Richard M. Effros

Dilution of respiratory solutes in exhaled condensates.

Most exhaled water is produced as gaseous water vapor, which can be collected in cooled condensers. The presence of nonvolatile solutes in these condensates suggests that droplets of respiratory fluid (RF) have also been collected. However, calculation of RF solute concentrations from condensates requires estimation of the dilution of RF droplets by water vapor. We used condensate electrolyte concentrations to calculate the dilution of RF droplets in condensates from 20 normal subjects. The total ionic concentration (conductivity) was 497 plus minus 68 (mean plus minus SEM) muM. Of this, 229 plus minus 43 muM was NH(4)(+), but little NH(4)(+) was collected from subjects with tracheostomies, indicating oral formation. The Na+ concentration in condensate ([Na+](cond)) averaged 242 plus minus 43 muM. Large variations in [Na(+)](cond) correlated well with variations of K+ in condensate ([K+](cond)) and Cl-) in condensate ([Cl-](cond)), and were attributed to differences in respiratory droplet dilution. Dividing condensate values of ([Na+] + [K+] ) by those of plasma indicated that RF represented between 0.01% and 2.00% of condensate volumes. Calculated values for Na+, K+, Cl-, lactate, and protein in RF were [Na+](RF) = 91 +/- 8 mM, [K+](RF) = 60 +/- 11 mM, [Cl-](RF) = 102 +/- 17 mM, [lactate](RF) = 44 +/- 17 mM, and [protein](RF) = 7.63 +/- 1.82 g/dl, respectively.

Recent Developments in Pulmonary Edema

Research on lung fluid balance and pulmonary edema has increased during the last decade. New approaches have led to insights into the role of each component of the alveolar-capillary barrier. The capillary endothelium is the first line of defense against lung fluid accumulation. The interstitium may play a more important role in lung fluid balance than previously appreciated. Active and passive transport properties of alveolar epithelium may be important in the pathogenesis and resolution of alveolar edema. New methods for the determination of epithelial permeability and lung water are being evaluated. The developments reviewed here may have an impact on the institution of new diagnostic and therapeutic approaches to pulmonary edema during the next decade.

Utility of exhaled breath condensates in chronic obstructive pulmonary disease: a critical review

Purpose of review Evaluation of the utility of exhaled breath condensates in chronic obstructive pulmonary disease. Recent findings Exhaled breath condensates have recently been introduced as a simple, noninvasive method of sampling respiratory fluid in inflammatory lung disorders, including chronic obstructive pulmonary disease. Increases in condensate concentrations of at least 12 markers of inflammation have been reported in these disorders. Furthermore, condensate pH appears to be decreased in both chronic obstructive lung disease and bronchial asthma. This has been referred to as acidopnea and could reflect airway acidification by inflammatory cells. Although safer and more convenient than bronchoalveolar lavage, interpretation of condensate data is complicated by uncertainty regarding the source of condensate solutes and by variable dilution of respiratory droplets from condensed water vapor, which represents more than 99.9% of condensate volumes. This dilution can be estimated from the dilution of plasma constituents such as urea or electrolytes. Because the principal buffer in condensate is NH4+, much of which is derived from bacterial degradation of urea in the mouth, condensate pH measurements may not provide accurate estimates of airway pH. Nevertheless, acidification of condensate may be indicative of gastroesophageal reflux, which frequently occurs in obstructive lung diseases and may contribute to cough and bronchospasm. Summary It is too early to tell how useful condensate studies will be to pulmonary investigators and clinicians. Realization of the enormous potential of this approach will require a thorough understanding of the manner in which these solutions are generated and how they should be analyzed.

Airway synthesis of 20-hydroxyeicosatetraenoic acid: Metabolism by cyclooxygenase to a bronchodilator

Rabbit airway tissue is a particularly rich source of cytochrome P-4504A protein, but very little information regarding the effect(s) of 20-hydroxyeicosatetraenoic acid (20-HETE) on bronchial tone is available. Our studies examined the response of rabbit bronchial rings to 20-HETE and the metabolism of arachidonic acid and 20-HETE from airway microsomes. 20-HETE (10(-8) to 10(-6) M) produced a concentration-dependent relaxation of bronchial rings precontracted with KCl or histamine but not with carbachol. Relaxation to 20-HETE was blocked by indomethacin or epithelium removal, consistent with the conversion of 20-HETE to a bronchial relaxant by epithelial cyclooxygenase. A cyclooxygenase product of 20-HETE also elicited relaxation of bronchial rings. [14C]arachidonic acid was converted by airway microsomes to products that comigrated with authentic 20-HETE (confirmed by gas chromatography-mass spectrometry as 19- and 20-HETE) and to unidentified polar metabolites. [3H]20-HETE was metabolized to indomethacin-inhibitable products. These data suggest that 20-HETE is an endogenous product of rabbit airway tissue and may modulate airway resistance in a cyclooxygenase-dependent manner.

Response of the lungs to aspiration

Aspiration of acid from the stomach and water from the mouth can cause significant lung injury. Animal experiments suggest that acid entering the lungs is normally neutralized by bicarbonate derived from the plasma. It is hypothesized that this process may be impaired in patients with cystic fibrosis and that some of the airway injury that they experience may be related to this defect. This disease is characterized by abnormalities in the cystic fibrosis transmembrane conductance regulator, which normally conducts bicarbonate and chloride exchange. Evidence is discussed regarding the role of water channels (aquaporins) in transporting water from the airspaces into the vasculature.

Asthma: new developments concerning immune mechanisms, diagnosis and treatment.

Purpose of review This brief review discusses how recent research may modify our understanding of the immunology of asthma. Consideration is given to the possible impact that these observations may have upon diagnostic and therapeutic strategies. Recent findings New studies indicate that current conceptions regarding the balance between Th1 and Th2 systems may need modification. The relationship between infection and the development of asthma in children has proven to be much more complex than originally suggested by the ‘hygiene hypothesis’. In addition, important genetic differences have been found in the response of asthmatic patients to therapeutic agents. Summary Greater insight into the mechanisms responsible for asthma and the introduction of new drugs will depend in part upon the development of reliable and simple methods for detecting airway inflammation. As the immunologic aspects of asthma are dissected, we can expect that many more potential targets for treatment will be discovered, but treatment may have to be individualized for genetic differences between different individuals.

Exhaled breath condensates: analyzing the expiratory plume.

The mixture of gases and droplets released during exhalation can be hydrodynamically designated as a “plume.” Traditionally, pulmonologists focused on respiratory gases in these plumes, but recent identification of biomarkers in exhaled breath condensates (EBCs) has initiated vigorous efforts to harness this simple, noninvasive approach for diagnostic purposes (>700 citations in PubMed since 2000). A typical condenser removes about half of the approximately 45 μl of liquid water that can theoretically be extracted from each liter of saturated exhaled air. Nearly all of this water evaporates from lung surfaces, but approximately 1/10,000 of the condensate (∼4.5 nl) represents droplets released from the airway lining fluid (ALF). The assumption that these droplets are released from airways seems justified because alveolar convection is probably negligible and oral contributions can be minimized. ALF droplets can be generated by detachment from airways or rupture of bubbles from previously occluded airways (1), perhaps associated with audible rales. Passage of exhaled air through the mouth at a site near the condenser makes it difficult to avoid contamination of EBC with salivary fluid. To ensure that saliva represents less than 10% of ALF volume, less than 10 nl of saliva should be collected in each milliliter of EBC, a daunting requirement. However, this was successfully accomplished in 36 of 40 samples when the mouthpiece was separated from the condenser, as judged by a sufficiently sensitive amylase assay (2). Nonvolatile biomarkers (e.g., cytokines, ions, and urea) found in EBCs are exclusively derived from ALF droplets (Figure 1) (3–8). Dilution (D) of ALF by water vapor is generally quite variable, but D can be estimated with nonvolatile “dilutional” standard indicators (S), selected to have similar concentrations in ALF and plasma, for example, urea, cations, or conductivity following lyophilization: [nonvolatile]ALF = D × [nonvolatile]EBC, where D = [S]ALF/[S]EBC = [S]plasma/[S]EBC. Figure 1. Contributions of diffusion and convection to formation of the expiratory plume. Rapid diffusion of water (evaporation) occurs from the fluid lining the surfaces of the airways (e.g., bronchi), the airspaces (e.g., alveoli), and mouth into the expiratory ... Lyophilization is used to extract water and volatiles (e.g., NH3 and CO2) from ice at temperatures below −50°C, and should not be confused with centrifugal evaporation at warmer temperatures, which may denature proteins. The EBC samples can be concentrated by reconstituting the dried samples in smaller volumes of pure water. D must be determined before conclusions can be reached about concentrations of nonvolatiles in ALF from EBC measurements. For example, increases in EBC nonvolatile concentrations may reflect collection of more ALF droplets rather than increased ALF concentrations, unless it can be shown that EBC dilution remains relatively unchanged. The reliability of specific dilutional indicators can be checked by showing that alternative markers yield similar values of dilution (3) and by documenting that incorporation of dilutional indicators reduces data variability (5–8). Volatile biomarkers and water directly diffuse as gases into the expiratory airflow from fluid covering both airspaces and airways (epithelial lining fluid). Volatile solutes with tissue–air partition coefficients above 100 (e.g., acetone) are lost primarily from airways, whereas those with lower coefficients are mostly lost from alveoli (9). Correction for dilution of droplets by water vapor is unnecessary if most of the biomarker is gaseous. However, gas phase measurements may be preferable to EBC studies because the distribution of volatiles between exhaled gas, lung fluid, and condensates can be quite variable. Droplet formation can be minimized by keeping exhaled air warm and measuring partial pressures in the gas phase (e.g., with gas chromatography/mass spectrometry). Volatile biomarkers diffuse more rapidly through lipid membranes separating gas, tissue, and blood compartments of the lungs than nonvolatile indicators and are consequently much more likely to be influenced by plasma concentrations and extrapulmonary inflammation. Because they can also diffuse directly from the oral, nasal, and gastrointestinal tracts into the exhaled breath, increases in EBC concentrations of volatile markers may be unrelated to pulmonary disorders. Diffusion and convection play additive roles in the transport of partially ionized acids and bases in exhaled air. However, diffusion of uncharged molecules of many volatile acids and bases overwhelms transport of ions, which is limited by the small volume of airway droplets in the EBC. For example, though concentrations of NH3 are less than 1% those of NH4+ in saliva, diffusion of gaseous NH3 from saliva accounts for most of the NH4+ found in the EBC (3, 10), and this transport promotes alkalinization of EBC. High salivary concentrations of NH4+ and NH3 are maintained by bacterial degradation of urea in a pool of fluid relatively remote from the circulation. Any NH3 that is inhaled from the mouth or produced locally in the lungs should rapidly diffuse into the pulmonary and bronchial microvasculature (11). Early studies suggested that EBC acidification is characteristic of asthma, indicating airway inflammation (12). The fundamental error in most EBC pH studies is the failure to measure the buffering capacities (β) of the ALF and EBC. These are essential for calculating ALF pH. NH4+ is overwhelmingly the most abundant cation in EBC, and it is associated with nearly equivalent concentrations of HCO3− in samples exposed to room air or 5% CO2 (3, 10, 13). Addition of weak or strong nonvolatile acids from the ALF generally has comparatively modest effects on EBC pH, which are difficult to detect. EBCs remain relatively alkaline unless acid concentrations in EBCs approach those of NH4+ (14), which seldom occurs in oral EBCs. A report that EBC pH is not influenced by NH4+ concentrations (15) appears untenable and was not confirmed in subsequent studies (16, 17). Controversy persists regarding the best PCO2 at which pH should be measured, but conventional flushing with argon does not remove all of the CO2, fails to raise pH of EBC to expected levels, and can remove variable amounts of water and other acid–base pairs (10, 16). These problems may explain why EBC acidification was not confirmed in a recent large, multicenter study of individuals with asthma (18) and suggest that conventional studies of EBC pH should be abandoned. In conclusion, EBC studies are best used for investigations of nonvolatile constituents, for example, electrolytes, nongaseous neutral molecules (e.g., sugars and urea), and macromolecules (e.g., cytokines and nucleic acids) in the ALF. In theory, this vast family of potential biomarkers can also be recovered by impaction, bubbling through water, or filtration. The initial enthusiasm regarding EBCs dimmed somewhat when the marked dilution and variability of EBC concentrations were fully appreciated. Although estimates of dilution may reduce variability found in EBC data, progress will require more sensitive and reproducible assays of all indicators. Nevertheless, it would be difficult to exaggerate the importance of noninvasively measuring airway biomarkers, and investigators should be encouraged to perfect analytical and collection techniques.

Protection of the lungs from acid during aspiration.

Unlike the thick mucosa that normally covers the upper gastrointestinal tract, the membranes that cover the distal surfaces of the lungs are remarkably attenuated. This permits rapid exchange of gases between the airspaces and pulmonary vasculature, and may make the lungs more susceptible to acid challenges associated with acid reflux and aspiration. Any injury to the alveolar epithelium could result in the movement of solute and water into the airspaces (chemical pneumonia) and impair gas exchange. In this study, we used a fluorescent approach to compare the relative permeability of the apical basolateral surfaces of the lungs to the exchange of the ionic forms of acids and bases. The apical membranes proved to be much less permeable to NH(4)(+) and HCO(3)(+) than the basolateral membranes. This asymmetry in permeability should enhance resistance of the epithelium to inspired acidic challenges by slowing entry of acid into the cells and by linking the intracellular pH of the alveolar cells to that of the plasma, which is a relatively large, well-buffered compartment. Evidence also was obtained that the acid is secreted by the membranes covering the lungs.

Exhaled breath condensates: a potential novel technique for detecting aspiration

There is an urgent need for diagnostic procedures that can detect aspiration of oral and gastrointestinal (GI) secretions into the respiratory tract. Current approaches are limited by poor sensitivity and specificity. These techniques include (1) adding indicators to feedings; (2) recovery of lipid-filled macrophages in respiratory secretions; (3) measurement of changes in the pH of the upper GI and respiratory tracts; (4) endoscopic visualization of reflux events; and (5) measurement of increased glucose concentrations in respiratory secretions. Ideally, specific markers from various sites in the oral and GI tracts might be discovered in respiratory secretions, but conventional bronchoalveolar lavage for sampling respiratory secretions is not practical and involves some risk. Noninvasive measurements of indicators in the exhaled breath condensates could be used to detect aspiration, but a number of theoretical and practical aspects of such studies must be considered before this approach can be applied to the problem of aspiration.

The Effect of Inhaled Nitric Oxide and Oxygen on the Hydroxylation of Salicylate in Rat Lungs

Inhaled nitric oxide (iNO) is used as a selective pulmonary vasodilator, and often under conditions when a high fraction of inspired oxygen is indicated. However, little is known about the potential toxicity of iNO therapy with or without concomitant oxygen therapy. NO can combine with superoxide (O2−) to form peroxynitrite (ONOO−), which can in turn decompose to form hydroxyl radical (OH·). Both OH· and ONOO− are involved in various forms of lung injury. To begin evaluation of the effect of iNO under either normoxic or hyperoxic conditions on OH· and/or ONOO− formation, rats were exposed for 58 h to either 21% O2, 21% O2 + 10 parts per million (ppm) NO, 21% O2 + 100 ppm NO, 50% O2, 90% O2, 90% O2 + 10 ppm NO, or 90% O2 + 100 ppm NO. We used a salicylate hydroxylation assay to detect the effects of these exposures on lung OH· and/or ONOO− formation measured as the appearance of 2,3-dihydroxybenzoic acid (2,3-DHBA). Exposure to 90% O2 and 90% O2 + 100 ppm NO resulted in significantly (p < 0.05) greater lung wet weight (1.99 ± 0.14 g and 3.14 ± 0.30 g, respectively) compared with 21% O2 (1.23 ± 0.01 g). Exposure to 21% O2 + 100 ppm NO led to 2.5 times the control (21% O2 alone) 2,3 DHBA formation (p < 0.05) and exposure to 90% O2 led to 2.4 times the control 2,3-DHBA formation (p < 0.05). However, with exposure to both 90% O2 and 100 ppm NO, the 2,3-DHBA formation was no greater than the control condition (21% O2). Thus, these results indicate that, individually, both the hyperoxia and the 100 ppm NO led to greater salicylate hydroxylation, but that the combination of hyperoxia and 100 ppm NO led to less salicylate hydroxylation than either did individually. The production of OH· and/or ONOO− in the lung during iNO therapy may depend on the ratio of NO to O2.