Antioxidant activity of bronchoalveolar lavage fluid in the adult respiratory distress syndrome MICHAEL G. LYKENS, W. BRUCE DAVIS, AND ERIC R. PACHT Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Ohio State University, Columbus, Ohio 43210 Lykens, Michael G., W. Bruce Davis, and Eric R. Pacht. Antioxidant activity of bronchoalveolar lavage fluid in the adult respiratory distress syndrome. Am. J. Physiol. 262 (Lung CeLL. Mel; Physiol. 6): L169-Ll75,1992.-Bronchoalveolar lavage (BAL) fluid from normal subjects is a potent inhibitor of lipid peroxidation. This antioxidant activity (AOA) of BAL fluid is primarily due to the serum proteins transferrin and ceruloplasmin. In the adult respiratory distress syndrome (ARDS), there is an influx of protein-rich edema fluid into the alveolar space that may increase antioxidant activity and provide protection against further oxidant-mediated lung injury. To test this hypothesis, the AOA of BAL fluid was measured in patients with ARDS (n = 11) and normal subjects (n = 12). When compared with normal subjects, BAL fluid from ARDS patients had a significantly higher concentration of total protein (2536.8 t 408.2 pg/ml vs. 77.3 t 7.0 pg/ml, P < 0.005). When compared at several volumes, BAL fluid from ARDS patients was a more potent inhibitor of lipid peroxidation than BAL fluid from normals. In addition, when AOA was determined on equal milligram amounts of BAL fluid protein from ARDS patients and normal subjects, ARDS BAL fluid protein had a significantly higher AOA. Consistent with its higher AOA, ARDS BAL fluid contained increased concentrations of both transferrin (77.8 t 15.3 pg/ml vs. 2.78 t 0.3 pg/ml, P < 0.05) and ceruloplasmin (36.5 t 5.6 pg/ml vs. 0.26 t 0.02 pg/ ml, P < 0.005) compared with normal subjects. The importance of both ceruloplasmin and transferrin in the enhanced AOA of ARDS BAL fluid was further demonstrated by studies that showed a significant decrement in AOA when the antioxidant functions of these two proteins were selectively blocked. These findings suggest that the BAL fluid in ARDS patients contains large amounts of serum proteins which possess antioxidant properties. Thus, the high permeability pulmonary edema of ARDS, although detrimental to gas exchange, may be beneficial in preventing further oxidant-mediated lung injury. lipid peroxidation;

ceruloplasmin;

transferrin;

iron

ALVEOLAR epithelial surface is coated with a thin layer of plasma ultrafiltrate and locally secreted substances. This fluid bathes and protects the alveolar surface while allowing for normal gas exchange. A wide spectrum of antioxidants are contained in this fluid (9), including serum proteins such as transferrin and ceruloplasmin (27), vitamin E (28), vitamin C (23, 31), and reduced glutathione (6). One index of lung oxidant stress is peroxidation of lipids and in that respect bronchoalveolar lavage (BAL) fluid obtained from normal subjects has been shown to be a potent inhibitor of lipid peroxidation. This antioxidant activity (AOA) appears to be mediated primarily through the serum proteins transferrin and ceruloplasmin (27). Thus, the lower respiratory tract, which is continuously exposed to a variety of oxidant stresses, is afforded antioxidant protection by the alveolar fluid. The composition of the alveolar fluid is dramatically

THE NORMAL

1040-0605/92

$2.00 Copyright

altered in the adult respiratory distress syndrome (ARDS), a form of noncardiogenic pulmonary edema. ARDS is characterized by increased pulmonary vascular permeability resulting in protein and fluid flux into the pulmonary interstitial and alveolar spaces accompanied by an acute inflammatory reaction (19, 37). The character of this edema fluid has previously been explored, and more recently several laboratories have begun to address the clearance mechanisms of pulmonary edema in ARDS (11, 22). However, little attention has been focused on the antioxidant function of pulmonary edema fluid in ARDS. Because normal alveolar fluid possesses potent antioxidant activity, we hypothesized that ARDS BAL fluid would have even greater AOA due to the substantial influx of serum proteins. The present study demonstrates that ARDS BAL fluid possessessignificant AOA and its ability to inhibit lipid peroxidation is primarily mediated through transferrin and ceruloplasmin. 1

1

METHODS Subjects. Eleven patients with ARDS were recruited from the Medical Intensive Care Unit at The Ohio State University Hospital. There were six females and five males with an age range of 23-77 yr (mean 46.5 t 5.5 yr). Diagnostic criteria for ARDS (19, 37) included 1) acute hypoxemic respiratory failure requiring mechanical ventilation, 2) diffuse bilateral alveolar infiltrates on chest X-ray,,3) refractory hypoxemia (fraction of inspired OZ of 0.60 to maintain partial arterial O2 pressure > 60 mmHg), 4) recognized appropriate clinical setting or risk factor for the development of ARDS, and 5) evidence of lowpressure, high-permeability pulmonary edema. Nine of the 11 patients had sepsis or sepsis syndrome (4); the remaining 2 had evidence of diffuse lung injury secondary to aspiration of gastric contents. The control groups consisted of 12 young, healthy, nonsmoking subjects (9 males and 3 females) whose mean age was 24.7 t 1.5 yr. Normal volunteers were screened with a medical history and physical examination. All subjects were studied under an approved protocol of The Ohio State University Human Subjects Review Committee. Subjects underwent simultaneous collection of serum and BAL fluid (see 23AL fluid processing). Samples were stored at -80°C until analysis. BAL. All subjects underwent bronchoscopy with BAL. Patients with ARDS underwent BAL within 36 h of diagnosis, with 9 of 11 patients undergoing BAL within 12 h. After topical lidocaine anesthesia, a 4.8-mm-diam flexible fiberoptic bronchoscope (model BF-B2, Olympus New Hyde Park, NY) was passed through the endotracheal tube of ARDS patients (transnasally in normal volunteers) and wedged into a right middle lobe or lingular subsegment. Five successive 20-ml aliquots of 0.9% saline (room temperature) were instilled and immediately aspirated with low suction. BAL fluid processing. BAL fluid samples were strained through monolayers of coarse mesh surgical gauze and centrifuged at 500 g for 15 min. One molar Tris HCl (pH 7.4) was added to the decanted supernate to achieve a final concentra-

0 1992 the American

Physiological

Society

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IN

tion of 0.05 M Tris. After passage through a 0.45pm filter, the cell-free BAL supernates were pressure concentrated to 1.5-2.5 ml over a YM5 membrane (Amicon, Lexington, MA). Small volume aliquots of the concentrated fluid representing large volumes of unconcentrated BAL fluid were stored at -80°C for use in the lipid peroxidation assay. Protein loss on the membrane was negligible, since total protein determinations before and after pressure concentration (corrected for volume) were within 5%. The cellular pellet was resuspended and washed twice in Hanks’ balanced salt solution without calcium or magnesium. A small aliquot was then cytocentrifuged at 35 g for 10 min, air dried, and stained by a modified Wright-Giemsa stain. A differential count was performed on a minimum of 200 cells. Measurement of BAL fluid proteins. Total protein concentration was determined utilizing a modification of the Bio-Rad protein microassay procedure (Bio-Rad Laboratories, Richmond, CA) (19). BAL concentrations of ceruloplasmin &g/ml) and transferrin (pg/ml) were determined by enzyme-linked immunoassay (ELISA). Briefly, an inhibition ELISA was established where transferrin (Sigma) was bound to a Dynatech 96 well plate for 24 h. A goat anti-human transferrin antibody (Sigma) was incubated for 24 h with either known concentrations of antigen (transferrin standards) or the sample antigen in BAL fluid. The following day, the mixture of antigen and antibody was incubated with the transferrin bound to the Dynatech plates. Residual free goat anti-human transferrin antibody was allowed to bind to the transferrin attached to the plate. A second antibody, rabbit anti-goat IgG peroxidase conjugate (Sigma) was added and bound to the residual antibodyantigen complex. An enzyme substrate (O-phenylenediaminephosphate citrate buffer) was utilized to react with the peroxidase conjugated on the second antibody, thus allowing for the product of the reaction to be measured. By comparing the amount of product with standard antigen samples, unknown concentrations of transferrin in BAL fluid were measured. A similar indirect ELISA was developed and utilized to measure ceruloplasmin. Lipidperoxidation assay. Brain tissue is a rich source of lipid and has previously been used as a substrate for assays of lipid peroxidation (32). Male Sprague-Dawley rats (450-600 g) were anesthetized with ether and exsanguinated via heart puncture. The brain was exposed, removed intact, and washed in an icecold 0.15 M saline solution to remove all visible blood and blood clots. The tissue was then chopped into small pieces in four times its weight of ice-cold phosphate-buffered saline (40 mM KH2P04-K2HP04, pH 7.4 in 0.142 mM NaCl). This mixture was homogenized in a Kontes homogenizer (Kontes Glass, Vineland, NJ) for 3 min. The resulting homogenate was centrifuged for 15 min at 1,000 g, and the supernate was divided into small-volume aliquots and stored at -80°C until use. Lipids in brain homogenates are known to undergo spontaneous autooxidation when exposed to room air at 37°C (3, 32). Thus rat brain homogenates were thawed, and 300 ~1 aliquots were placed in borosilicate glass tubes and immediately diluted with 700 ~1 of phosphate-buffered saline for a total volume of 1 ml. As expected, the final reaction mixtures underwent autooxidation when incubated for 1 h at 37OC, as detected by the formation of malondialdehyde (MDA) (see MDA determination). While the 1 ml final volume of the assay was preserved, different amounts of BAL fluid were added prior to the 1 h incubation in order to assess their ability to inhibit the spontaneous autooxidation of lipids. After demonstrating that ARDS BAL fluid was a potent blocker of lipid peroxidation, additional experiments were performed to determine which components of the fluid were primarily responsible for its antioxidant activity. BAL fluid from patients with ARDS was preincubated with excess FeC13 (750

ARDS

PM) for IO min to saturate the iron binding site on transferrin. Previous studies (25, 27) demonstrating that iron loading both ablated the AOA of transferrin and did not interfere with the assay system were confirmed for this investigation (data not shown). In another set of experiments, BAL fluid from patients with ARDS was preincubated with 100 mM sodium azide. Previous studies (13, 27) demonstrating that sodium azide inactivates the ferroxidase activity of ceruloplasmin and abolishes its antioxidant activity without interfering with the assay system were confirmed for this investigation (data not shown). A IOO-~1 aliquot of ARDS BAL fluid preincubated with either FeCls or sodium azide was added to the brain homogenate system before the l-h incubation and AOA was determined. The l-ml total volume of the assay system was preserved. All reaction mixtures were briefly mixed and a “0-min” 400~1 aliquot was quickly withdrawn. The 0-min aliquot was covered with aluminum foil and stored under NP at 4°C until MDA concentration was determined. The remaining reaction mixture was allowed to incubate at 37°C for 1 h. A “60-min” 400-~1 aliquot was then removed. The 0- and 60-min tubes were immediately assayed for MDA concentration (see MDA determination). MDA determination. MDA production is directly related to the amount of lipid peroxidation occurring during the l-h incubation (3, 17). Before the MDA assay, a standard thiobarbituric acid reagent mixture was prepared. In a loo-ml flask, 15 g of trichloroacetic acid (Fisher Scientific, Fair Lawn, NJ), 0.375 g of 2thiobarbituric acid (Sigma), and 4.16 ml of 6 N HCl (Fisher Scientific) were combined with distilled water to a final volume of 100 ml. This solution was mixed vigorously, and 2.0 ml were added to both the 0- and 60-min tubes. These tubes were covered with aluminum foil, vortexed, and heated at 97°C for 20 min. After 20 min, the tubes were cooled on ice, centrifuged (1,700 g for 30 min), and the absorbance of the supernates was measured at 532 nm on a spectrophotometer (Beckman Instruments, Palo Alto, CA). A standard curve using known amounts of MDA (Aldrich Chemical, Milwaukee, WI) was used to determine amounts of MDA in each test sample. The amount of MDA produced during the l-h incubation was calculated by subtracting the 0-min tube from the 60-min tube. The AOA of a particular test substance was calculated as follows AOA

(%) = 1 -

nmol of MDA/ml (test solution) nmol of MDA/ml (control solution)

x 100%

Thus, AOA was expressed as the percent inhibition of spontaneous autooxidation of the control brain homogenate. MDA was determined on at least three control brain homogenates with every experiment. An AOA of 100% meant that lipid peroxidation was completely inhibited, whereas an AOA of 0% meant that none of the lipid peroxidation was inhibited. Statistical methods. All data were expressed as means t SE. Data were compared using the student’s t test and differences were assumed significant if P < 0.05. RESULTS

ARDS patient data. Eleven patients with ARDS were studied as shown in Table 1. The group included 6 females and 5 males, ranging from 23 to 77 yr old with a mean of 46.5 t 5.5 yr. Three patients had bacterial sepsis documented by positive blood cultures, whereas another six patients had the sepsis syndrome (4) as their clinical risk factor for ARDS. Two patients developed acute diffuse lung injury after aspirating gastric contents. The mean acute lung injury score (26) was 2.57 t 0.15 indi-

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Ll71

AOA IN ARDS

transferrin concentration, when expressed as a percentage of total protein, was not significantly increased in Lung Total BAL ARDS patients compared with normal subjects (3.0 t Risk Patient Sex/Age Injury Protein, Outcome Factor 0.3 vs. 3.5 t 0.4%, P > 0.10). In contrast, BAL fluid Score /G/ml concentrations of ceruloplasmin, when expressed as a LA 2.67 1,196 D M/59 Sepsis (pneumococcal) percentage of total protein, were significantly increased SW 2.75 2,184 S F/46 Sepsis syndrome in ARDS patients compared with normal subjects (1.6 t FB F/21 Sepsis syndrome 2.67 2,818 S 0.1 vs. 0.4 t 0.05%, P < 0.005). These values are also DL M/49 Sepsis (staphylococcal) 1.67 1,244 D LE M/66 Sepsis syndrome 2.33 1,154 D similar to previously published results (19). HE F/59 Sepsis (bacteroides) 3.33 4,925 S Antioxidant activity. Previous investigations in our GW M/41 Aspiration 2.33 4,172 S laboratory have demonstrated that BAL fluid from norBC F/46 Aspiration 2.50 1,978 S mal subjects is a potent inhibitor of lipid peroxidation LJ F/23 Sepsis syndrome 3.33 3,702 D GW M/25 ’ Sepsis syndrome 2.00 1,193 S (27). Therefore, aliquots of concentrated BAL fluid repLC F/77 Sepsis syndrome 2.67 3,439 D resenting known volumes of unconcentrated BAL fluid and known milligram amounts of BAL fluid protein from Values for lung injury score are from Ref. 26. ARDS, adult respiratory distress syndrome; BAL, bronchoalveolar lavage; D, died; S, sur- ARDS patients and normal subjects were tested for their vived. ability to inhibit lipid peroxidation in the rat brain homogenate assay system. ARDS BAL fluid, expressed eating extensive lung injury. All individuals had markas milliliters of unconcentrated fluid, possessed far edly increased total protein concentrations in their BAL greater antioxidant activity than normal BAL fluid (Fig. fluid, further substantiating the extent of their pulmo1). In normal subjects, BAL volumes greater than 15 ml nary capillary permeability defect. Five of the 11 patients were required to completely inhibit lipid peroxidation in died for an overall mortality of 45%. There was no the assay system. However, in ARDS patients, much correlation between lung injury score and mortality (data smaller volumes (~2 ml) of BAL fluid completely inhibnot shown). ited lipid peroxidation (Fig. 1). Thus, at low BAL fluid BAL. At the time of bronchoscopy the appearance of volumes, ARDS BAL fluid was much more potent at the trachea and major bronchi appeared essentially nor- inhibiting lipid peroxidation than normal BAL fluid. mal with no evidence of purulent secretions or airway Iron loading and sodium azide studies. Once it was hemorrhage. BAL was well tolerated by all subjects, and demonstrated that BAL fluid of ARDS patients was a there was no significant difference in the percent of more potent inhibitor of lipid peroxidation than BAL instilled saline recovered between the two groups (Table fluid of normal subjects, further experiments were per2). The total cell count, expressed as cells x lo3 per formed to delineate the mechanism responsible for the milliliter was significantly higher in the ARDS group enhanced AOA. From prior investigations (27), cerulocompared with normal subjects (Table 2). As expected, plasmin and transferrin seemed to be likely candidates the differential cell count of the BAL fluid revealed a for the increased AOA. To test this hypothesis, concenhigh percentage of neutrophils in the ARDS group com- trated BAL fluid from patients with ARDS was initially pared with normal subjects. Neutrophils comprised 66.6 preincubated with excess Fe”+ to saturate the transferrin t 9.1% of recovered cells from ARDS patients compared iron binding sites. The previous studies in our laboratory with only 0.5 -t 0.2% of recovered cells from normal (27) demonstrating that iron loading ablated all of transsubjects (P < 0.005) (Table 2). These differential cell ferrin’s antioxidant properties and had no effect on the counts are similar to previous studies of ARDS (37). assay system were confirmed for this investigation (data Protein studies. Patients with ARDS had more than a not shown). When ARDS BAL fluid was preincubated 3O-fold increase in BAL fluid total protein compared with Fe3+ (n = 3), antioxidant activity of the fluid was with normal subjects (2536.8 t 408.2 pg/ml vs. 77.3 +- essentially abolished (69.0 t 5.7% without preincubation 7.0 pg/ml, P < 0.005, Table 3). Both transferrin and with iron to 2.2 t 2.1% with preincubation with iron, P ceruloplasmin levels were markedly increased in the < 0.05, Table 4). A second set of experiments involved preincubating ARDS BAL fluid with sodium azide (n = ARDS group. Transferrin concentrations in BAL fluid were 77.8 t 15.3 pg/ml in the ARDS patients compared 8). Previous studies in our laboratory (27) demonstrating with only 2.78 t 0.3 pg/ml in normal subjects (P < 0.05, that sodium azide abolished the ferroxidase activity of Table 3). Ceruloplasmin levels were 36.5 t 5.6 pg/ml in ceruloplasmin and had no effect on the assay system were confirmed for this investigation (data not shown). ARDS BAL fluid compared with only 0.26 t 0.02 pg/ml AOA decreased -37% following preincubation with soin normal BAL fluid (P < 0.005, Table 3). BAL fluid Table 1. Clinical profile of ARDS patient group

Table 2. Bronchoalveolar lavage fluid results Group

% Recovered Fluid

Cell Count/ml BAL Fluid

Differential, Neut

AM

%

Lymph

Eos

22Ok36 x lo3 0.5t0.2 91.8t0.8 7.1t0.9 0.4t0.2 Normal 55.7k2.2 704t200 x 1O”Jf 66.6+9.1$ 23.6&7.3$ 7.623.4 2.0+0.6-f ARDS 57.4t3.5 Values are means k SE; n = 9 normal subjects and n = 11 adult respiratory distress syndrome (ARDS) patients. BAL, bronchoalveolar lavage; Neut, neutrophils; AM, alveolar macrophages; Lymph, lymphocytes; Eos, eosinophils. “f P < 0.05 compared with normal subjects; $ P < 0.005 compared with normal subjects.

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L172

AOA

Table

3. Protein Group

concentrations

Total

Protein, #%/ml

in BAL fluid Transferrin, l-%/ml

Ceruloplasmin, r.Lglml

Normal

77.3t7.0

2.78t0.30

0.26t0.02

ARDS

(12) 2,536.8+408.2"c

(4) 77.8tl5.3*

(4) 36.5+5.6-j-

WI

IN

(11)

(11)

Values are means t SE; nos. in parentheses are no. of subjects. BAL, bronchoalveolar lavage; ARDS, adult respiratory distress syndrome. * P < 0.05 compared with normal subjects; t P < 0.005 compared with normal subjects.

100 -

ARDS

ARDS BAL protein was more potent at inhibiting lipid peroxidation than normal BAL protein (Fig. 2). At low amounts of total protein (3 mg or less), AOA was significantly greater with ARDS BAL protein compared with normal BAL protein. Thus ARDS BAL protein was a more potent inhibitor of lipid peroxidation than normal BAL protein. Since equal milligram amounts of BAL protein from the two patient groups contained equal milligram amounts of transferrin (i.e., transferrin as a percentage of total protein was unchanged in the two groups), these findings suggest an important role for ceruloplasmin. DISCUSSION

The current study substantiates the fact that ARDS patients have altered lung permeability, as noted by more than a 30-fold increase in total BAL fluid protein concentration. Further analysis of the specific proteins in = Normal the BAL fluid revealed that transferrin and ceruloplasmin were present at markedly increased levels. More q ARDS importantly, despite the marked inflammatory cell influx in lungs of ARDS patients and the subsequent release of oxidants and proteases, these serum proteins retained * p