Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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Surfactant dysfunction in acute lung injury has been postulated as a result of free radical damage to lipid and protein components. This study examines whether transition metals with different redox potentials and different binding affinities for lipids and proteins affect interfacial properties differently. Purified whole calf lung surfactant (CLS) was incubated with 0.125 mM Fe2+, Fe3+, Fe3+-EDTA complex, or Cu2+ either alone or with 0.25 mM H2O2 or H2O2 plus 0.25 mM ascorbate for 4 and 24 h. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS), and free radical-mediated alterations in protein structure were assessed by fluorescamine assay and Western blot analysis. Function was assayed by pulsating bubble surfactometry. Lipid peroxidation was detected in samples incubated with Fe2+, Fe3+, and Fe3+-EDTA but not with Cu2+. All transition metal-based free radical systems affected surfactant protein composition by fluorescamine assay, indicating free radical-mediated modification of protein side chains. Western blot analysis demonstrated surfactant protein A modification, with the generation of higher- and lower-molecular-mass immunoreactive products. Despite biochemical evidence of lipid and protein modification, surfactant dysfunction was minimal and was manifest as an increase in the compression ratio required to achieve surface tension < 1 dyn/cm. This dysfunction was readily reversed by the addition of 5 mM Ca2+ either before or after oxidation. These data indicate that copper- and iron-based free radical-generating systems modify the lipid and protein components of surfactant differently but suggest that these changes have little effect on surfactant function.
acute lung injury; lipid oxidation; protein oxidation
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INTRODUCTION |
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SURFACTANT DYSFUNCTION is an important factor contributing to the pathophysiology of acute respiratory distress syndrome (ARDS) (10, 13, 18). Several mechanisms have been proposed to account for the observed changes in interfacial properties, including biophysical interactions with serum proteins in edema fluid and hyaline membranes, decreased surfactant apoprotein content as a result of degradation mediated by leukocyte proteases, and altered surfactant lipid and protein composition resulting from chemical modifications caused by reactive free radicals (19, 23). A better understanding of the relative contributions of these distinct mechanisms would be useful for developing new strategies for ARDS treatment and for directing future research efforts. The present study focuses on free radical-based contributions to this process and specifically characterizes the biochemical alterations in whole surfactant that result from direct exposure to reactive free radicals and how these alterations affect interfacial properties.
Both in vitro and in vivo data suggest that free radicals may be important in the pathophysiology of acute lung injury (18, 26). The activated neutrophil generates several reactive free radical oxygen species including hydrogen peroxide, hydroxyl radicals, and superoxide anions, each of which can interact with surfactant lipid and/or protein components to alter function (4, 23). Samples from patients with acute lung injury show biochemical evidence of lipid peroxidation in the form of volatile exhaled hydrocarbons, suggesting free radical-mediated chemical alterations in lung surfactant (3, 19). In animal models of ARDS, significant increases in lipid hydroperoxides have been detected compared with control animals, further supporting the argument that free radical products generated during acute lung injury may be important in mediating changes in surfactant lipids and possibly in surfactant function (15, 16). Finally, case reports (2, 24) of an improved outcome in ARDS patients treated with antioxidant therapy suggested that the generation of free radicals may be important in the pathogenesis of lung dysfunction in this syndrome.
Transition metals have been implicated as potential contributors to free radical-mediated surfactant injury because they can promote formation of reactive chemical species. These ions, either bound to or released from proteins, may contribute to free radical generation by catalyzing Haber-Weiss reactions in which a reactive hydroxyl radical is generated from either superoxide anion or hydrogen peroxide. Transition metals present in biological systems that could participate in Haber-Weiss reactions include Cu2+, Fe2+, and Fe3+, and all have been used in vitro with H2O2 and ascorbate to generate free radicals. The present study examines the effects of free radicals catalyzed by each of these three ions on surfactant lipid and protein biochemistry and on surfactant dynamic interfacial properties.
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METHODS |
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Calf lung surfactant isolation. Calf
lungs were lavaged with 4 liters of a 0.15 M NaCl solution at
20-25 cmH2O infusion pressure within 4 h of procurement. Bronchoalveolar lavage fluid was collected by gravity drainage, and cells and debris were removed by low-speed centrifugation (450 g at 4°C for 8 min). The cell-free supernatants were pooled, and crude surfactant was
isolated by centrifugation at 30,000 g. The pellets were resuspended in
2-3 ml of saline by repeated injection through a 25-gauge needle,
layered over an 0.8 M sucrose solution, and centrifuged at 30,000 g for 20 min. The pellicle containing
purified surfactant was aspirated, washed twice with 0.15 M NaCl, and
resuspended in 0.15 M NaCl containing 0.1% sodium azide. Purified
surfactant was stored under nitrogen in 10-mg aliquots at
20°C. Biochemical and functional integrity have been
demonstrated for up to 6 mo with this isolation and storage procedure.
Isolation of purified surfactant protein
A. A 10-mg aliquot of calf lung surfactant (CLS) in 0.5 ml of an aqueous solution was injected through a 25-gauge needle into
45 ml of 1-butanol and vortexed for 1 min. Aqueous soluble components
including surfactant protein (SP) A and serum proteins were isolated by
centrifugation (20,000 g at 4°C
for 20 min). Purified SP-A was obtained by resuspending this pellet in
20 mM -octylglucopyranoside and centrifuging at 10,000 g for 4°C for 45 min. The
resulting pellet containing SP-A was dialyzed for 48 h against a 5 mM
Tris buffer, pH 7.4, to remove residual detergent. SP-A content was
determined by bicinchoninic acid assay, and sample purity was
demonstrated by 12% SDS-PAGE.
Oxidation of CLS. Stock solutions of
aqueous 50 mM FeCl2, 50 mM
FeCl3, 50 mM
Fe3+-EDTA, 100 mM
CuCl2, 100 mM
H2O2,
and 50 mM ascorbic acid were prepared fresh on the day of each
experiment immediately before use. Concentrated stocks of superoxide
dismutase (SOD) and catalase (Cat) were prepared and stored under
nitrogen at 20°C. For each transition metal, reaction
mixtures containing 1 mg/ml of whole CLS were prepared with 125 µM
transition metal alone, 125 µM transition metal plus 250 µM
H2O2,
and 125 µM transition metal plus 250 µM H2O2
plus 250 µM ascorbate. These concentrations of reactants have been
shown to effectively catalyze formation of hydroxyl radical in aqueous
solutions in vitro and have been previously demonstrated to promote
peroxidation of lipid surfactant components (6, 9). Control incubations
were performed for each reaction with the antioxidant pair SOD (3 U/ml
final concentration) and Cat (2,000 U/ml final concentration).
Additional control incubations were performed with surfactant alone,
surfactant plus
H2O2,
or surfactant plus
H2O2
plus ascorbate. Samples were incubated for 4 and 24 h at 37°C and
divided for subsequent thiobarbituric acid-reactive substances (TBARS)
assay, fluorescamine assay, and functional assay by pulsating surfactometry.
TBARS assay measurement of lipid peroxidation products. TBARS assay was performed according to standard protocol (8). Briefly, surfactant samples were incubated with a 1:1:1 mixture of 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.01% butylated hydroxytoluene in 0.25 N HCl at 95°C for 25 min; cooled to room temperature; centrifuged at 8,000 g for 2 min to remove debris; and examined by absorbance spectrophotometry at 535 nm. Total TBARS levels were determined for samples containing 500 µg of lipid. Standards were prepared with 1,1,3,3-methylmalonaldehyde. Additional control standard curves were performed with 125 µM each transition metal to demonstrate no direct effect of reagents on assay measurements.
Fluorescamine assay of free amino groups. Fluorescamine assay was performed to provide an index of the extent to which free radical reactants had altered SP components. This assay specifically detects free radical-mediated nucleophilic modification of exposed amino groups on lysine, arginine, asparagine, and glutamine residues. Assay methodology is as previously described (22). In brief, surfactant samples containing 500 µg of lipid were diluted to a total volume of 3 ml in 0.05 M phosphate buffer. One milliliter of 0.03 g of fluorescamine in 100 ml of spectrophotometric grade dioxane was added, and samples were vortexed vigorously for 30 s. Fluorescence was measured within 30 min of sample preparation at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. Excitation and emission slit widths were adjusted to give a maximum signal-to-noise ratio. Standard curves were performed with increasing concentrations of albumin, albumin exposed to a CuCl2 plus H2O2 free radical-generating system, and albumin dissolved in a 1 mg/ml aqueous surfactant solution to assess how lipids would affect the ability to detect free radical modifications of exposed amino groups.
Fluorescamine assay was performed on samples of whole surfactant, on the aqueous-soluble protein fraction of whole surfactant isolated by butanol precipitation after free radical exposure, and on purified SP-A exposed to free radicals in aqueous Tris buffer (5 mM, pH 7.45). This approach allows for the assessment of the effects of free radicals on SP-A in both the presence and absence of lipids. Control assays were performed with protein alone, protein plus H2O2, and protein plus H2O2 plus ascorbate.
SDS-PAGE and Western blot analysis. In preparation for SP-A Western blot analysis, 12% SDS-PAGE was performed according to standard protocol for both purified aqueous SPs (1 mg/lane) and whole lung surfactant (10 mg/lane). Separation was performed at constant voltage (100 V) at 4°C. Samples were blotted onto polyvinylidene difluoride filter paper by electrotransfer (Bio-Rad, Hercules, CA). Whole rabbit serum against purified calf SP-A (1:1,000 dilution) served as the primary antibody. The detection system was sensitive to 50 ng of SP-A and demonstrated no cross-reactivity to albumin, hemoglobin, or the hydrophobic surfactant apoproteins B and C. Overnight blocking was performed with 5% dried milk in Tris-buffered saline. Signal amplification was performed with avidin-linked goat anti-rabbit antibody. Blots were developed with horseradish peroxidase-tetramethylbenzidine reagent.
Bicinchoninic acid protein assay. Protein contents of purified SP fractions and whole surfactant samples were determined by bicinchoninic assay (32). The protein concentration was measured with respect to an albumin standard dissolved in 0.5% SDS buffer. Samples were similarly dissolved in 0.5% SDS buffer to ensure homogeneity. Standards for the samples containing whole surfactant were prepared in a 1 mg/ml sonicated aqueous dipalmitoylphosphatidylcholine solution to simulate the effects of surfactant lipid components on the assay results.
Ammonium molybdate-inorganic phosphate assay. Phospholipid (PL) composition was determined by ammonium molybdate-inorganic phosphate assay on organic extracts of surfactant samples that were separated by thin-layer chromatography and extracted from the silica gel into chloroform (1). Briefly, organic surfactant extracts prepared by extraction with the method of Folch et al. (7) were dried to a final volume of 100 µl. Thirty microliters of 10% Mg(NO3)2 were added to each sample, which was then flamed to dryness. The samples were resuspended in 3 ml of 0.5 M HCl and incubated for 15 min at 95°C. Then, 0.7 ml of a mixture of 1 part 10% ascorbic acid and 6 parts 0.42% ammonium molybdate was added, and the samples were incubated for an additional 20 min at 50°C. The inorganic phosphate concentration is proportional to absorbance at 820 nm and was measured spectrophotometrically, referencing a dipalmitoylphosphatidylcholine standard curve.
Thin-layer chromatography separation of surfactant PL subtypes. Organic extracts of selected samples were prepared according to the methodology of Folch et al. (7). The samples were spotted onto CHCl3-methanol-washed silica G/H plates (Fisher Scientific, Pittsburgh, PA) along with authentic PL standards, and PL subtypes were separated with a CHCl3-methanol-propanol-H2O-triethylamine mobile phase (42:12.6:9.8:35:35). Spots corresponding to each individual PL subtype were detected under low ultraviolet light after being stained with a 1,3,5-diphenylhexatriene solution. Samples were scraped, and the PLs were extracted from the silica powder into a CHCl3 solution by vigorous vortexing for 1 h at room temperature. The powder was then removed by centrifugation, and total PL content was determined by inorganic phosphate assay as described in Ammonium molybdate-inorganic phosphate assay.
Pulsating surfactometer studies.
Measurements of surface tension () versus surface area
(A) were recorded by pulsating
bubble surfactometry (PBS; Electronetics, Amherst, NY). The PBS system was calibrated statically and dynamically with distilled water before
and after each experiment. Sample recordings were performed at a 1 mg/ml bulk-phase concentration in 0.15 M NaCl at 37°C and 20 cycles/min. Bubble radius was cycled between 0.32 and 0.5 mm. The
samples were loaded with the modified technique of Putz et al. (21) to
prevent capillary tube leakage. Leakage, which did occasionally occur,
was detected by direct observation with the microscope objective of the
system and by examination of surface tension profiles. Samples in which
leakage was detected were restudied.
Dynamic surface properties were assessed for all samples after the addition of CaCl2 (5 mM final concentration). Any sample demonstrating evidence of dysfunction was also studied after the addition of purified SP-A (5% by weight) to assess whether apoprotein reconstitution might restore function. Concentrated SP-A was added as an aqueous mixture, and measurements were recorded after low-speed vortexing at room temperature. The functional integrity of SP-A was demonstrated by its ability to augment adsorption of hydrophobic surfactant extracts in vitro (20).
-A profiles represent steady-state
behavior, defined as that for which the shape of the
-A loops no longer changed from one
cycle to the next. This typically took between 30 and 60 s to achieve.
Statistics. Results of the TBARS assays, fluorescamine assays, and PL assays are presented as means ± SD. Comparisons of values measured for each sample at multiple time points were performed by ANOVA for repeated measurements. Comparisons among different samples were performed by one-way ANOVA, and post hoc assessment of significance was performed by the method of Tukey (28). Comparisons of unpaired samples were performed by Student's t-test analysis where appropriate. Significance was defined as P value < 0.05.
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RESULTS |
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TBARS assay results. Figure
1 summarizes the results of TBARS assays
after exposure of intact lung surfactant to each of the transition
metal-based free radical-generating systems
(n = 4 samples). Results
are expressed as the absolute amount of TBARS products assayed above
that observed in surfactant control samples incubated for the same
period of time (i.e., sample signal control signal). At 4 h
(Fig. 1A), samples containing
Fe3+ in both the chelated and
nonchelated forms had increased levels of TBARS compared with
Fe2+ and
Cu2+ samples. Addition of
H2O2
or
H2O2
plus ascorbate did not significantly increase TBARS levels in samples
containing Fe3+ but did result in
significant TBARS generation in samples containing Fe2+. None of the samples
containing Cu2+ had demonstrable
TBARS products after 4 h of incubation.
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After 24 h (Fig. 1B), TBARS increased two- to threefold in samples containing Fe3+-EDTA plus H2O2, demonstrating a progressive and significant increase in TBARS from 4 h. TBARS products remained increased in samples incubated with Fe3+ and Fe3+-EDTA but were not different from those measured at 4 h. Samples incubated with Fe2+ alone also had significantly increased levels of TBARS at 24 h. TBARS levels in samples incubated with Fe2+ plus H2O2 or Fe2+ plus H2O2 plus ascorbate were not increased above levels observed after 4 h of incubation. TBARS in Cu2+ samples remained virtually undetectable.
Incubation with the free radical scavenger pair SOD and Cat (Fig. 1C) significantly reduced the level of TBARS at 24 h in all iron-containing samples except those containing FeCl3 plus H2O2.
TBARS standard curves were performed with 125 µM each transition metal and with SOD plus Cat to ensure that changes in absorbance were not due to a direct interaction between any of the ions or free radical scavenger proteins and the assay reagents. Slopes and intercepts for each of the five standard curves (Fe2+, Fe3+, Fe3+-EDTA, Cu2+, and SOD-Cat) were found to be nearly identical.
Fluorescamine assay results for whole surfactant
samples. The effect of free radical exposure on SP
components assessed by fluorescamine assay is summarized in Fig.
2 (n = 4 samples). Results are expressed as the percent decrease in fluorescence
signal relative to the control signal, where signal attenuation is due
to oxygen radical-mediated modification of exposed amino groups on
proteins. After 4 h of incubation (Fig.
2A), most samples demonstrated
evidence of free radical-related protein modification, with significant decreases in fluorescence intensity. Incubation with
Fe2+ alone generated a small,
insignificant decrease in fluorescence attenuation, whereas samples
incubated with Fe2+ plus
H2O2
or Fe2+ plus
H2O2
plus ascorbate displayed large effects, consistent with extensive
protein modification. Samples incubated with
FeCl3 alone or in combination with
H2O2
or
H2O2
plus ascorbate showed significant fluorescence attenuation as well.
After 4 h of incubation, the
Fe3+-EDTA complex affected the
fluorescence signal only in samples containing both
H2O2
and ascorbate. In contrast to its negligible effect on lipid
peroxidation, all Cu2+-containing
samples caused large attenuations in fluorescence. After 24 h (Fig.
2B), fluoresence attenuation
patterns were unchanged from those observed at 4 h. Incubation with SOD
plus Cat prevented fluorescence signal attenuation in all
iron-containing samples but had only a partial effect in
Cu2+-containing samples (Fig.
2C).
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A fluorescamine assay standard curve was performed with purified human albumin in Tris buffer and human albumin in a saline solution containing 1 mg/ml of native surfactant to verify the accuracy of the assay in a lipid environment. A linear relationship between fluorescence signal attenuation and protein concentration was observed for samples containing 0 and 50 µg/ml of albumin in both solvent preparations. Coincubation with SOD plus Cat resulted in complete abolition of fluorescence attenuation, confirming that the changes in fluorescence were free radical mediated.
Effects of
Cu2+ and
Fe2+ free
radical systems on SP-A in intact surfactant and purified SP-A in
aqueous buffer.
To assess whether interactions with lipid surfactant components afford
protection to SP-A against the effects of free radicals, fluorescamine
assay results performed on SP-A samples isolated by butanol
precipitation from whole native surfactant after incubation with free
radicals for 24 h were compared with the results from assays performed
on SP-A first purified from surfactant and then incubated with free
radicals in aqueous buffer (n = 3 samples). FeCl2 and
CuCl2 free radical systems, which
produced large fluorescamine signals after incubation with whole
surfactant, were examined. The results are summarized in Fig.
3. The percent decrease in fluorescence for
SP-A after incubation with and without lipids was similar. Samples
incubated with CuCl2 alone, with
H2O2,
and with
H2O2
plus ascorbate for 24 h all demonstrated a significantly greater
decrease in fluorescence than the corresponding samples incubated with
FeCl2.
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DISCUSSION |
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Free radical species have been shown to affect the interfacial
properties of various surfactant preparations in vitro, and it has been
postulated that similar mechanisms contribute to the pathophysiology of
lung dysfunction in acute lung injury (18, 25, 26). Previous studies
(8, 11) have demonstrated that organic extracts of surfactant display
abnormal behavior, measurable lipid peroxidation products, and
altered SP components after exposure to oxygen free radicals. The
present study examined the biophysical and biochemical consequences of
free radical exposure on intact whole lung surfactant rather than on an
organic extract. The results demonstrate that surfactant lipid and
protein components, specifically SP-A, are chemically altered by
exposure to oxygen free radicals in vitro and that radical species
generated with different transition metals can modify surfactant lipids
and proteins differently. Despite evidence for free radical lipid and
protein modifications, all surfactant samples tested at a 1 mg/ml bulk
concentration displayed nearly identical equilibrium
values and
reached minimum
values of <1 dyn/cm during PBS within seconds of
initiating oscillation, similar to control samples.
Interpretation of biochemical changes resulting from free radical exposure. The findings presented here are largely consistent with and expand on previously published results. An elevation in TBARS and a decrease in unsaturated lipids have been observed by Gilliard et al. (9) after incubation of surfactant organic extracts, which do not contain SP-A, with FeCl2 plus H2O2. Haddad et al. (12) have also observed lipid peroxidation after exposure of organic surfactant extracts to free radicals generated by exposure to peroxynitrite and hydroxyl radical. In the present study, additional insight is provided regarding the specific free radical pathways likely to be involved in these observations. After 4 h, TBARS were increased in samples containing Fe2+ plus H2O2 either alone or with ascorbate without a further increase for up to 24 h. These results are most consistent with lipid peroxidation consequent to hydroxyl radical formation via a Haber-Weiss pathway because only samples containing both reduced iron and hydroxyl radical had measurable TBARS. However, samples incubated with Fe2+ alone, which had failed to show evidence of lipid peroxidation after 4 h, did demonstrate a significant increase in TBARS by 24 h (0.27 ± 0.57 vs. 1.52 ± 0.26 nM; P < 0.05 by ANOVA). This observation is most consistent with an autoxidation process in which Fe2+ reduces triplet oxygen to superoxide anion, undergoes dismutation to hydrogen peroxide, and then participates in a conventional Haber-Weiss reaction. This pathway is known to proceed spontaneously in aqueous solutions at neutral pH in the presence of molecular oxygen.
The temporal pattern and magnitude of TBARS production in samples containing Fe3+ and Fe3+-EDTA conjugate were quite distinct from those observed in Fe2+ samples. Significant increases in TBARS were observed by 4 h, independent of whether exogenous H2O2 and ascorbate were added. Additional increases in TBARS levels after incubation for up to 24 h occurred only in samples containing unconjugated Fe3+, conjugated Fe3+, and conjugated Fe3+ with H2O2. Because Fe3+ cannot directly catalyze a Haber-Weiss reaction, alternative pathways must account for these findings. One potential mechanism is through the reduction of Fe3+ to Fe2+ (6) by aqueous reducing compounds in solution, with subsequent participation in a conventional Haber-Weiss reaction. Alternatively, generation of reactive ferryl ion as a result of the interaction between Fe3+ and hydroxyl radical has been proposed (27). This second pathway is important in membrane lipid peroxidation in vitro and thus may be specifically relevant to reactions involving surfactant lipids (14).
The addition of SOD and Cat to the incubation mixtures resulted in incomplete suppression of TBARS production among FeCl3 samples, suggesting site-specific catalysis of free radical reactivity (17). In this process, the transition metal catalyst associates closely with the free radical target compound and is relatively inaccessible to aqueous soluble inhibitors. Our observations suggest that ferric ion complexes with either surfactant anionic PLs (31) or SP components and subsequently catalyzes formation of radical species within the lipid environment.
In contrast to the results observed in Fe2+ and Fe3+ samples, no TBARS formation was measured after exposure of whole surfactant to any Cu2+ free radical systems. Cu2+ either alone or together in aqueous solution with hydrogen peroxide is thought to catalyze formation of hydroxyl radical via a conventional Haber-Weiss pathway. The redox potential for the reactions of Cu2+, Fe2+, and Fe3+ with H2O2 are similar and are dictated primarily by the favorable energetics of electron transfer to H2O2. Therefore, the explanation for differences in lipid reactivity between copper- and iron-catalyzed reactions is not due to differences in reactivity related to redox potential. Furthermore, Gilliard (9) did, in fact, observe TBARS formation after 24 h of incubation of hydrophobic surfactant extracts with aqueous CuCl2, indicating that Cu2+-mediated free radical reactions involving lipid components can occur under appropriate conditions. Taken together, these findings suggest that Cu2+, like Fe3+, interacts with surfactant components in a site-specific fashion. Partitioning of metalloligand complexes between aqueous and lipid phases is known to be influenced by the biophysical properties of the ligand. In water, CuCl2 forms homogeneously dispersed hydroxy-chloro complexes (6). These metallocomplexes have been shown to selectively associate with peptide side groups of proteins, after which their reactivity is quite different from unbound Cu2+ in solution. In this situation, free radical-mediated reactions occur in immediate proximity to the ion complex and would therefore be restricted to the protein components of surfactant. Failure of SOD and Cat to completely inhibit these chemical alterations also favors site-specific reactivity involving Cu2+.
Although TBARS are the primary lipid breakdown products formed after exposure of surfactant to oxygen free radicals, nucleophilic free radical addition at the sn-1 and sn-2 carbons involved in acyl-ester linkages of surfactant PLs has also been reported. This reaction results in generation of free acyl alcohols and lysophospholipids (22) and is thought to account for reported "phospholipase-like" activity of certain free radical compounds. In the present study, however, thin-layer chromatography followed by quantitative PL extraction failed to reveal generation of lysophospholipid compounds (limit of detection 5 µg, total sample analyzed 400 µg) after surfactant exposure to free radicals, arguing against a role for this pathway in the present study.
Fluorescamine results demonstrate that free radical generation mediated by all of the transition metals can affect protein structure. In control studies with aqueous albumin, protein modification was observed after 4 h of incubation. This was prevented by coincubation with SOD plus Cat, indicating that the changes in fluorescence signal were specific to reactive oxygen radical species and were not the result of a change in solution ionic strength, which could cause charge-related alterations in protein tertiary structure resulting from internalization of free amino groups. Fluorescence attenuation was observed in surfactant samples after 4 h of exposure to each of the transition metal systems, with no further change for up to 24 h.
To investigate whether interactions with surfactant lipids might influence the susceptibility of SP-A to free radical modification, we compared fluorescamine signals of aqueous soluble SPs isolated by butanol precipitation after free radical exposure of whole surfactant with those measured in samples containing purified aqueous SP-A exposed to Cu2+ plus H2O2 and Fe2+ plus H2O2 in the absence of lipids. No difference was observed between the two preparations, suggesting that lipid binding does not significantly influence the susceptibility of SP-A to free radical attack.
Western blot analysis demonstrated modification of SP-A structure after free radical exposure. Intact surfactant, butanol precipitates of surfactant, and aqueous SP-A all showed similar changes by Western blot. Both smaller- and larger-molecular-mass aggregates were observed after overnight incubation, similar to observations previously reported by Haddad et al. (11). These alterations in SP-A structure are thought to result from free radical-mediated oxidation-reduction of the intrachain disulfide bonds that maintain the complex hexameric configuration of native SP-A (29).
Biophysical consequences of free radical-mediated
biochemical changes assessed in terms of interfacial
properties. Although these data clearly demonstrate
that free radical exposure affects the biochemical composition of lipid
and protein surfactant components, the changes appear to have minimal
effects on the interfacial properties at a bulk-phase lipid
concentration of 1 mg/ml. -A profiles of all exposed samples demonstrated minimum
values of <1
dyn/cm and maximum
values of 28-30 dyn/cm. In the majority of
samples, minimum
was reached after 30-35% compression,
similar to control samples. After 24 h of incubation, only surfactant samples incubated with FeCl2 plus
H2O2
and CuCl2 plus
H2O2
demonstrated any abnormalities, manifest as an increase in the
compression ratio to achieve minimum
from 30 to >70%.
Dysfunction in both samples was reversed by the addition of
CaCl2 after completion of free
radical exposure and was likewise prevented by the addition of 5 mM
CaCl2 to the surfactant-free
radical mixture before incubation. Although
CaCl2 restored function, it did
not prevent TBARS formation, fluorescamine changes, or Western blot
changes in SP-A structure observed in
Ca2+-free samples. Addition of 5%
purified SP-A restored function to
FeCl2 plus
H2O2-exposed
samples but not to CuCl2 plus
H2O2-exposed samples, suggesting that although both free radical systems affect SP-A, the specific sites of reactivity may be different. The
Ca2+-dependent changes in
surfactant function reported here may relate to the effects of this
divalent cation on SP-A conformation and on its ability to promote PL
aggregation (30).
Alterations in surfactant interfacial properties caused by serum proteins, membrane lipids, or lysophospholipid products have been reported in vitro, and each of these factors has been proposed as a cause of surfactant dysfunction in ARDS (12, 17). The majority of previous studies (5, 33) have involved organic extracts of surfactant rather than whole surfactant, however. The results presented here involving whole surfactant highlight the potential differences that can exist between extracted and intact surfactant with respect to susceptibility to inactivation. In those studies that utilized whole surfactant, dysfunction from these mechanisms has been either minimal or observed in dilute samples of surfactant with concentrations below those likely to be present in the normal lung. Together, these findings suggest that, in vitro, whole lung surfactant is relatively resistant to inactivation by any one of the many mechanisms proposed to account for inactivation in vivo during acute lung injury and that multiple mechanisms contributing simultaneously may be required to generate dysfunction in the intact lung.
In summary, our results demonstrate that exposure of surfactant to oxygen free radicals can result in changes in both lipid and protein constituents. Our results expand on previous findings by demonstrating that these effects are dependent on the specific in vitro free radical system used. Iron-based systems caused significant biochemical changes in both SP-A and surfactant lipids, whereas copper-based systems affected only SP-A, suggesting site-specific reactivity. Furthermore, although biochemical changes in surfactant were observed, these changes had minimal consequences on surfactant interfacial properties, in contrast to functional studies previously reported after exposure of surfactant extracts to free radicals (9, 12, 25).
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FOOTNOTES |
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Address for reprint requests: E. P. Ingenito, Pulmonary and Critical Care Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: epingenito{at}bics.bwh.harvard.edu).
Received 31 December 1997; accepted in final form 17 November 1998.
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