Adenosine triphosphate in exhaled breath condensate of healthy subjects and patients with chronic obstructive pulmonary disease.

OBJECTIVES: The effect of hypoxic relapse of chronic obstructive pulmonary disease (COPD) on lung adenosine triphosphate (ATP) concentration was studied measuring ATP in exhaled breath condensate (EBC). SUBJECTS: Thirty COPD patients with severe exacerbation, thirteen healthy non-smokers and thirteen healthy smokers. METHODS: ATP was detected using a luciferin-luciferase assay, dilution of airway droplets in EBC was assessed measuring sample conductivity. RESULTS: ATP concentrations were similar in COPD patients, non-smoking and smoking healthy individuals (141 +/- 44, 115 +/- 21 and 90 +/- 15 pM; p = 0.66). After treatment oxygenation of COPD patients improved (6.85 +/- 1.29 kPa vs. 8.20 +/- 1.28 kPa, p < 0.001), but EBC ATP concentration was similar to that of admission (p = 0.84). There was no correlation between EBC ATP concentration and airway droplet dilution. CONCLUSION: ATP detected in EBC indicates the presence of ATP in airway lining fluid. Lack of difference in ATP concentration between health and COPD suggests that airway ATP level is under complex control of multiple factors.

Effect of exercise on levels of cyclo-oxygenase mediators in exhaled breath condensate in elite athletes.

AIM: Physical exercise requires adaptation from the airways, which includes bronchodilation. Prostaglandins are involved in airway regulation and their plasma level changes during exercise. The purpose of this study was to investigate the effect of symptom-limited exercise on the levels of prostaglandin E(2) (PGE2) and thromboxane B2 (TXB2) in the airways of elite sportsmen. METHODS: Thirty healthy judo competitors, 15 women and 13 men, aged between 16 and 30 years, participated in this study. Subjects completed a standardized maximal treadmill exercise test. Exhaled breath condensate was collected for non-invasive sampling of the airway lining fluid before and immediately after the exercise. PGE2 and TXB2 levels were determined by commercially available radioimmunoassay. Data are given as median (range). RESULTS: Baseline levels of PGE2 and TXB2 were not different between male and female subjects. Exercise caused a significant increase both in PGE2 and TXB2 concentrations in male subjects (from 180 [100-350] to 240 [115-720] pg/mL, P<0.01 and from 24 [0-80] to 37 [0-110] pg/mL, P<0.05, respectively), but not in female subjects. CONCLUSION: Our data indicate that physical exercise modulates the airway level of PGE2 and TXB2 in healthy subjects. These changes may play an important role in the airway adaptation to exercise.

The effect of allergic rhinitis on adenosine concentration in exhaled breath condensate.

BACKGROUND: Patients with allergic rhinitis (AR) frequently develop asthma. This initiating inflammation in the lower airways may result in increased levels of inflammatory mediators such as adenosine in the exhaled breath. OBJECTIVE: We compared adenosine levels in exhaled breath condensate (EBC) and both exhaled and nasal nitric oxide (NO) levels of AR patients and healthy control subjects. We also tested whether inhalation through inflamed nasal cavity during EBC sampling influences adenosine concentrations in exhaled air. METHODS: Exhaled and nasal NO levels were measured and EBC samples (at oral inhalation) were collected from 27 patients and 15 healthy controls. EBC collection was repeated after 15 min with subjects inhaling through their nose. Adenosine was measured by HPLC and NO was determined by chemiluminescence. RESULTS: The concentration of EBC adenosine was higher in patients with AR than in healthy controls (12.4+/-1.3 nM vs. 6.5+/-0.7 nM, P=0.0019) and this was accompanied by an increase in the concentration of exhaled NO (10.2+/-1.3 ppb vs. 5.3+/-0.5 ppb; P=0.0099, respectively). No difference in nasal NO was detected. EBC adenosine concentration showed a significant positive correlation with the level of exhaled NO. In contrast to healthy control subjects, patients with rhinitis had higher levels of exhaled adenosine when inhaling via the nose instead of the mouth (17.7+/-2.8 nM, P=0.007). CONCLUSION: When compared with healthy subjects, patients with AR exhibit an increased concentration of exhaled adenosine and a related increase in exhaled NO concentration. EBC adenosine is further increased when rhinitis patients inhale through their nose than via their mouth. Our data suggest that non-asthmatic patients with rhinitis may have subclinical inflammation in their lower airways.

Comparative measurement of thromboxane A2 metabolites in exhaled breath condensate by different immunoassays.

OBJECTIVE: Differences between detection techniques may be partly responsible for variable mediator concentrations reported in exhaled breath condensate (EBC). We compared two types of immunoassays to estimate thromboxane A(2) (TxA(2)) concentration. MATERIALS AND METHODS: Thromboxane B(2) (TxB(2)) levels were measured by enzyme immunoassay (EIA) and TxB(2)/2,3-dinor TxB(2) by radioimmunoassay (RIA) in 10 healthy subjects and 13 asthmatic patients. 2,3-Dinor TxB(2) was also determined by a separate EIA. RESULTS: Thromboxane was detected in all samples by RIA, but only in about 75% of samples by EIA. 2,3-Dinor TxB(2) was detected in most samples. There was no agreement between the results of the different immunoassays. As compared to healthy subjects, exhaled breath condensates of asthmatic patients contained significantly more immunoreactivity by RIA and TxB(2) EIA (but not by 2,3-dinor TxB(2) EIA). CONCLUSION: RIA and EIA resulted in vastly different absolute values. The difference found between healthy volunteers and asthmatic patients however, suggests an increased level of TxA(2) in the airways of asthmatics.

Adenosine level in exhaled breath increases during exercise-induced bronchoconstriction.

In asthmatic patients, airway obstruction provoked by exercise challenge is accompanied by an increase in plasma adenosine level. In this study, the current authors investigated if exercise-induced bronchoconstriction was associated with local changes of adenosine concentration in the airways. Oral exhaled breath condensate (EBC) collection (5-min duration) and forced expiratory volume in one second (FEV1) measurements were performed at rest (baseline) and 4-8 times after treadmill exercise challenge in healthy and asthmatic subjects. Adenosine concentration in EBC was determined by HPLC. Observations indicated that physical exercise results in bronchoconstriction together with a significant increase of adenosine level in EBC in asthmatic patients (mean+/-sd maximal fall in FEV1 27+/-13%; associated increase in adenosine 110+/-76% as compared to baseline), but not in healthy control subjects. Exercise-induced changes in adenosine concentration correlated significantly with the fall in FEV1 values in asthmatic patients. In conclusion, the observed increase in adenosine concentration of oral exhaled breath condensate most probably reflects changes in the airways during exercise-induced bronchoconstriction. Due to its known bronchoconstrictor property in asthma, adenosine may contribute to the development of bronchospasm.

Adenosine in exhaled breath condensate in healthy volunteers and in patients with asthma.

Persistent airway inflammation may require the use of different markers for monitoring airway inflammation. In this study, the authors investigated whether adenosine, which may be produced in allergic inflammatory conditions, could be measured with good reproducibility in exhaled breath condensate (EBC), and whether its concentration was elevated in patients with asthma. EBC adenosine and exhaled nitric oxide (eNO), a noninvasive marker of asthmatic airway inflammation, were measured in 40 healthy volunteers and 43 patients with allergic bronchial asthma. Repeatability of adenosine measurement was checked in 20 pairs of samples collected from healthy control subjects. Adenosine was detectable in all EBC samples by the applied high-performance liquid chromatographic method. The mean difference between repeated measurements of adenosine was -0.1 nM and all differences were within the coefficient of repeatability. Adenosine concentration was higher in steroid-naive patients (n=23) compared with healthy control subjects and steroid-treated patients (n=20). In patients with worsening symptoms of asthma (n=23), adenosine concentration was elevated compared with those in a stable condition (n=20). Furthermore, adenosine concentrations were related to eNO levels in asthmatic patients. These results, showing good reproducibility of adenosine measurements and increased adenosine concentrations in steroid-naive patients and in patients with worsening of asthmatic symptoms, indicate that adenosine measurement in exhaled breath condensate might be an acceptable novel method to investigate the role of local production of adenosine in the airways.

Coronary metabolic adaptation restricted by endothelin in the dog heart.

Endothelin elicits long-lasting vasoconstriction in the coronary bed. This remarkable spastic response raises the question whether or not the metabolic adaptive mechanisms of the coronaries are activated under endothelin effect. The role of the compensatory mediators adenosine and inosine was investigated before and after intracoronary (i.c.) administration of endothelin-1 (ET-1, 1.0 nmol) using 1-min reactive hyperemia (RH) tests on in situ dog hearts (n=15) with or without blocking the ATP-sensitive potassium (K+(ATP)) channels by glibenclamide (GLIB, 1.0 micromol min(-1), i.c.). The release of adenosine and inosine via the coronary sinus was measured by HPLC during the first minute of RH. Endothelin-1 reduced baseline coronary blood flow (CBF) and RH response (hyperemic excess flow (EF) control vs. ET-1: 81.7+/-13.6 vs. 43.4+/-10.9 ml, P<0.01), while it increased the net nucleoside release (adenosine, control vs. ET-1: 58.9+/-20.4 vs. 113.7+/-39.4 nmol, P<0.05; inosine: 242.1+/-81.8 vs. 786.9+/-190.8 nmol, P<0.05). GLIB treatment alone did not change baseline CBF but also reduced RH significantly and increased nucleoside release (EF control vs. GLIB: 72.1+/-11.7 vs. 31.9+/-5.5 ml, P<0.01; adenosine: 18.8+/-4.6 vs. 63.0+/-24.8 nmol, P<0.05; inosine: 113.0+/-37.2 vs. 328.2+/-127.5 nmol, P<0.05). Endothelin-1 on GLIB-treated coronaries further diminished RH and increased nucleoside release (EF: 21.5+/-8.0 ml, P<0.05 vs. GLIB; adenosine: 75.3+/-28.1 nmol, NS; inosine: 801.9+/-196.6 nmol, P<0.05 vs. GLIB). The data show that ET-1 reduces metabolic adaptive capacity of the coronaries, and this phenomenon is due to decreased vascular responsiveness and not to the blockade of ischemic mediator release from the myocardium. The coronary effect of ET-1 may partially be dependent on K+(ATP) channels.

Enhanced accumulation of pericardial fluid adenosine and inosine in patients with coronary artery disease.

Adenosine and inosine are believed to have cardioprotective effects. However, little is known about their possible role in the metabolic autoregulation of human coronaries and in pathologic conditions with supply/demand imbalance of the heart such as coronary artery disease. Since these low molecular weight nucleosides freely diffuse through the monolayer of the visceral pericardium, adenosine and inosine concentrations in pericardial fluid may well reflect the conditions in cardiac interstitium. The pericardial fluid and systemic venous blood adenosine and inosine concentrations were measured in 98 human subjects undergoing heart surgery for coronary artery disease or valvular heart disease. Adenosine and inosine concentrations were measured by HPLC with UV detection. In subjects with coronary artery disease pericardial fluid nucleoside concentrations were significantly higher than in patients with valvular heart disease (adenosine: 1545 (996-3146) nmol/L [median (25th-75th quartiles)] vs. 738 (390-2527) nmol/L, P<0.01; inosine: 658 (321-1331) nmol/L vs. 347 (159-1037) nmol/L, P<0.05), while in both patient groups pericardial fluid nucleoside concentrations were higher by an order of magnitude than in venous plasma. Our results show the enhanced release of adenosine and inosine by the ischemic myocardium as a marker of supply/demand imbalance and support the hypothesis that these cardiac nucleosides may have an important role in the adaptation of coronary blood flow in human coronary artery disease.

Elevated circulating adenosine level potentiates antigen-induced immediate bronchospasm and bronchoconstrictor mediator release in sensitized guinea pigs.

BACKGROUND: Adenosine causes bronchoconstriction in asthmatic patients, and it is also accepted that adenosine influences histamine release from activated human mast cells and basophils in vitro. OBJECTIVE: In this study we tested the hypothesis that adenosine potentiates both the airway narrowing and the release of bronchoconstrictor mediators induced by ovalbumin challenge in sensitized guinea pigs. METHODS: After ovalbumin sensitization, 4 groups were studied: control group, adenosine group (ADO), ovalbumin group (OA), and adenosine plus ovalbumin group (ADO + OA). Changes in airway resistance were assessed from continuously recorded pulmonary insuffilation pressure (PIP). The concentration of histamine, PGD2, and thromboxane B2 were determined from bronchoalveolar lavage fluids. RESULTS: Adenosine alone (6 mg/kg intravenously) did not influence baseline values of PIP and the mediator concentrations; however, ovalbumin (10 mg/kg intravenously) increased both the PIP and the levels of the measured mediators compared with the control and ADO groups. When ovalbumin challenge was preceded by adenosine administration, both PIP and mediator levels were significantly enhanced compared with values obtained after simple ovalbumin provocation (ADO + OA vs OA: P <.05). CONCLUSION: These results suggest that adenosine potentiates the airway narrowing induced by ovalbumin challenge and that this effect may develop through facilitation of the release of bronchoconstrictor mediators during the immediate airway response.

Compensation of endothelin-1-induced coronary vasoconstriction.

The vasodilator capacity of the coronaries was determined by the reactive hyperemia (RH) test in open-chest anesthetized dogs. The myocardial release of adenine nucleosides (adenosine and inosine) was measured by the HPLC-UV method. In group I (n = 9) after the control RH test, a bolus injection of endothelin-1 (ET-1; 1.0 nmol i.c.) was administered and was followed by a second RH test. In group II (n = 9), glibenclamide (GLIB) was infused continuously (1.0 mumol/min i.c.) and RH tests were performed during the control period and then before and after bolus injection of ET-1. In contrast to the significant reduction of the RH response after ET-1 in group I and after GLIB in group II, the nucleoside release into the coronary sinus during the first minute of the RH test was significantly higher (adenosine release 0.05 +/- 0.02 vs. 0.10 +/- 0.04 mumol, and 0.02 +/- 0.00 vs. 0.08 +/- 0.02 mumol; p < 0.05). Injection of ET-1 did not result in further RH reduction in GLIB-pretreated dogs (group II) but significantly increased nucleoside release. High doses of ET-1 activated the metabolic compensatory mechanisms of the myocardium and thereby increased the release of adenine nucleosides into the venous blood of the heart. However, whether these metabolites can exert any significant compensatory vasodilator effects appears doubtful.

Effects of arachidonic acid and prostaglandin F2 alpha in isolated rat lungs.

Isolated rat lungs were ventilated and perfused by saline-Ficoll perfusate at a constant flow. The baseline perfusion pressure (PAP) correlated with the concentration of 6-keto-PGF1 alpha the stable metabolite of PGI2 (r = 0.83) and with the 6-keto-PGF1 alpha/TXB2 ratio (r = 0.82). A bolus of 10 micrograms exogenous arachidonic acid (AA) injected into the arterial cannula of the isolated lungs caused significant decrease in pulmonary vascular resistance (PVR) which was followed by a progressive increase of PVR and edema formation. Changes in perfusion pressure induced by AA injection also correlated with concentrations of the stable metabolites (6-keto-PGF1 alpha: r = -0.77, TxB2: -0.76), and their ratio: (6-keto-PGF1 alpha/TXB2: r = -0.73). Injection of 10 and 100 micrograms of PGF2 alpha into the pulmonary artery stimulated the dose-dependent production of TXB2 and 6-keto-PGF1 alpha. No significant correlations were found between the perfusion pressure (PAP) which was increased by the PGF2 alpha and the concentrations of the former stable metabolites. The results show that AA has a biphasic effect on the isolated lung vasculature even in low dose. The most potent vasoactive metabolites of cyclooxygenase, prostacyclin and thromboxane A2 influence substantially not only the basal but also the increased tone of the pulmonary vessels.

Effects of ketotifen and clenbuterol on beta-adrenergic receptor functions of lymphocytes and on plasma TXB-2 levels of asthmatic patients.

Clinical observations indicate that beta-adrenergic drugs may increase bronchial reactivity in asthmatics. To find out possible reasons for this phenomenon the beta-adrenergic receptor function of isolated lymphocytes of asthmatic patients treated with clenbuterol alone or with ketotifen and clenbuterol together were studied. The cAMP levels of lymphocytes stimulated by different doses of isoproterenol were measured by radioimmunoassay and have been compared in the groups of healthies, and asthmatic patients after 3-months running of clenbuterol (Spiropent, Sandoz), as well as in the same asthmatics after one-week running of parallel administration of ketotifen and clenbuterol. There was no difference between the beta-adrenergic receptor function in asthmatic patients treated with clenbuterol alone vs. untreated healthies. Applying ketotifen and clenbuterol together the beta-adrenergic receptor function increased compared to the values obtained after application of clenbuterol alone (intraindividual-control) as well as vs. the group of healthies (control). Data presented support the view that therapeutic doses of selective beta 2-agonists do not lead to damage of the beta-adrenoceptor function. The improvement of receptor function after parallel administration of clenbuterol and ketotifen may be a consequence of the participation of ketotifen in the control of beta-adrenergic receptor system. Thus it seems unlikely that down-regulation of beta-adrenergic receptors is responsible for the beta-agonist induced bronchial hyperreactivity. That's why TXB-2 levels in the plasma of the same asthmatic patients and healthy volunteers were determined by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of propranolol, nalorphine and haloperidol on the breathing and on the metabolism of PGI2 and TXA2 of normotensive and spontaneously hypertensive rats.

The influence of propranolol, nalorphine and haloperidol on the breathing pattern and on the blood levels of cyclooxygenase products of anaesthetized spontaneously-breathing normotensive Wistar rats (WR) and of spontaneously hypertensive rats (SHR) were investigated. The respiratory rate was higher and the effective lung resistance was smaller in the SHR than in the WR. Breathing frequency decreased after nalorphine in both groups, while only in SHR after haloperidol. Propranolol augmented the dynamic lung resistance in both groups. The blood 6-keto-PGF1 alpha level was higher and the TXB2 level was lower in the SHR than in the WR. The central inspiratory activity as well as the levels of peripherally acting substances involved in the regulation of respiration and in the control of bronchial smooth muscle tone are different in the SHR and WR.

Breathing pattern of spontaneously hypertensive rats.

The trachea of rats anaesthetized with sodium pentobarbitone was cannulated and the air flow velocity and the pressure of the oesophagus were measured. In the spontaneously hypertensive rats the breathing frequency was higher, the tidal volume and the effective lung resistance were smaller than that of the normotensive Wistar rats. It seems that the neurohumoral control of respiration in SHR animals differs from that of normotensive rats.

The beta-adrenoceptors of lymphocytes in asthmatic patients treated with beta-agonists.

Numbers of beta-adrenoceptors in intact human lymphocytes of asthmatics treated continuously with the usual doses of beta-agonists have been estimated. Lymphocytes were isolated by density gradient centrifugation as described by BOYUM [4]. The binding experiment was performed with (-)3H-dihydroalprenolol which is a high affinity beta-adrenoceptor antagonist. There was no difference between the mean values of the binding sites of asthmatic patients and those of the normal volunteers. In asthmatic patients, a significant correlation was found between the density of receptors and the percentage increase of FEV1 after salbutamol inhalation. Symptoms of drug tolerance were not observed in patients treated with therapeutic doses of beta-agonists.

Effect of enzyme inducer flumecinolum (Zixoryn) on aminophylline metabolism in rats.

The effect of the enzyme inducer flumecinolum, m-trifluoromethyl-alpha-ethylbenzhydrol (Zixoryn), on aminophylline metabolism was examined in rats. Aminophylline plasma levels were determined by HPLC. Aminophylline T1/2 was 2.85 hours (r = 0.9353) in the pretreated group and 3.75 hours (r = 0.9471) in the untreated control group. Flumecinolum was found to accelerate the elimination of aminophylline and the effect became significant 3.92 hours after the administration of aminophylline.