Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects.

The exhaled breath condensate (EBC) method represents a new, noninvasive way to detect inflammatory and metabolic markers in the fluid that covers the airways [epithelial lining fluid (ELF)]. However, respiratory droplets represent only a very small and variable fraction of the EBC, most (approximately 99.99%) of which is water vapor. Our objective was to show that ELF concentrations could be calculated from EBC values by using any of three dilutional indicators (urea, total cations, and conductivity) in nine normal and nine chronic obstructive lung disease (COPD) subjects. EBC concentrations of Na(+), K(+), Ca(2+), Mg(2+), total cations, urea, and conductivity varied over a 10-fold range among individuals, but concentrations of these constituents (except Ca(2+)) remained well correlated (r(2) = 0.44-0.83, P < 0.001). Dilution (D) of respiratory droplets in water vapor was calculated by dividing plasma concentrations of the dilutional indicators by EBC concentrations. Estimates of D were not significantly different among these indicators, and urea D averaged 10,800 +/- 2,100 (SE) in normal and 12,600 +/- 3,300 in COPD subjects. Although calculated Na(+) concentrations in the ELF were less than one-half those in plasma, and concentrations of K(+), Ca(2+), and Mg(2+) exceeded those in plasma, total cation concentrations in ELF were not significantly different from those in plasma, indicating that ELF is isotonic in both normal and COPD subjects. EBC amylase concentrations (measured with an ultrasensitive procedure) indicated that saliva represented <10% of the respiratory (ELF) droplets in all but three samples. Dilutional and salivary markers are essential for interpretation of EBC studies.     

Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled Breath Condensate pH Levels in Atopic and Healthy Children

Endotoxin, a component of the cell walls of gram-negative bacteria, is a contaminant in organic dusts (house dust) and aerosols. In humans, small amounts of endotoxin may cause a local inflammatory response. Exhaled nitric oxide (eNO) levels, an inflammation indicator, are associated with the pH values of exhaled breath condensate (EBC). This study evaluated seasonal changes on indoor endotoxin concentrations in homes and the relationships between endotoxin exposure and eNO/EBC pH levels for healthy children and children with allergy-related respiratory diseases. In total, 34 children with allergy-related respiratory diseases and 24 healthy children were enrolled. Indoor air quality measurements and dust sample analysis for endotoxin were conducted once each season inside 58 surveyed homes. The eNO, EBC pH levels, and pulmonary function of the children were also determined. The highest endotoxin concentrations were on kitchen floors of homes of children with allergy-related respiratory diseases and healthy children, and on bedroom floors of homes of asthmatic children and healthy children. Seasonal changes existed in endotoxin concentrations in dust samples from homes of children with allergic rhinitis, with or without asthma, and in EBC pH values among healthy children and those with allergy-related respiratory diseases. Strong relationships existed between endotoxin exposure and EBC pH values in children with allergic rhinitis.     

Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution

Bronchoalveolar lavage is a powerful technique for sampling the epithelial lining fluid (ELF) of the lower respiratory tract but also results in a significant dilution of that fluid. To quantify the apparent volume of ELF obtained by bronchoalveolar lavage, urea was used as an endogenous marker of ELF dilution. Since urea diffuses readily through the body, plasma and in situ ELF urea concentrations are identical; thus ELF volume can be calculated using simple dilution principles. Using this approach, we determined that with a standard lavage procedure, the volume of ELF recovered from a normal human is 1.0 +/- 0.1 ml/100 ml of recovered lavage fluid. Time course experiments in which the saline used for lavage was permitted to remain in the lower respiratory tract for various "dwell times" suggested that diffusion of urea from sources other than recovered ELF can contribute to the total urea recovered resulting in an overestimate of the volume of ELF recovered. Thus, while reasonably accurate, the volume of ELF determined by urea must be considered an overestimate, or "apparent" volume. The ELF albumin concentration based on the apparent ELF volume was 3.7 +/- 0.3 mg/ml, a value that is in good agreement with direct measurements made by other techniques in experimental animals. The density of all inflammatory and immune effector cells on the epithelial surface of the lower respiratory tract, based on the apparent ELF volume, was 21,000 +/- 3,000 cells/microliter, a value that is twofold greater than that in blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138-624 nmol l^-1 EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC-H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC-pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

A feasibility study into adenosine triphosphate measurement in exhaled breath condensate: a potential bedside method to monitor alveolar deformation

Recent research suggested an important role for pulmonary extracellular adenosine triphosphate (ATP) in the development of ventilation-induced lung injury. This injury is induced by mechanical deformation of alveolar epithelial cells, which in turn release ATP to the extracellular space. Measuring extracellular ATP in exhaled breath condensate (EBC) may be a non-invasive biomarker for alveolar deformation. Here, we study the feasibility of bedside ATP measurement in EBC. We measured ATP levels in EBC in ten subjects before and after an exercise test, which increases respiratory parameters and alveolar deformation. EBC lactate concentrations were measured as a dilution marker. We found a significant increase in ATP levels in EBC (before 73 RLU [IQR 50–209] versus after 112 RLU [IQR 86–203]; p value 0.047), and the EBC ATP-to-EBC lactate ratio increased as well (p value 0.037). We present evidence that bedside measurement of ATP in EBC is feasible and that ATP levels in EBC increase after exercise. Future research should measure ATP levels in EBC during mechanical ventilation as a potential biomarker for alveolar deformation.     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

Abstract

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the ‘ECoScreen’ in healthy calves (body weight 63–98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system ‘ECoCheck’. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138–624 nmol l^?1 EBC or 0.10–0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC–H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC–pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC–pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

Exercise changes volatiles in exhaled breath assessed by an electronic nose

Exercise-caused metabolic changes can be followed by monitoring exhaled volatiles; however it has not been previously reported if a spectrum of exhaled gases is modified after physical challenge. We have hypothesized that changes in volatile molecules assessed by an electronic nose may be the reason for the alkalization of the exhaled breath condensate (EBC) fluid following physical exercise.Ten healthy young subjects performed a 6-minute running test. Exhaled breath samples pre-exercise and post-exercise (0 min, 15 min, 30 min and 60 min) were collected for volatile pattern ("smellprint") determination and pH measurements (at 5.33 kPa CO2), respectively. Exhaled breath smellprints were analyzed using principal component analysis and were related to EBC pH.Smellprints (p=0.04) and EBC pH (p=0.01) were altered during exercise challenge. Compared to pre-exercise values, smellprints and pH differed at 15 min, 30 min and 60 min following exercise (p<0.05), while no difference was found at 0 min post-exercise. In addition, a significant correlation was found between volatile pattern of exhaled breath and EBC pH (p=0.01, r=-0.34).Physical exercise changes the pattern of exhaled volatiles together with an increase in pH of breath. Changes in volatiles may be responsible for increase in EBC pH.     

Inflammatory mediators in exhaled breath condensate of healthy donors and exacerbated COPD patients

Samples of exhaled breath condensate (EBC) provide a convenient and non-invasive method to study inflammation in lung diseases. The aim of the present study was to evaluate and compare the inflammatory protein mediator levels in EBC from healthy donors (HD) and from patients with exacerbation of chronic obstructive pulmonary disease (COPD) using an EBC collection device with and without a coating of albumin as a carrier. We studied 13 HD and 26 patients with exacerbation of COPD. The concentrations of myeloperoxidase (MPO), IFNγ and secretory leukocyte protease inhibitor (SLPI) in EBC were measured by immunoassays. The EBC samples from HD and COPD patients showed higher concentrations of MPO when samples were recovered with an albumin-coated device. Furthermore, levels of MPO in COPD patients were significantly higher than in HD. An inverse correlation was observed between MPO and spirometric parameters (FVC and FEV1). Almost all samples collected with the albumin-coated device showed higher amounts of IFNγ and SLPI than those collected with the uncoated device. The levels of SLPI in COPD patients were significantly higher than in HD. A direct correlation was observed between FVC% predicted and SLPI. We concluded that coating the collection device with albumin increased the sensitivity of the technique, at least for measurements of MPO, SLPI and IFNγ. Furthermore, the higher levels of MPO and SLPI and lower levels of IFNγ in EBC from COPD patients could reflect the immunological status and the response of lung parenchyma to treatment during the exacerbation of the illness.     

pH-dependent effects of Cr(NH3)2ATP on kinetics of yeast hexokinase PII. Relationship to the slow transition mechanism.

The effect of Cr(NH3)2ATP, a virtually inert, inner sphere metal-ligand complex, on the kinetics of purified yeast hexokinase PII has been studied at pH 6.5 and pH 7.5. At pH 6.5, where the normal assays exhibit a slow burst-type transient, low concentrations of Cr(NH3)2ATP were found to activate both phii, the initial velocity, and phiII, the steady state velocity. At higher concentrations, Cr(NH3)2ATP was found to be a competitive inhibitor versus MgATP for both phii and phiII. The apparent Ki values for both velocities were the same. The inhibition by Cr(NH3)2ATP at pH 6.5 was found to be a slow process with half-times similar to those found for the normal burst-type transient at this pH value. At pH 7.5, where normal assays exhibit linear progress curves, Cr(NH3)2ATP behaved similarly to that observed before at pH 7 (Danenberg, D. D., and Cleland, W. W. (1975) Biochemistry 14, 28-39), i.e. it was a competitive inhibitor versus MgATP and it caused a slowing of the reaction rate over the first several minutes. The apparent Ki for the initial velocity was 8-fold higher than the apparent Ki for the steady state velocity, suggesting tighter binding of Cr(NH3)2ATP with time. Preincubation experiments indicated that the normal pH 6.5 burst-type transient could be eliminated by appropriate preincubation with Cr(NH3)2ATP and a sugar. In agreement with Danenberg and Cleland (1975), similar preincubations have been shown to produce linear assays at pH 7.5 in the presence of Cr(NH3)2ATP. Similar results were seen with MgITP as the nucleotide substrate, where a burst-type transient is not seen at either pH value under normal assay conditions. At pH 7.5, a slow decrease in the reaction rate is seen over the first several minutes in the presence of Cr(NH3)2ATP. The apparent Ki for phii was 7-fold higher than the apparent Ki value for phiII, again suggesting a tighter binding of Cr(NH3)2ATP with time. A similar observation was made at pH 6.5, but the Ki values for phii and phiII were the same, suggesting no tightening of the binding of Cr(NH3)2ATP with time at this pH value. These results suggested that both slow processes reflect the same basic molecular change, but the consequences are different at the two pH values, presumably because of the difference in the charge of the enzyme. The Cr(NH3)2ATP kinetics at pH 6.5 have been interpreted in terms of a modification of the slow transition mechanism for hexokinase (Shill, J. P., and Neet, K. E. (1975) J. Biol. Chem. 250, 2259-2268). It is postulated that glucose and Cr(NH3)2ATP induce the same slow conformational change at pH 6.5 as that induced by glucose and MgATP, which gives rise to the normal burst-type transient. This suggests that Cr(NH3)2ATP may be a useful tool for physical studies to determine the cause of the slow transition of yeast hexokinase. Activation by low concentrations of Cr(NH3)2ATP was interpreted as binding of the nucleotide to an activator site on the enzyme, causing a shift in the distribution of enzyme towards the more active form.     

Evaluation of methodological and biological influences on the collection and composition of exhaled breath condensate

The purpose of this inter-species comparison (calves and pigs) was to identify methodological and biological influences on the collection and composition of exhaled breath condensate (EBC). A total of 352 EBC samples were collected, whilst variables of ventilation were registered in parallel. Partial pressure of carbon dioxide (pCO₂) and pH were analysed in non-degassed EBC samples. The concentration of total protein in EBC was measured colorimetrically. In both species, lung function was evaluated before and after EBC collection. Statistical analyses were performed to study the effect of EBC collection on lung function and to identify the influence of ventilatory variables on the collection and composition of EBC. Collection of EBC did not affect lung function. Despite the volume of EBC collected per unit time being primarily dependent on ventilation per unit time, species-specific conditions during the EBC collection process resulted in different dependences of EBC collection from other variables of ventilation (i.e. maximal airflow during expiration or expired tidal volume kg^-1 body weight). The concentration of protein ml^-1 EBC increased with the expired volume per min and with peak expiratory flow. Although the pCO₂ in fresh EBC was significantly negatively dependent on the duration of collection, comparable pHs (5.6 - 6.2) were measured in EBC of both calves and pigs. The obtained data may help one standardize EBC collection in different species.     

Evaluation of methodological and biological influences on the collection and composition of exhaled breath condensate

Abstract

The purpose of this inter-species comparison (calves and pigs) was to identify methodological and biological influences on the collection and composition of exhaled breath condensate (EBC). A total of 352 EBC samples were collected, whilst variables of ventilation were registered in parallel. Partial pressure of carbon dioxide (pCO₂) and pH were analysed in non-degassed EBC samples. The concentration of total protein in EBC was measured colorimetrically. In both species, lung function was evaluated before and after EBC collection. Statistical analyses were performed to study the effect of EBC collection on lung function and to identify the influence of ventilatory variables on the collection and composition of EBC. Collection of EBC did not affect lung function. Despite the volume of EBC collected per unit time being primarily dependent on ventilation per unit time, species-specific conditions during the EBC collection process resulted in different dependences of EBC collection from other variables of ventilation (i.e. maximal airflow during expiration or expired tidal volume kg^?1 body weight). The concentration of protein ml^?1 EBC increased with the expired volume per min and with peak expiratory flow. Although the pCO₂ in fresh EBC was significantly negatively dependent on the duration of collection, comparable pHs (5.6???6.2) were measured in EBC of both calves and pigs. The obtained data may help one standardize EBC collection in different species.     

Breath condensate hydrogen peroxide correlates with both airway cytology and epithelial lining fluid ascorbic acid concentration in the horse

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and alpha-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0 +/- 0.5 micromol/l. 0.4 +/- 0.2 micromol/l and 0.9 +/- 0.2 micromol/l H2O2, respectively; p < 0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r = -0.80; p < 0.0001) and correlated positively with TW inflammation score (r = 0.76, p < 0.0001) and BAL neutrophil count (r = 0.80, p < 0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.     

Exhaled breath condensate pH as a biomarker of COPD severity in ex-smokers

Endogenous airway acidification, as assessed by exhaled breath condensate (EBC) pH, is present in patients with stable COPD. The aim of this study was to measure EBC pH levels in a large cohort of COPD patients and to evaluate associations with functional parameters according to their smoking status.EBC was collected from 161 patients with stable COPD and 112 controls (current and ex-smokers). EBC pH was measured after Argon deaeration and all subjects underwent pulmonary function testing.EBC pH was lower in COPD patients compared to controls [7.21 (7.02, 7.44) vs. 7.50 (7.40, 7.66); p < 0.001] and ex-smokers with COPD had lower EBC pH compared to current smokers [7.16 (6.89, 7.36) vs 7.24 (7.09, 7.54), p = 0.03]. In ex-smokers with COPD, EBC pH was lower in patients with GOLD stage III and IV compared to patients with stage I disease (p = 0.026 and 0.004 respectively). No differences were observed among current smokers with different disease severity. EBC pH levels in ex-smokers were associated with static hyperinflation (as expressed by IC/TLC ratio), air trapping (as expressed by RV/TLC ratio) and diffusing capacity for carbon monoxide, whereas no associations were observed in current smokers.Endogenous airway acidification is related to disease severity and to parameters expressing hyperinflation and air trapping in ex-smokers with COPD. The possible role of EBC pH in COPD needs to be further evaluated in longitudinal studies.     

Solute exchange between the plasma and epithelial lining fluid of rat lungs.

Although the transport of solutes from air spaces to plasma has been extensively studied, comparatively little information is available concerning solute equilibration between the plasma and the epithelial lining fluid (ELF) of air-filled lungs. In the present study, 11 lipophobic indicators varying in molecular mass between 22 and 80,000 Da were injected intravenously and/or intramuscularly into anesthetized rats in a manner designed to keep blood concentrations constant. The animals were killed by rapid lavage of their lungs at various intervals up to 120 min after the injections had been made. Indicator concentrations in the bronchoalveolar lavage (BAL) fluid and plasma were determined, and BAL-to-plasma concentration ratios were calculated for indicators that were injected (exogenous: [14C]urea, 22Na+, [3H]mannitol, 99mTc-diethylenetriaminepentaacetate (a chelate), 51Cr-(ethylene dinitrilo)tetraacetate (a chelate), 113mIn-transferrin, human albumin, and Evans blue-labeled rat albumin) and those that were already present from the plasma and ELF (unlabeled urea, rat albumin, and rat transferrin). Leakage of exogenous indicators in the blood into the BAL fluid was observed during the lavage procedure. Leakage of [14C]urea, 22Na+, and [3H]mannitol exceeded that of the heavier solute molecules. Diffusion of proteins and the labeled chelates into the ELF before lavage occurred at similar rates, suggesting vesicular transport. Use of rapidly diffusible solutes such as urea for determining dilution of ELF by BAL should be accompanied by intravascular injections of labeled solutes to correct for diffusion from the blood during lavage. Alternatively, labeled chelates or serum proteins can be used to estimate dilution of ELF by BAL. Interstitial sampling may be inevitable if the epithelium has been injured before lavage.     

The relation between intracellular pH and rate of protein synthesis in sea urchin eggs and the existence of a pH-independent event triggered by ammonia

The relation between rate of protein synthesis and intracellular pH (pHi) was investigated in the eggs of the sea urchin Strongylocentrotus purpuratus. Increasing external pH (pHo) resulted in raising pHi of eggs and also in increased rate of protein synthesis. Similarly, at constant pHo, adding various concentrations of NH4Cl to eggs caused graded increases of both pHi and protein synthesis. Using various concentrations of NH4Cl at a low pHo and incubating eggs at high pHo, we compared protein synthesis under similar pHi conditions and this revealed that at least half the increased protein synthesis stimulated by NH4Cl is independent of induced rise of pHi, as also seems to be chromosome condensation which was never observed in eggs incubated at high pHoS. The additional pH-independent event triggered by NH4Cl does not appear related to elevated free Ca2+, since protein synthesis and chromosome condensation do not require external Ca2+ and no increases of free Ca2+ sufficient to activate the Ca2+-calmodulin-mediated enzyme NAD kinase occurred. Monensin disrupts intravesicular pH gradients but does not stimulate protein synthesis, indicating that this local effect, also promoted by NH4Cl, is not involved in ammonia-induced increase of protein synthesis. Using two other amines which have low pKa values, benzocaine and tricaine, we observed 2-fold increases in protein synthesis rates, even though pHi was lowered. While the exact nature of the pH-independent event(s) triggered by NH4Cl, and possibly by other amines, remains unidentified, its possible involvement in normal mitosis is stressed.     

Increase of pro-oxidants with no evidence of lipid peroxidation in exhaled breath condensate after a 10-km race in non-athletes

It is a well-established fact that exercise increases pro-oxidants and favors oxidative stress; however, this phenomenon has been poorly studied in human lungs. Pro-oxidative generation (H₂O₂, NO₂ ^?), lipid peroxidation markers (MDA), and inflammation (pH) in exhaled breath condensate (EBC) have been determined through data from 10 active subjects who ran 10 km; samples were obtained immediately before, at 20, and at 80 min post-exertion. In EBC, the concentration of H₂O₂ at 80 min post-exertion was increased. NO₂ ^? concentration showed a tendency to increase at 80 min post-exertion, with no variations in MDA and pH. No variations of NO₂ ^? were found in plasma, while there was an increase of NO₂ ^? at 80 min post-exertion in the relation between EBC and plasma. NO₂ ^? in EBC did not correlate to plasmatic NO₂ ^?, while it did correlate directly with H₂O₂ in EBC, suggesting a localized origin for the exercise-related NO₂ ^? increase in EBC. MDA in plasma did not increase nor correlate with MDA in EBC. In conclusion, high-intensity exercise increases lung-originated pro-oxidants in non-athlete subjects with no evidence of early lipid peroxidation and changes in the pH value in EBC.     

Simultaneous determination of 3-nitrotyrosine, tyrosine, hydroxyproline and proline in exhaled breath condensate by hydrophilic interaction liquid chromatography/electrospray ionization tandem mass spectrometry

The analysis of biomarkers from exhaled breath condensate (EBC) is a non-invasive but challenging method for the detection of pulmonary diseases. The amino acids L-proline (Pro) and l-tyrosine (Tyr) are precursors for two important metabolites, trans-L-4-hydroxyproline (trans-L-4-hydroxypyrrolidin-2-carboxylic acid, t-Hyp) and nitrotyrosine (NT). Whereas t-Hyp is supposed to be a biomarker for lung fibrosis, NT is a promising biomarker for inflammation in airway diseases. Analysis of EBC requires extremely sensitive methods, because the epithelial lining fluid of the lung and upper airway is highly diluted in EBC. The high intra- and interindividual variation of this dilution implicates additional problems for sample collection and the interpretation of EBC results. Hence, our aim was to work out a method that would compensate for these possible dilution effects. We have developed a new, reliable and very sensitive method for the simultaneous determination of Pro, t-Hyp, Tyr and NT from EBC. Except for t-Hyp, we used labelled internal standards (IS) L-proline (13)C(5), (15)N (Pro (13)C(5)), L-tyrosine-(13)C(9) (Tyr (13)C(9)), (13)C(9)-3-nitrotyrosine (NT(13)C(9)), IS for t-Hyp was cis-4-hydroxy-L-proline, which were added to the samples before they were lyophilised for concentration. For the separation of the analytes we used hydrophilic interaction liquid chromatography (HILIC), coupled to tandem-mass-spectrometry (MS/MS). The limit of detection (LOD) was 0.5 microg/l for Pro and Tyr and 5 ng/l for t-Hyp and NT. The relative standard deviation (RSD) of the precision from day to day was between 2.6 and 8.0% at spiked concentrations between 4 and 25 microg/l for Pro and between 4.2 and 7.3% for Tyr. The RSD of the precision from day to day was between 7.5 and 13.2% at spiked concentrations between 40 and 250 ng/l for t-Hyp and between 3.5 and 8.2% for NT. The method was established using 27 healthy subjects with a median age of 46 years. Concentrations ranged from 2.8 to 51.9 microg/l for Pro, from <5 to 516.5 ng/l for t-Hyp, from 2.4 to 99.1 for Tyr and for NT concentration ranged between <5 and 1686.5 ng/l.     

Response of rat liver glutaminase to pH, ammonium, and citrate. Possible regulatory role of glutaminase in ureagenesis

Liver glutaminase is stimulated by an increase in NH4+ concentration and NH4+ is an absolute requirement for activity at approximate physiological concentrations of phosphate and glutamine. Increases in the concentration of NH4+ cannot, however, overcome the inhibitory effect of a decrease in pH. In addition, the concentration of NH4+ required for half-maximal rate decreases as pH increases. This decrease is the result of two factors: a direct effect of pH on the apparent affinity of the enzyme for NH4+, and an indirect effect of pH brought about by an increase in the apparent affinity of the enzyme for phosphate which results in a further decrease in the M0.5 for NH4+. In addition, liver glutaminase responds strongly to the concentration of citrate over a physiologically relevant range at approximate physiological concentrations of NH4+, phosphate, and glutamine. An increase in citrate concentration stimulates glutaminase by increasing the affinity of the enzyme for glutamine. The apparent affinity of the enzyme for citrate increases as pH increases. The strong response of liver glutaminase to pH, NH4+, and citrate and the fact that the hydrolysis of glutamine can supply metabolites and effectors for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Biomarkers in breath condensate: a promising new non-invasive technique in free radical research

Oxidative stress is associated with a range of inflammatory lung diseases including asthma, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, lung transplantation, chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis and lung cancer. Increased concentrations of reactive oxygen species (ROS) in the airways of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples, or examine the compartments. As a consequence, there is a need for less invasive approaches to measure oxidative stress. Analysis of breath hydrocarbons has partly fulfilled this need, however only gas phase volatile constituents can be assessed by this approach. The collection of exhaled breath condensate (EBC) is a simple, non-invasive approach, which comprehensively samples the lower respiratory tract. It is currently used as a research and diagnostic tool in the free radical field, yielding information on redox disturbance and the degree and type of inflammation in the lung. With further technical developments, such an approach may ultimately have a role in the clinic, in helping to diagnose specific lung diseases. EBC can be exploited to assess a spectrum of potential biomarkers, thus generating a “finger print” characteristic of the disease. By assessing the nature of oxidative stress in this manner, the most appropriate therapy can be selected and the response to treatment monitored.     

ReviewBiomarkers in Breath Condensate: A promising New Non-invasive Technique in Free Radical Research

Oxidative stress is associated with a range of inflammatory lung diseases including asthma, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, lung transplantation, chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis and lung cancer. Increased concentrations of reactive oxygen species (ROS) in the airways of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples, or examine the compartments. As a consequence, there is a need for less invasive approaches to measure oxidative stress. Analysis of breath hydrocarbons has partly fulfilled this need, however only gas phase volatile constituents can be assessed by this approach. The collection of exhaled breath condensate (EBC) is a simple, non-invasive approach, which comprehensively samples the lower respiratory tract. It is currently used as a research and diagnostic tool in the free radical field, yielding information on redox disturbance and the degree and type of inflammation in the lung. With further technical developments, such an approach may ultimately have a role in the clinic, in helping to diagnose specific lung diseases. EBC can be exploited to assess a spectrum of potential biomarkers, thus generating a “finger print” characteristic of the disease. By assessing the nature of oxidative stress in this manner, the most appropriate therapy can be selected and the response to treatment monitored.