Hydrogen Peroxide–Scavenging Properties of Normal Human Airway Secretions

Division of Pulmonary and Critical Care Medicine, Department of Anesthesiology, and Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida

	ABSTRACT

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To examine the antioxidant capacity of normal human airway secretions and to characterize its molecular components, tracheal lavages were obtained from eight patients intubated for elective surgery and free of lung disease. These samples (20 µl, approximately 6.8 µg of protein) scavenged 0.57 ± 0.09 nmol of added 0.96 nmol hydrogen peroxide (H₂O₂) within 10 minutes at room temperature (n = 8). The scavenging activity was inhibited 60 ± 4% by azide (an inhibitor of heme-containing peroxidases and catalase) and 42 ± 9% by dapsone (an inhibitor of lactoperoxidase). Mercaptosuccinic acid (an inhibitor of glutathione peroxidase) did not significantly inhibit H₂O₂ scavenging by these secretions. Fourfold diluted secretions showed only nonenzymatic scavenging activity, but the addition of thiocyanate to these samples (0.4 mM; substrate for lactoperoxidase) restored their ability to scavenge H₂O₂. The addition of reduced glutathione (8 µM) only enhanced nonenzymatic scavenging activity. These data provide evidence that multiple enzymatic and nonenzymatic systems coexist in human airway secretions that contribute to H₂O₂ scavenging. It appears, however, that H₂O₂ is mainly consumed by the lactoperoxidase system.

　

Key Words: oxidants • peroxidase • hydrogen peroxide • antioxidants

Reactive oxygen species (ROS) are produced by airway epithelial cells and seem to be specifically released toward the luminal side of the airways (1). Although they likely serve specific functions (e.g., host defenses and signaling), ROS can also damage or adversely affect airway epithelial cells. Naturally occurring antioxidants thus play a major role in protecting the cells from damage and possibly regulating ROS-mediated signaling. Several enzymatic and nonenzymatic ROS scavenging mechanisms have been implicated and identified in airway epithelial surface liquids. Enzymes that provide such a function include airway lactoperoxidase (LPO) (2), glutathione peroxidase (GPx) (3, 4), superoxide dismutase (5), and catalase (6). Nonenzymatic components such as mucins (7) and low molecular weight compounds such as vitamin E (8) may also play a role. Antioxidant functions of the airway surface liquid may be even more important in airway diseases when increased ROS production occurs. For instance, hydrogen peroxide (H₂O₂) was detected at sixfold higher levels in breath condensates from patients with asthma (9) and at 20-fold higher levels in patients with chronic obstructive pulmonary disease (COPD) compared with normal subjects (10).

Although several articles have investigated the role of single enzymes in scavenging ROS and H₂O₂ in particular, no investigation to date has looked at the contribution of each system to the total antioxidant capacity of airway secretions. Furthermore, most studies of human samples have used bronchoalveolar lavages. Although the interpretation of such studies is complicated by contamination with antioxidants derived from the alveolar space, they suggested that GPx is an important scavenger in the airway (3, 4). On the other hand, airway LPO has been found to be the most important H₂O₂ scavenger in sheep airway mucus when only tracheal secretions were collected and examined (2, 11).

Thus, there is uncertainty about the role of each of these enzymatic and nonenzymatic antioxidant defense molecules in the airways. The balance of these systems in protecting the airway epithelium from H₂O₂ (downstream from superoxide dismutase) will depend on both enzyme as well as specific substrate availability. Because catalase is not a secreted extracellular enzyme, H₂O₂ scavenging in normal conditions in which peroxidases from inflammatory cells are absent (such as myeloperoxidase and eosinophil peroxidase) will mainly depend on the availability of GPx and its substrate, reduced glutathione (GSH), as well as on airway LPO and its substrate, thiocyanate (SCN^-). In addition, nonspecific scavengers such as sulfhydryl groups may play a role as well. The purpose of this study was to examine the contribution of the different components of normal human airway secretions (not contaminated by products from the alveolar space) to H₂O₂ scavenging. The results suggested that LPO is the major consumer of H₂O₂ in normal human airway secretions.

	METHODS

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Chemicals
Unless specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Subject Selection and Tracheal Secretions Collection
Eight adults (seven females and one male; age range, 24–64 years), all nonsmokers, without a history of airway or lung disease, receiving no medications used to treat airway or lung disease and undergoing elective outpatient surgery requiring intubation, gave their consent for collecting tracheal secretions. The protocol was approved by the University of Miami Institutional Review Board. Immediately after intubation for surgery, 3 ml of saline was instilled into the trachea through the endotracheal tube and suctioned back with a small catheter into a trap. Recovered secretions were cleared from debris by centrifugation at 16,000 g for 20 minutes at 4°C. The supernatant was aliquoted and stored at -80°C for later analysis.

H₂O₂ Scavenging
The scavenging ability of human tracheal secretions was tested using the phenol red assay as described (12). Briefly, a small amount of tracheal secretions (usually 20 µl, average of 6.8 ± 0.6 µg protein) was placed in a well of a 384-well microtiter plate, and H₂O₂ (0.96 nmol) in phosphate-buffered saline was added for a final concentration of 10 µM. The mixture was allowed to react for 10 minutes at room temperature. After this incubation period, horseradish peroxidase (final concentration, 10 U/ml) and phenol red (final concentration, 1.35 µM) were added. The reaction was stopped 2 minutes later with NaOH (0.01 N final concentration). Absorbance was measured at 610 nm in a microplate reader (Softmax Pro; Molecular Devices, Sunnyvale, CA), and the H₂O₂ concentration was calculated by interpolation from a standard curve. This standard curve showed that the assay was linear between 1 and 15 µM H₂O₂. All assays were performed in duplicates.

Determination of Total Protein and LPO Activity
LPO activity was assayed by oxidation of 3,3',5,5'-tetramethylbenzidine (TMB [2]). To assess the contribution of LPO and MPO to the TMB oxidation activity, dapsone (4,4'-diaminodiphenyl sulfone) was prepared in 0.46 N of HCl and was added to the assay to reach a final concentration of 100 µM, whereas the buffer was adjusted to maintain a pH of 5.2. At this concentration and pH, dapsone inhibits LPO but not MPO (2, 13). Thus, dapsone addition to this assay allowed the distinction between LPO and MPO activity in the samples.

Protein assays were performed using the bicinchoninic acid assay according to the manufacturer's instructions (Pierce, Rockford, IL).

Determination of GPx Activity
GPx activity was determined using a kit obtained from Calbiochem (San Diego, CA). The assay followed the oxidation of nicotinamide adenine dinucleotide phosphate at 340 nm over 3 minutes at room temperature in a microplate using 15 µL of lung lavage in a total volume of 240 µL and tert-butyl hydroperoxide as a substrate.

Immunoblotting
Protein (120 µg as estimated by bicinchoninic acid assay) from two additional lavages was precipitated with 80% acetone and analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions (100 mM of dithiothreitol) according to the method of Laemmli (14). Gels were then electrotransferred (15) to Immobilon-P membrane, blocked with 5% bovine serum albumin, 0.2% Tween-20 in Tris-buffered saline for 1 hour, washed with 0.1% Tween-20 in Tris-buffered saline for 10 minutes three times. The blot was then incubated for 1 hour with rabbit antisheep airway LPO antibody (previously immunoselected against bovine LPO) at a concentration of 1 µg/ml in 100% human serum or with rabbit antihuman MPO (Abcam, Cambridge, UK) at a concentration of 85 µg/ml. After three washes with 0.1% Tween 20 in Tris-buffered saline, the blot was incubated for 1 hour with the secondary antibody goat antirabbit (Kirkegaard and Perry Laboratories, Gaithersburg, MD) at a dilution of 1:10,000 and at 1:25,000 in 100% human serum for the anti-LPO and the anti-MPO antibody, respectively. After three washes with 0.1% Tween-20 in Tris-buffered saline, the blot was developed with Nitro Blue Tetrazolium and 5-Bromo-4-Chloro-3-Indolyl-Phosphate according to the manufacturer's instructions. A control using only secondary antibody was also performed.

Statistical Analysis
One-way analysis of variance was used to compare the means of more than two groups using JMP software from SAS Institute Inc. (Cary, NC). If a significant difference was found, a group-by-group comparison was done using the Tukey-Kramer honestly significant difference test; p < 0.05 was considered significant. Data were expressed as mean ± SEM, with n representing the number of samples.

	RESULTS

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H₂O₂ Scavenging Activity in Airway Secretions
We have previously shown that sheep airway secretions scavenged H₂O₂ (2). To assess similar capabilities of human airway secretions, secretions were diluted into 10 µM of H₂O₂, as no H₂O₂ was detectable in these samples, as assessed by the phenol red assay (less than 1 µM). Then H₂O₂ disappearance was measured. Twenty microliters of human secretions scavenged 0.57 ± 0.09 nmol of H₂O₂ within 10 minutes (n = 8), reducing the original H₂O₂ concentration to 4.1 ± 0.9 µM .

fig.ommitted TABLE 1. Hydrogen peroxide scavenging by human tracheal secretions

 
To assess the contribution of nonenzymatic scavenging, samples were heated to 100°C for 5 minutes. Heat treatment reduced H₂O₂ scavenging activity of airway secretions by 80 ± 3% (n = 8; p < 0.05 compared with no inhibitor). This suggested that a substantial amount of H₂O₂ scavenging activity (approximately 20%) was heat insensitive and presumably nonenzymatic  .

fig.ommitted Figure 1. H2O2 scavenging by normal human airway secretions. Scavenging activity measured by the phenol red assay is displayed in relation to activity measured in the absence of any inhibitors (no inhibitor). Other bars indicate scavenging in the presence of 10 µM azide (azide), 100 µM dapsone (dapsone), 100 µM mercaptosuccinic acid (mercaptosuccinic acid), and after heating to 100°C for 5 minutes (heat); *p < 0.05 compared with no inhibitor. H2O2 scavenging is reduced by heme-peroxidase inhibitors and heat but not by a GPx inhibitor.

 
To differentiate the enzymatic components involved in scavenging and to determine the contribution of each of them to the total scavenging activity, the influence of the following inhibitors was tested: azide (10 µM; a broad inhibitor of heme-containing peroxidases and catalase but not GPx [16]), dapsone (100 µM; an inhibitor of heme-containing peroxidases at the assay pH of 7.4, but not catalase or GPx [13]), and mercaptosuccinic acid (100 µM; an inhibitor of selenium containing GPx [17]). Standard curves for these assays were generated by adding the inhibitors to the control sample buffer that did not contain human secretions. The inhibitors were also tested in specific peroxidase activity assays (see METHODS) and in the phenol red assay using, instead of secretions, purified bovine milk LPO, canine MPO (a gift of Dr. Roger Fenna, University of Miami), and gluthatione peroxidase, respectively  .

fig.ommitted Figure 2. Characterization of different inhibitors on LPO and GPx activity. Shown is the amount of H2O2 scavenged after 10 minutes of incubation assayed by phenol red. LPO (6 ng) was incubated with SCN- (0.4 mM) and 10 µM H2O2 in the absence of any inhibitor (LPO), in the presence of 100 µM of mercaptosuccinic acid (LPO + mercapto), 100 µM dapsone (LPO + dapsone), or 10 µM azide (LPO + azide). The same experiments were performed when GPx (20 mU) was incubated for 10 minutes in the presence of 8 µM GSH and 10 µM H2O2 without any inhibitors (GPx), with 100 µM mercaptosuccinic acid (GPx + mercapto), and with 10 µM azide (GPx + azide). Azide inhibited LPO while having no effect on GPx. Dapsone at 100 µM and pH 7.4 inhibited 70% of LPO. Mercaptosuccinic acid (100 µM) inhibited GPx but did not affect LPO.

 
Azide (10 µM), a broad inhibitor of heme-containing peroxidases and catalase (but not the selenium-containing GPx), reduced the H₂O₂ scavenging activity of airway secretions by 60 ± 4% (n = 8, p < 0.05 compared with no inhibitor; ). Dapsone, an inhibitor of both LPO (70%) and MPO (100%) at pH 7.4 and at 100 µM  (18), reduced the H₂O₂ scavenging activity of airway secretions by 42 ± 9% (n = 8, p < 0.05 compared with no inhibitor; ). No statistically significant difference between azide and dapsone inhibition of H₂O₂ scavenging activity was found.

Mercaptosuccinic acid (100 µM), an inhibitor of GPx with no effect on LPO or MPO , did not influence scavenging activity of airway secretions. The samples still scavenged 0.5 ± 0.08 nmol of H₂O₂ (n = 8, p > 0.05 compared with no inhibitor), decreasing the original scavenging activity by only 12 ± 14% within 10 minutes .

Peroxidase Activities of Human Airway Secretions
TMB oxidation assays were used to assess total heme-containing peroxidase activity in these samples and to quantify LPO activity separate from MPO activity (only LPO is sensitive to 100 µM dapsone at pH 5.2). The average total heme-containing peroxidase activity was 0.29 ± 0.06 ng/µl of lavage of which 0.25 ± 0.06 ng/µl or 86 ± 21% was sensitive to dapsone and thus due to LPO (n = 8; ) . The average protein concentration was 0.34 ± 0.03 µg/µl (n = 8). When comparing the LPO activity with the protein content in each sample, there was a weak correlation with an r² value of 0.51  .

fig.ommitted Figure 3. Relationship between total peroxidase and LPO concentration in normal human airway secretions. Shown is a comparison of total peroxidase concentration, as estimated from TMB oxidation, with the LPO concentration as estimated from dapsone-inhibited TMB oxidation at pH 5.2. The mean total peroxidase concentration was 0.29 ± 0.06 ng/µl and the mean LPO concentration was 0.25 ± 0.06 ng/µl (closed squares).

 

fig.ommitted   Figure 4. Relationship between peroxidase activity and total protein concentration in normal human airway secretions. Peroxidase concentration of each human airway secretion (ng/µl) was measured by TMB oxidation and was plotted against total protein concentration as measured by bicinchoninic acid (µg/µl). The line represents linear regression fit (r2 = 0.51).

 
GPx activity in airway secretions was measured by following the oxidation of nicotinamide adenine dinucleotide phosphate. The lower detection limit of this assay was 5 mU per assay with 1 U of activity defined as the oxidation of 1 µmol nicotinamide adenine dinucleotide phosphate per minute using an extinction coefficient of 6.22 x 10⁶ x M^-1 x cm^-1 for nicotinamide adenine dinucleotide phosphate (19). No GPx activity was detected in any of the eight samples. The lack of measured activity was not due to the absence of substrate because GSH was provided and continuously replenished in these assays (see METHODS). Therefore, the secretions contain less than 0.33 mU/µl GPx if any. These data suggested that virtually all of the measurable peroxidase activity in airway secretions was due to the presence of LPO.

Reconstitution of Scavenging Activity after Dilution by Replenishing Different Substrates
To confirm the data obtained with inhibitors, samples were diluted to reduce substrate availability, and then substrates specific for the different enzymatic activities were readded to assess whether scavenging could be recovered. By diluting samples an additional fourfold, their scavenging ability decreased to 0.24 ± 0.03 nmol of H₂O₂ in 10 minutes , reducing the original 10 µM of H₂O₂ to only 7.5 ± 0.3 µM (n = 8). Heat treatment of fourfold diluted samples did not change scavenging activity (118 ± 8% of diluted, nonheated samples, n = 8, p > 0.05), indicating that diluted samples scavenged in a purely nonenzymatic fashion.

To evaluate whether enzymatic scavenging activity could be restored by specific substrate repletion, SCN^- or GSH was added to the diluted samples to levels thought to be present in airway secretions, and H₂O₂ scavenging was re-evaluated. H₂O₂ scavenging in diluted samples in which SCN^- was restored to 0.4 mM increased to 209 ± 19% of unreplenished, diluted samples (n = 8, p < 0.05; ) . Azide (10 µM) addition to SCN^--replenished samples again reduced H₂O₂ scavenging activity to 96 ± 20% of unreplenished, diluted samples (n = 8, p > 0.05; ). Thus, SCN^- replenishment of diluted samples restored an enzymatic scavenging activity consistent with LPO. This was further supported by the fact that a linear correlation existed between the recovered, SCN^--dependent scavenging activity and the TMB-assessed LPO activity in these samples (r² = 0.74; ) .

fig.ommitted Figure 5. H2O2 scavenging activity of diluted human airway secretions. Human airway secretions were diluted fourfold and H2O2 scavenging was measured by the phenol red assay. Activity is shown normalized to the activity measured in the absence of any inhibitors (No inhibitor). Samples were replenished with 0.4 mM SCN-, 8 µM GSH, 10 µM azide plus SCN-, and azide plus GSH. Heat-treated diluted samples with no inhibitors (no inhibitor + heat) or heat-treated plus SCN- or GSH; *p < 0.05 compared with no inhibitor. The data show that diluted secretions replenished with SCN- recover enzymatic scavenging activity consistent with LPO, whereas the scavenging activity recovered with GSH is nonenzymatic in nature.

 

fig.ommitted Figure 6. Relationship between peroxidase activity and H2O2 scavenging in diluted human airway secretions replenished with SCN- H2O2 scavenging by diluted human airway secretions replenished with SCN- (0.4 mM), the LPO substrate, plotted against peroxidase concentration as measured by TMB oxidation. The line represents linear regression fit (r2 = 0.74).

 
To demonstrate further the enzymatic nature of the recovered scavenging activity by replenishing fourfold diluted tracheal lavages with 0.4 mM of SCN^-, samples were heated to 100°C for 5 minutes. Heated and diluted samples, replenished with SCN^-, scavenged 131 ± 11% of nonheated diluted control subjects (n = 8, p > 0.05 compared with both nonheated, diluted samples and heated, diluted samples; p < 0.05 compared with nonheated, SCN^--replenished, diluted samples; ). Thus, SCN^- replenishment of diluted samples restored an enzymatic scavenging activity consistent with LPO.

Diluted samples in which GSH was replenished to 8 µM also revealed increased scavenging activity: These samples scavenged 171 ± 8% of nonheated, diluted samples (n = 8, p < 0.05). This activity could not be due to GPx because it would have reflected an amount of enzyme 40 times the detection limit of the specific GPx assays discussed previously here. Because no GPx activity was detected by direct enzyme assay, the H₂O₂ scavenging ability recovered with GSH replenishment was not due to GPx. Scavenging was also not due to direct reaction of H₂O₂ with GSH because standard curves were performed in the presence of GSH, and thus, any direct H₂O₂ consumption by GSH was already accounted for. Others have shown that LPO can use GSH as a substrate (20). Thus, diluted and GSH replenished samples were assayed in the presence of 10 µM of azide. The measured scavenging in these samples was 166 ± 17% of nonheated, diluted samples (n = 8), a value not significantly different from samples replenished with GSH but with no added azide. Thus, the activity recovered after GSH replenishment was not due to LPO .

To see whether the GSH-dependent, recovered activity was enzymatic at all, samples were heated to 100°C for 5 minutes. These samples still scavenged 171 ± 13% of nonheated, diluted samples (n = 8, p > 0.05 compared with samples with GSH replenishment). Thus, scavenging activity recovered by replenishing GSH to 8 µM was nonenzymatic in nature .

Immunoblotting
To provide a third independent assessment that LPO was the major peroxidase found in these secretions, we analyzed two additional lavages that contained approximately 10-fold higher protein concentrations than the eight analyzed previously here. This higher protein content allowed precipitation of sufficient amounts of protein (approximately 120 µg) for Western blot detection of both MPO and LPO ( lanes 4 and 5;  lanes 3 and 4). The lavages contained 8.7 and 4.6 ng/µl of total peroxidase activity, respectively, 90% of which was due to LPO based on its sensitivity to dapsone. Western blotting with anti-MPO antibody  detected immunoreactive bands at approximately 55 kD in the sample with an estimated 44 ng of MPO but not in the sample with an estimated 23 ng of MPO. This result was supported by the appearance of an immunoreactive band in a control lane containing 60 ng of human MPO but not in the lane containing 18 ng of MPO. A second visualized band at 50 kD was nonspecific . These samples contained an estimated 400 ng and 200 ng of LPO, respectively. Immunoreactive bands at approximately 90 kD were consistent with the cross-reactivity of this antibody with human LPO  (21).

fig.ommitted Figure 7. Western blot of human airway secretions. Human lavages (120 µg of protein) were run on 8% polyacrylamide gels and transferred to Immobilon-P membranes. (A) Incubation with anti-MPO. (B) Control using only secondary antibody. (C) Incubation with anti-LPO. Lane 1 (A–C), molecular weight markers. (A, B) Lane 2: human MPO control 18 ng; lane 3: human MPO control 60 ng; lanes 4 and 5: human secretions. (C) Lane 2: human MPO control 60 ng; lanes 3 and 4: human secretions; lane 5: bovine milk LPO 100 ng. Immunoreactive bands are consistent with MPO marked with small arrows (A, C), and bands consistent with LPO are marked with arrowheads (A, C).

 
To show that these bands were indeed human LPO (because the anti-MPO antibody did not detect bovine milk LPO), Western blots using anti-LPO antibody were examined showing again an immunoreactive band at approximately 90 kD, consistent with LPO (21). In addition, human MPO control subjects (60 ng) cross-reacted with the anti-LPO antibody. Because the concentration of the secondary antibody was higher compared with the anti-MPO blots, the nonspecific bands seen at approximately 50 kD were stronger .

These data confirm the estimated amounts of MPO and LPO in these samples as measured in enzymatic assays: Western blotting showed more prominent bands for LPO than for MPO, consistent with the MPO and LPO control lanes.

	DISCUSSION

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The surfaces of the airways are naturally exposed to a variety of potentially injurious airborne materials, including inorganic and organic particulate and gaseous matter. Of specific interest is the exposure of the airways to ROS, which can adversely affect tracheal epithelial cells (6). In light of their adverse effects, it may be unexpected on first thought that superoxide radicals (O₂^-) as well as H₂O₂ are produced by the airway epithelium (22, 23). Although the levels of H₂O₂ encountered in the airway are not expected to be antibacterial (10^-6 M or less), H₂O₂ is a required substrate for LPO antibacterial activity, and thus, it is logical that these oxidants are produced in the airway. Not surprisingly, the airway surface liquids have antioxidant properties to protect airway epithelial cells from injury. Because no data are available on the balance of these systems in normal human airway, this article examined the balance of enzymatic and nonenzymatic processes to scavenge H₂O₂ (downstream from superoxide dismutase). The results indicated that LPO was the major enzymatic H₂O₂ consumer in normal airway secretions and that approximately 20% of the total activity appeared to be nonenzymatic in nature under the conditions of this experimental model.

Prior studies addressing H₂O₂ scavenging by human airway secretions used secretions collected by bronchoalveolar lavage and found it to be rich in GSH and GPx (24). Because bronchoalveolar lavage is contaminated with antioxidant substances from alveoli that are usually not present in the airways, data obtained with such collection methods may not accurately reflect the situation in the airway. Although extracellular GPx is produced by bronchial epithelial cells (4), it is not clear whether GPx and GSH levels measured in BAL reflect levels in the airways. To our knowledge, only one study has been published in which GSH was directly assessed in airways and found it to be around 2 µM (25); however, the studied species was the rhesus monkey. Indirect data using induced sputum from healthy human subjects showed a GSH concentration of 5–10 µM (26). Thus, GSH levels may be much lower in the airway than previously thought using bronchoalveolar lavage. We therefore used for our study small-volume lavages from human trachea avoiding contamination of samples with products from the alveolar space or saliva.

Our results indicated that approximately 80% of the measured H₂O₂ consumption was enzymatic in nature. Previous data from our laboratory suggested that LPO was the major enzymatic H₂O₂ scavenger in sheep airways (2, 11), and our current data also support this notion for human airways: azide, an inhibitor of heme-containing peroxidases and catalase but not GPx (16), decreased the scavenging ability of tracheal lavages by approximately 60%, whereas mercaptosuccinic acid, an inhibitor of GPx, had no significant effect despite GPx production by cultured human airway epithelial cells (3). Although azide also inhibits catalase, catalase is not a secreted enzyme (27) and is not present in significant concentrations in the airways (11). This notion is further supported by data obtained with dapsone, an inhibitor of heme peroxidases but not catalase: Dapsone, which inhibits 70% of LPO and all of MPO activity at the used pH of 7.4 , decreased the H₂O₂ scavenging by approximately 45%. Taking the incomplete inhibition of LPO by dapsone under the assay conditions and the TMB data into account, we concluded that LPO accounts for approximately 60% of the total scavenging activity and for approximately 80% of the enzymatic H₂O₂ scavenging activity in normal human airway secretions.

These results could be influenced by dilution, unduly lowering substrate concentrations for certain enzymes such as GPx. To address this question and to confirm the inhibitor data, samples were further diluted until only nonenzymatic scavenging could be detected (heat treatment of the diluted samples did not further decrease scavenging activity). By adding specific substrates, we would be able to distinguish different enzymatic activities without the use of inhibitors. SCN^- is the substrate for LPO and is found in sheep airways at a concentration of 0.16 mM (28). In addition, active transport of SCN^- by human airway epithelial cells has been established (29), and unpublished data from our laboratory show concentrations of SCN^- in human airways to be 0.4 mM. Replenishing SCN^- to 0.4 mM in such diluted samples restored scavenging by LPO, a finding supported by the fact that the SCN^- dependent, rescued scavenging activity was both azide and heat sensitive. The recovered activity also had a linear correlation with the amount of peroxidase activity calculated from TMB oxidation, further supporting the fact that the SCN^--dependent H₂O₂ scavenging was due to LPO .

GSH supplementation of diluted samples also restored H₂O₂ scavenging activity, despite the fact that the undiluted samples did not reveal significant GPx activity. If this recovered activity was due to GPx, we should have been able to measure GPx activities in our specific assays as the recovered activity was higher than the assay's lower detection limit of 5 mU/ml. We therefore considered that the activity rescued with addition of GSH was due to LPO, but scavenging was not inhibited by azide. The rescued activity was also heat insensitive, excluding an enzymatic process.

Although we were mainly interested in the enzymatic aspects of H₂O₂ consumption by airway surface liquids, we found that approximately 20% of the total scavenging activity was nonenzymatic in nature. Interestingly, we found that a fourfold dilution of the samples increased the apparent nonenzymatic scavenging, perhaps because of less competition for the substrate H₂O₂. Without knowing more about the nonenzymatic nature of scavenging, such unexpected changes in nonenzymatic scavenging activity will have to remain unexplained.

In summary, normal human tracheal secretions scavenge H₂O₂ both in a nonenzymatic (approximately 20%) and enzymatic (approximately 80%) fashion. The major enzymatic H₂O₂ consumption is due to LPO. Surprisingly, GPx plays only a minor role. It is interesting to speculate that H₂O₂ found in the airway lumen is produced as a substrate for LPO as LPO's oxidative function uses H₂O₂.

It is well known that there is an increase in H₂O₂ concentrations in airway diseases (9, 10), and an H₂O₂ concentration of greater than 10 µM can be detrimental to airway epithelial cells (30). Studies have shown that pretreating sheep airways with a H₂O₂ scavenger such as catalase can significantly decrease bronchial hyperresponsiveness in response to allergen (31). In addition, the presence of pathophysiologically relevant levels of peroxidases and H₂O₂ reversibly inhibited nitric oxide–dependent bronchodilation of preconstricted tracheal rings (32). Therefore, it is clear that ROS play a major role in airways diseases, and a better understanding of the mechanisms by which the airways scavenge H₂O₂ in normal conditions is crucial, as it will help to understand certain disease states and possibly lead to novel therapeutic approaches.

Received in original form June 8, 2002; accepted in final form November 8, 2002

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