Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid.

Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.     

Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.     

The mechanism of oleic acid nitration by *NO(2)

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).     

Reactive absorption of nitrogen dioxide by pulmonary epithelial lining fluid

In a previous study (J. Appl. Physiol. 68: 594-603, 1990) in isolated rat lungs, we suggested that the rate of pulmonary air space absorption of inhaled NO2 is limited, in part, by chemical reaction(s) rather than by physical solubility. Because the initial site of primary absorption interactions involves the epithelial lining fluid (ELF), we investigated whether ELF-NO2 interactions could account for pulmonary NO2 reactive absorption. Rat ELF, obtained by bronchoalveolar lavage (BAL), was compared with a model chemical system (reduced glutathione, GSH). In vitro exposures (NO2-air) used constant gas flow and planar gas-liquid interfaces. 1) Solvent pH notably altered NO2 uptake by GSH but to a lesser extent by BAL. 2) Uptake displayed [GSH]-dependent saturation. [ELF] in BAL was augmented by sequential lavage (lavagate reuse) of multiple lungs. Uptake was proportional to [ELF] but did not saturate under these exposure conditions. 3) The uptake rate exhibited [NO2] dependence. However, relative to increasing [NO2], fractional uptakes decreased for BAL and 1 mM GSH but not for 10 mM GSH. 4) Altered convective gas flow produced nonlinear increments in uptake (10 mM GSH) and substantial decrements in fractional uptake. 5) Arrhenius plots [ln(r) vs. 1/T, where r is reaction rate and T is absolute temperature (degree K)] for BAL and 1 mM GSH yielded respective activation energies of 4,952 and 4,149 kcal.g-1.mol-1 and degree of change in the rate of NO2 uptake per 10 degrees C (Q10) of 1.32 and 1.25. These results imply that the rate of NO2 uptake into rat ELF, like intact lung, is limited, in part, by chemical reaction(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal alveolar epithelial lining fluid contains high levels of glutathione

The epithelial cells on the alveolar surface of the human lower respiratory tract are vulnerable to toxic oxidants derived from inhaled pollutants or inflammatory cells. Although these lung cells have intracellular antioxidants, these defenses may be insufficient to protect the epithelial surface against oxidants present at the alveolar surface. This study demonstrates that the epithelial lining fluid (ELF) of the lower respiratory tract contains large amounts of the sulfhydryl-containing antioxidant glutathione (GSH). The total glutathione (the reduced form GSH and the disulfide GSSG) concentration of normal ELF was 140-fold higher than that in plasma of the same individuals, and 96% of the glutathione in ELF was in the reduced form. Compared with nonsmokers, cigarette smokers had 80% higher levels of ELF total glutathione, 98% of which was in the reduced form. Studies of cultured lung epithelial cells and fibroblasts demonstrated that these concentrations of reduced glutathione were sufficient to protect these cells against the burden of H2O2 in the range released by alveolar macrophages removed from the lower respiratory tract of nonsmokers and smokers, respectively, suggesting that the glutathione present in the alveolar ELF of normal individuals likely contributes to the protective screen against oxidants in the extracellular milieu of the lower respiratory tract.     

Ethanol- and Fe(+2)-induced membrane lipid oxidation is not additive in developing chick brains

In order to study the effects of exogenous EtOH and/or Fe(+2) on membrane lipid peroxidation, exogenous EtOH, FeCl(2), FeCl(2) & EtOH, NaCl and NaCl & EtOH were injected into fertile chicken eggs. Controls were either shams or injected with saline. These injections were made at 0 days or 0-2 days of development and tissue removed at stage 37 (11 days of development). Embryonic exposure to exogenous EtOH and/or Fe(+2) promoted decreased brain mass, decreased levels of brain membrane polyunsaturated fatty acids, elevated levels of brain lipid hydroperoxides, and elevated levels of Fe(+2) within embryonic brain and liver. These alterations were more severe in triple-injected embryos (E0-2/E11) as compared to single-injected embryos (E0/E11). While exogenous treatments of either EtOH and/or FeCl(2) promoted increased levels of endogenous brain Fe(+2), the effects were not additive. These observations are consistent with the hypothesis that embryonic exposure to exogenous EtOH and/or Fe(+2) promotes brain membrane lipid peroxidation.     

Influence of exogenous iron and ascorbate on H2O2-induced glutathione oxidation in red cells

The effective fall in cytosolic reduced glutathione levels in intact red cells exposed to exogenous oxidant stress in the form of Fe2+, H2O2 and ascorbate was caused by H2O2 alone. Relatively high concentrations of Fe2+ had no contributory effect on the oxidizing capacity of H2O2. Ascorbate, at physiological levels, showed no protection whereas glucose was totally protective. Since glucose, via hexose monophosphate shunt, is the only source of reducing equivalent in red cells, the NADPH/NADP+ redox role in the diminution of intracellular reduced glutathione.     

Solute concentrations of the pulmonary epithelial lining fluid of anesthetized rats

Uncertainty persists concerning the best method of estimating the volume and solute concentrations of the pulmonary epithelial lining fluid (ELF) recovered during bronchoalveolar lavage (BAL). In the present study, measurements were made of the BAL-to-plasma concentration ratios of a variety of solutes in an anesthetized rat model. One minute after an intravenous injection of labeled Na+ and urea, 5 ml of isotonic mannitol, saline, or glucose were injected into the trachea and an initial aliquot of the BAL was immediately removed. Initial BAL-to-plasma concentration ratios of urea, Na+, Cl-, Ca2+, and total protein were similar (ranging from 0.013 to 0.017) after BAL with mannitol, but albumin and transferrin ratios were approximately 60% lower and K+ ratios were five times greater. Lavage with saline yielded BAL-to-plasma urea concentration ratios similar to those obtained with mannitol lavage. The BAL-to-plasma specific activity of urea was about twice that of Na+, indicating that urea diffused into the ELF more rapidly than Na+ during the 70 s that elapsed between the time the radioactive urea and Na+ were injected into the circulation and the time when lavage was complete. Subsequent lavage samples also indicated that urea rapidly diffuses into the fluid-filled lungs. These experiments suggest that isotonic mannitol may be a useful solution for lavage, because it allows use of Na+ and perhaps Cl- as additional indicators of ELF dilution by BAL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers

Background

Lung epithelial lining fluid (ELF)—sampled through sputum induction—is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood.

Objectives

To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort.

Methods

Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes.

Results

The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender.

Conclusions

We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

     

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox)

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.     

Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid

Lung epithelial lining fluid (ELF) is a thin layer of plasma ultrafiltrate and locally secreted substances that may provide antioxidant protection and serve as a "front-line" defense for the lower respiratory tract epithelium. To characterize the antioxidant properties of ELF, young, healthy, nonsmoking volunteers underwent bronchoalveolar lavage with determination of ELF volumes and ELF proteins. ELF (greater than 0.4 ml) is a potent inhibitor of lipid peroxidation as measured by malondialdehyde (MDA) production in an in vitro iron-dependent assay system. Two serum proteins, transferrin and ceruloplasmin, were quantitated in ELF and found to be potent inhibitors of lipid peroxidation. Other ELF components, including vitamin E, vitamin C, and albumin, did not function as antioxidants in this system. Several experimental observations suggest that ELF transferrin was more important than ceruloplasmin in inhibiting lipid peroxidation: 1) ELF concentrations of transferrin were 20-fold higher than those for ceruloplasmin; 2) ELF antioxidant activity was abolished by preincubation with Fe3+; 3) ELF antioxidant activity was minimally affected by sodium azide, which is known to inhibit ceruloplasmin ferroxidase activity; and 4) ELF ceruloplasmin ferroxidase activity was virtually nondetectable. ELF possesses a significant antioxidant activity that may be important in vivo in protecting the lung from oxidant injury.     

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.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.     

Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2)

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H(2)O(2)) toxicity and protect cells against H(2)O(2) toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H(2)O(2) toxicity in cultured liver endothelial cells over a wide range of NO and H(2)O(2) concentrations. NO was generated by spermine NONOate (SpNO, 0.001-1 mM), H(2)O(2) was generated continuously by glucose/glucose oxidase (GOD, 20-300 U/l), or added as a bolus (200 microM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H(2)O(2)-induced cell death. SpNO concentrations >0.1 mM were injurious with low H(2)O(2) concentrations, but protective at high H(2)O(2) concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H(2)O(2) steady-state levels in line with inhibition of H(2)O(2) degradation. Thus, the overall effect of NO on H(2)O(2) toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H(2)O(2) levels and enhancement being predominant with high NO and low H(2)O(2) levels.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

Influence of salt concentration on membrane lipids of halophilic bacteria

Abstract A review of salt-dependent changes in membrane lipid composition of halotolerant, moderately halophilic, and extremely halophilic bacteria is presented. The biosynthetic and regulatory mechanisms underlying the observed changes are discussed. Possible implications for the evolution of extreme halophiles and other Archaebacteria are also discussed.