published data showed that the erythropoietic response did not correlate with the severity of hypoxia, hypoxemia, or erythrocytosis Mediators of Inflammation 72.4 ± 5.81 pO 2 (mean ±95% Cl), in mmHg 68.5 ± 3.25 pCO 2 (mean ±95% Cl), in mmHg 34.7 ± 1.67 * patients with arterial hypertension (n = 2), aortic valve stenosis (n = 1), atrial fibrillation (n = 2), and stable coronary heart disease (n = 3). of the anaemic patients with COPD elevated EPO levels were found The main purpose of our study was to investigate whether EPO is generally detectable in EBC, as EPO could be a versatile marker for various pathologic conditions, as well as for detection of exogenous supply of EPO in competitive sports or for further treatment strategies in advanced COPD. METHODS Subjects Over a period of 6 weeks, 22 consecutive unselected in-and outpatients of the Section of Pulmonary Medicine, University of Ulm, Germany, were enrolled into the study. All patients were recruited from the lung function lab while routine tests. A detailed overview on patient characteristics is given in Exhaled breath condensate collection EBC was collected according to the ATS/ERS Task Force • C. Figure 1: TNF-α-and EPO levels in exhaled breath condensate (EBC) from 13 nonhypoxic subjects in which both markers could be detected simultaneously, horizontal line = mean. Cytometric bead array A multiplex chemiluminescent immunoassay system (Immulite, DPC Biermann, Germany) was used to detect EPO and TNF-α. The measurement was done according to the manufacturer's protocol. The use of cytometric bead arrays has been validated for the use of EBC previously [4]. The detection limit was 0.24 mIU/mL for EPO and 1.7 pg/mL for TNF-α. Lung function measurement To measure lung function, a bodyplethysmograph was used (MasterScreen Body; Jaeger GmbH, Germany) and for blood gas analysis, a radiometer was used (ABL555, Radiometer A/S, Denmark). Lung function (spirometric parameters: forced expiratory volume in 1 second, FEV 1 , and inspiratory vital capacity, VC) was measured according to European Respiratory Society (ERS) recommendations Statistical analysis Data were expressed as mean values and standard errors of mean (SEM). To compare EPO-and TNF-α levels between subgroups of patients, the Mann-Whitney U test was utilized. A value of P < .05 was considered statistically significant. Statistical analysis was performed with the Statistica software package (StatSoft Inc Tulsa, USA). Graphs were compiled using the GraphPad Prism Software (GraphPad Software, Inc, San Diego, USA). RESULTS EPO was detected in 15 and TNF-α in 13 out of 22 patients. In 13 cases, EPO and TNF-α could be detected simultaneously (EPO 0.21 ± 0.03 U/mL, TNF-α 34.6 ± 4.2 in pg/mL; mean ± SEM), see hypoxia and the smoking status did not influence EPO levels in EBC. In 2 subjects in which EPO was detectable, TNF-α was not traceable. In COPD, TNF-α levels were significantly higher than in OSA and lung healthy patients (pooled as non-COPD, P < .05), see Lung function measurement showed normal lung functions in the majority of individuals, as displayed in DISCUSSION The use of EBC to monitor inflammation in airways is well documented, and might be expanded to other fields of diagnosis In patients with COPD, we noted a trend towards higher EPO levels than those with OSA or in lung healthy patients (pooled as non-COPD), however this was statistically not significant. These were comparable to normal reference values of EPO in serum (differences were not significant). Higher EPO levels in COPD might be expected due to the nature of disease leading to chronic hypoxia. However, it is unclear if EPO or TNF-α can be expected in EBC in all individuals at all times. Furthermore, no reliable data exist concerning the right point in time to measure biomarkers in EBC. TNF-α levels in COPD patients were significantly higher than in OSA patients or lung healthy individuals (pooled as non-COPD). No significant correlation of EPO levels with TNF-α was found. Our results concerning TNF-α were higher but still in the range of data described previously EPO could not be detected in 7 subjects, whereas TNF-α was not measured in 2 cases in which EPO was found. Even though the sampling of EBC was strictly performed according to the ATS/ERS Task Force Report on EBC, the inconstant detection of EPO, respectively, TNF-α might be explained by unresolved and previously reported methodological problems like unknown dilution of each biomarker in EBC Reviewing the literature, some attempts have been made to assess the dilution of substances in EBC samples, which represent more than 99.9% of condensate volumes. Neither the measurement of exhaled volume, exhaled ions, urea, protein concentration nor the conductance of lyophilised samples and external dilution markers (for internal or external standards) could resolve this problem completely To improve the method, combined efforts of all persons involved in collecting and handling of EBC samples are needed. A standardization of the method to collect EBC was recently accomplished and published by the European Respiratory Society (ERS) In severe COPD, anaemia is present up to 23% and higher EPO levels are expectable as disease progresses There is ample evidence that a complex network of inflammatory cytokines and chemokines has a prominent role in mediation and perpetuation of the processes of acute lung injury Detection of EPO in EBC might further be a useful tool in a variety of diseases, however especially in conditions with reduced tissue oxygenation, for example, COPD, pulmonary hypertension, various states of shock and myocardial infarction, but might develop into a useful noninvasive tool for detection of exogenous supply of EPO in competitive sports on the one hand, and for monitoring training effects in legal sports on the other hand, after data on EPO detection are bolstered by other groups and larger trials. In this context, noninvasive measurement of EPO in EBC (eg, in mechanically ventilated patients) might provide new insights into mechanisms of disease and might develop into a tool which allows to early counteract undesirable effects of tissue hypoxia apart from clinical shock as, for example, induction of apoptosis or inflammation. For example, EPO has been linked to cardiac ischemia and reperfusion injury and it has been shown that administration either before or during myocardial ischemia-reperfusion can protect against myocardial cell apoptosis and decrease infarct size, resulting in enhanced cardiac function and improved left ventricular contractility in a rabbit model of infarction Mediators of Inflammation Our results are further in contrast to recent publications that measured levels of biomarkers in EBC vary among each other, due to variable dilution of the ELF droplets with condensed water vapour (approximately up to 20 000-fold) as compared to serum levels To the best of our knowledge, this is the first description of detection of EPO in EBC in patients with COPD and OSA, however the clinical significance remains unclear. ACKNOWLEDGMEN