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(Chest. 2002;121:573-581.)
© 2002 American College of Chest Physicians

Tissue Lipid Peroxidation and Reduced Glutathione Depletion in Hypochlorite-Induced Lung Injury*

Stefan Hammerschmidt, MD; Nicole Büchler and Hans Wahn, MD

* From the Department of Medicine, University Würzburg, Würzburg, Germany.

Correspondence to: Stefan Hammerschmidt, MD, Medizinische Universitätsklinik I, Johannisallee 32, D-04103 Leipzig, Germany; e-mail: stefan.hammerschmidt{at}t-online.de


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: Neutrophils are involved in acute lung injury during ARDS via several mechanisms. This study focuses on neutrophil-derived oxidative stress. Hypochlorite is a major neutrophil-derived oxidant. This study characterizes hypochlorite-induced acute changes in pulmonary circulation and the involvement of tissue lipid peroxidation (LPO) and reduced glutathione (rGSH) depletion.

Methods: Hypochlorite (500, 1,000, and 2,000 nmol/min) or buffer (control) were infused into isolated rabbit lungs. Pulmonary artery pressure (PAP), capillary filtration coefficient (Kf,c) [104/mL/s/cm H2O/g], and lung weight were measured. Experiments were terminated after 105 min or when fluid retention was > 50 g. Lung tissue was frozen immediately after termination of the experiments and analyzed for LPO products and rGSH (nanomoles per milligram of protein).

Results: Baseline PAP and Kf,c values averaged from 6.1 to 6.5 mm Hg and from 0.97 to 1.23, respectively, in all groups. Hypochlorite infusion of 500, 1,000, and 2,000 nmol/min (n = 5 to 7 per group) evoked an increase (mean ± SEM) in maximum PAP (PAPmax) [12.9 ± 2.1, 14.3 ± 1.7, and 13.3 ± 2.2 mm Hg], in maximum Kf,c (Kf,cmax) [1.9 ± 1.2, 6.34 ± 1.2, and >10.0], and in tissue LPO products (1.7 ± 0.06, 2.1 ± 0.06, and 2.3 ± 0.11 vs 1.4 ± 0.04 in controls), and a decrease in tissue rGSH (73.4 ± 8.7, 43.0 ± 9.6, and 50.4 ± 7.2 vs 139 ± 12.6 in controls). Parameters of lung injury (PAPmax and Kf,cmax) of each single experiment were closely correlated with tissue rGSH but did not correlate with tissue LPO products. All changes are significant (p < 0.05) vs control.

Conclusion: The neutrophil-specific oxidant hypochlorite induces acute lung injury, rGSH depletion, and LPO in isolated rabbit lungs. The lung injury correlates with rGSH depletion, suggesting an important mechanistic role in hypochlorite-induced acute lung injury.

Key Words: ARDS • hypochlorite • glutathione • lipid peroxidation • oxidative stress


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung injury during ARDS is characterized by noncardiogenic pulmonary edema due to increased vascular permeability and by increased pulmonary artery pressure (PAP).1 2 3 A great variety of mechanisms and mediators are involved in the pathogenesis of the lung injury during ARDS.3 4 5 Although acute lung injury is observed in neutropenic patients and in leukocyte-free animal models,6 7 polymorphonuclear leukocytes (PMN) play a crucial role in the pathogenesis of acute lung injury.4 8 9 10 The sequestration of PMN in the pulmonary microvasculature due to chemotactic stimuli is accompanied by a measurable increase in the count of PMN and of myeloperoxidase activity in the BAL fluid.11 12 Stimulated neutrophils may affect lung tissue via the release of proteolytic enzymes,10 13 the production of prostanoids,14 15 or the generation of highly reactive oxygen species.8 10 13

Oxidative stress derived from activated PMN and endothelial cells is considered to be one major final pathway in acute lung injury. Several studies deal with oxidative stress-induced acute lung injury, including lung injury due to complement activation or ischemia/reperfusion,16 17 endotoxin-induced,18 quartz-induced,19 cobra venom factor-induced or IgG immune complex-induced lung injury,20 and especially ARDS.21 22 These studies report tissue lipid peroxidation (LPO) as indicated by accumulation of LPO products, eg, malonaldehyde or 4-hydroxyalkenals, and/or tissue thiol and reduced glutathione (rGSH) depletion. They investigate the effects of oxidative stress in several models of acute lung injury but do not characterize the biochemical actions of single oxidants. Several highly reactive oxygen radicals and metabolites are generated during the respiratory burst of stimulated PMN.23 Hypochlorite is synthesized by a myeloperoxidase-mediated reaction from hydrogen peroxide and chloride. Whereas hydrogen peroxide, superoxide, and hydroxyl radical are also released from sources other than PMN,24 25 hypochlorite is a PMN-specific oxidant. Hypochlorite, as a nonradical oxidant, has been shown to cause oxidative modification of free functional groups of proteins and amino acids, especially of free sulfhydryl groups.26 27 Furthermore, hypochlorite has been shown to evoke changes in pulmonary microvasculature that are comparable to the effects of stimulated PMN.28 Whereas hydrogen peroxide, another nonradical oxidant, causes glutathione depletion and—via iron-dependent hydroxyl radical formation29 —tissue LPO in pulmonary microvascular cells,30 the effects of hypochlorite on lung tissue thiol or rGSH content and LPO have not been investigated. Hypochlorite causes oxidation of intracellular rGSH of erythrocytes.31 However, there is a controversy about the ability of hypochlorite to induce LPO. Whereas some studies report no LPO32 or only slow rates of LPO due to coincubation of human low-density lipoprotein with hypochlorite,33 other authors34 35 describe hypochlorite-induced LPO in liposomes and lipoproteins. Studies35 36 have elucidated a possible mechanism of hypochlorite-induced LPO and point out the importance of the reaction of hypochlorite with hydroperoxides that yields free radicals able to cause further oxidation of lipids. However, there are no data about hypochlorite-induced LPO in more complex experimental models, such as an isolated organ.

This study uses a model of hypochlorite-induced lung injury in isolated rabbit lungs as a model of neutrophil-derived oxidative stress-induced lung injury. It tests the hypothesis that the specific neutrophil-derived product, hypochlorite, may directly reduce tissue rGSH content and induce LPO. Furthermore, the study correlates indicators of acute lung injury, ie, the PAP and vascular permeability with changes in tissue rGSH and LPO product content.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolated Rabbit Lung Model
Preparation of isolated rabbit lungs was performed using the method described in detail by Seeger et al.37 Rabbits of either sex between 2.5 kg and 3.5 kg were used. The lungs were ventilated with 4% CO2, 17% O2, and 79% N2 (tidal volume, 30 mL; frequency, 30/min). Perfusion flow was gradually increased to the definite flow rate of 100 mL/min with recirculation of the buffer medium (Krebs-Henseleit buffer; total circulating volume, 300 mL). Perfusate temperature was 37°C. PAP, pulmonary venous pressure (PVP), inflation pressure, and the weight gain of the isolated organ were continuously recorded. After a steady-state period of 30 min, only those lungs were selected for the study that showed no signs of leakage at the catheter insertion sites, that had a homogenous white appearance with no signs of edema formation, and that had lost weight during the phase of temperature increase and were completely isogravimetric during the steady-state period. After steady state, the PAP ranged from 5 to 8 mm Hg. The inspiratory peak inflation pressure was adjusted to between 7 mm Hg and 9 mm Hg. The PVP was adjusted to 2 mm Hg.

Hydrostatic Challenge
The capillary filtration coefficient (Kf,c) was determined gravimetrically from the slope of lung weight gain after a sudden venous pressure elevation of 10 cm H2O for 8 min.38 Kf,c was related to wet weight lung, which was calculated from the body weight using the following equation: wet weight lung = body weight times 0.0024. Kf,c values are presented as 104/mL/s/cm H2O/g.

Vascular compliance is defined as the change in vascular volume divided by change in microvascular pressure. The total rapid change in weight over the first 1 to 2 min after onset of the venous pressure rise was taken as pure vascular filling and used for the calculation of compliance that was given in grams per centimeters of water column.38

Retention was determined as the remaining difference between weight before and after a hydrostatic challenge corresponding to the remaining interstitial fluid after normalization of venous pressure and equilibration of a new fluid balance. It was presented in grams.

Substances
All reagents were obtained from Sigma (Munich, Germany). The concentration of the hypochlorite stock solution was determined spectrophotometrically ({epsilon} 290 nm = 350 mol/cm) immediately before use.39

Analytical Methods
Frozen lung tissue was stored at - 80°C. For analysis, it was homogenized in ice-cold buffer. The homogenates were centrifuged at 3,000g for 5 min at 4°C. The supernatant was assayed for protein concentration, for LPO products, and for free thiols and rGSH.

Protein Concentration:
The homogenate protein concentration was measured in accordance with a method described by Lowry et al.40

LPO Products:
The tissue content of the LPO products (malonaldehyde and 4-hydroxyalkenals) was measured using an assay kit obtained from Calbiochem-Novabiochem GmbH (Schwalbach, Germany). This assay kit is based on a chromogenic reagent, which reacts with malonaldehyde and 4-hydroxyalkenals at 45°C yielding a stable chromophore with a maximum absorption at a wavelength of 586 nm. The concentration of LPO products in the tissue homogenate was related to homogenate protein concentrations. Thus, tissue LPO products were presented as nanomoles per milligram of protein.

rGSH and Free Sulfhydryl Groups:
The tissue rGSH content and the content of free functional sulfhydryl groups were assayed using an assay kit obtained from Calbiochem-Novabiochem GmbH. This kit uses a two-step reaction. Thioethers with a maximum absorption at 356 nm are formed as substitution products of all mercaptans in a first reaction. The second reaction step transforms specifically the substitution product obtained with rGSH into a chromophoric thione with a maximum absorption at 400 nm. The homogenate rGSH and free functional sulfhydryl concentrations were related to homogenate protein concentrations. Thus, the tissue content of reduced rGSH and free functional sulfhydryl groups were presented as nanomoles per milligram of protein.

Perfusate concentrations of potassium and lactate dehydrogenase (LDH) activity were determined to demonstrate that no severe cell damage had occurred. Measurement of potassium and LDH were performed by routine laboratory methods.

Experimental Protocol
The continuous infusion of hypochlorite into the arterial line of the system was started after a 30-min steady-state period (time = 0 min) and after a baseline hydrostatic challenge (time = - 15 min). Hypochlorite, which was diluted with perfusate in different concentrations, was infused with a constant flow rate of 0.5 mL/min, so that hypochlorite doses of 500, 1,000, and 2,000 nmol/min were administered (500 nmol/min, 1,000-nmol/min, and 2,000 nmol/min HOCl groups, respectively). PAP, PVP, and lung weight gain were continuously recorded. Hydrostatic challenges were performed at 30 min, 60 min, and 90 min. The experiments were terminated after 105 min or when lung weight gain was > 50 g due to severe edema formation. Lung tissue was rapidly frozen in liquid nitrogen immediately after termination of the experiment for later analysis.

The results were compared with two control groups. For control, the experiment was terminated after the 30-min steady state period (time = 0 min) [c-0] or the experiment was terminated after continuous buffer administration during the whole recording time of 105 min (c-105). Potassium concentration and LDH activity were measured immediately before hydrostatic challenges to show that no cell damage had occurred.

Statistical Methods
Data were given as mean ± SEM. Analysis for statistical significance was performed by the two-tailed Student’s t test for unpaired samples. Bonferroni’s correction for multiple testing was performed.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypochlorite-Induced Effects on Pulmonary Microvasculature
The hypochlorite-induced pressure response is summarized in Figure 1 . There are no differences in the baseline pressures of approximately 6 mm Hg in the groups. No pressure response was found in control experiments (c-105). Continuous infusion of hypochlorite causes a dose-dependent increase in PAP. The start of the pressure rise and the time of maximum PAP (PAPmax), which is given in Figure 2 , are dose dependent. Severe edema formation with an increase in lung weight gain of > 50 g results in premature termination especially in the 1,000 nmol/min HOCl and 2,000 nmol/min HOCl groups after mean recording times of approximately 80 min and 40 min, respectively, as shown in Figure 2 . The premature termination prevents a further pressure rise and may explain why PAPmax does not differ in the hypochlorite groups.



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  		Figure 1. Hypochlorite-induced pressure response. Upper panel: course of the increase in PAP due continuous infusion of hypochlorite over time. Lower panel: comparison of baseline PAP (PAPt = 0 min) and PAPmax of all experimental groups (*p < 0.05 vs c-105).

 


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  		Figure 2. Mean total recording (ttot) and mean time of PAPmax (tPAPmax) of all experimental groups are compared (*p < 0.05 vs c-105).

 
Table 1 summarizes the results calculated from the hydrostatic challenges. Kf,c, fluid retention, and vascular compliance average from 0.93 to 1.20 104/mL/s/cm H2O/g, from 1.2 to 2.0 g, and from 0.32 to 0.38 g/cm H2O, respectively. No significant differences are found under baseline conditions and in the c-105 group. Hypochlorite evokes dose-dependent effects on Kf,c and fluid retention. A twofold increase in Kf,c and fluid retention is caused by 500 nmol/min of hypochlorite after 90 min. Infusion of 1,000 nmol/min of hypochlorite results in significant changes in Kf,c after 30 min and 60 min and in fluid retention after 60 min. Because of an exponential increase in the weight gain over time, Kf,c is not calculated after 30 min in the 2,000 nmol/min HOCl group. This exponential time course of the weight gain, however, indicates a remarkable increase in Kf,c > 103/mL/s/cm H2O/g. The vascular compliance is found to remain unchanged during hypochlorite administration, indicating that the increase of the lung weight is due to changes in the microvascular permeability rather than to increased vascular filling. Because the PAP does not exceed 18 mm Hg, the formation of hydrostatic edema would not be expected. Perfusate concentrations of potassium and LDH (data not shown) show no significant change as well, indicating that the observed effects are not due to nonspecific cell damage.


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  		Table 1. Kf,c, Fluid Retention, and Vascular Compliance Measured During Hydrostatic Challenges*

 
Tissue rGSH and LPO Products
Figure 3 shows the tissue content of rGSH, free sulfhydryl groups, and of the LPO products (malonaldehyde and 4-hydroxyalkenals). There are no significant differences between both control groups in rGSH and free sulfhydryl groups. Tissue rGSH and free sulfhydryl group levels are decreased to about 55% of control group values after continuous infusion of 500 nmol/min of hypochlorite. Nearly similar rGSH and free sulfhydryl group levels of approximately 30 to 35% and approximately 35 to 40% of control group values are found in the 1,000 nmol/min HOCl and 2,000 nmol/min HOCl groups, respectively. The ratios between rGSH and total free nonprotein sulfhydryl groups are summarized in Table 2 . These ratios indicate that rGSH represents approximately 75 to 90% of the total, nonprotein free functional sulfhydryl groups. Hypochlorite infusion does not evoke significant changes of this ratio.



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  		Figure 3. Tissue concentration of rGSH and of free functional sulfhydryl (SH) groups (upper panel) and the tissue concentration of the LPO products malonaldehyde and 4-hydroxyalkenals (lower panel) of all experimental groups are shown (*p < 0.05 vs both controls).

 

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  		Table 2. Ratios Between rGSH and Total Free Nonprotein Sulfhydryl Groups*

 
Measurement of rGSH in perfusate samples of all groups indicates that no release of rGSH into the perfusate occurs (data not shown). The tissue content of LPO products does not differ between both control groups. Continuous hypochlorite administration causes a dose-dependent increase in these LPO products to 123%, 143%, and 164% of control group value in the 500 nmol/min, 1,000 nmol/min, and 2000 nmol/min HOCl groups, respectively.

Hypochlorite-induced changes in pulmonary microvasculature, ie, {Delta}PAPmax and maximum Kf,c (Kf,cmax), are plotted against tissue rGSH content and against tissue LPO products in Figures 4 , 5 . As the Kf,c of the 30-min hydrostatic challenge are not calculated in the 2,000 nmol/min HOCl group due to an exponential time course of lung weight gain, there is no plot for the Kf,cmax of the 2,000 nmol/min HOCl group. Figure 4 shows negative correlations between {Delta}PAPmax/Kf,cmax and the tissue rGSH content. These correlations are found within the data sets of each hypochlorite group as well as within the pooled data sets of all hypochlorite groups. They may indicate a functional connection between hypochlorite-induced deterioration of pulmonary microvasculature and pulmonary rGSH depletion. As demonstrated by Figure 5 , there is no correlation between changes in pulmonary microvasculature and tissue level of LPO products, suggesting that tissue LPO may be less important in the mediation of hypochlorite-induced effects on pulmonary microvasculature.



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  		Figure 4. Parameters of lung injury, ie, {Delta}PAPmax (upper panels) and Kf,cmax (lower panels), of the 500 nmol/min, 1,000 nmol/min, and 2,000 nmol/min HOCl groups are plotted against the corresponding tissue rGSH concentration of each individual experiment. The left column summarizes these correlations of the data for all three groups. The correlation coefficient and its p value are given.

 


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  		Figure 5. Parameters of lung injury, ie, {Delta}PAPmax (upper panels) and Kf,cmax (lower panels), of the 500 nmol/min, 1,000 nmol/min, and 2,000 nmol/min HOCl groups are plotted against the corresponding tissue LPO product concentration of each individual experiment. The correlation coefficient and its p value are given.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a prior study,28 we demonstrated that the neutrophil-derived oxidant hypochlorite causes an increase in PAP and vascular permeability. These changes are comparable to effects of stimulated neutrophils28 and are observed during ARDS.1 2 In the current study, we report that the effects of hypochlorite on PAP and vascular permeability are accompanied by an by an increase in the tissue content of LPO products and by rGSH depletion. LPO and rGSH depletion have been observed in clinical studies21 22 and in animal models of acute lung injury.16 17 18 19 20 As the present study uses a single oxidant in an isolated lung model, the effects on PAP and vascular permeability as well as on LPO and rGSH depletion may be directly attributed to hypochlorite without the interfering influences from systemic circulation. The study provides evidence that hypochlorite causes LPO and rGSH depletion. It shows a correlation between parameters of lung injury and rGSH depletion, which suggests a possible role of this effect in the pathogenesis of acute lung injury. This contributes to the explanation of the role of neutrophils in acute lung injury.

Stimulated neutrophils release 2 x 10-7 mol of hypochlorite per 106 cells during 2 h.41 Assuming this hypochlorite generation rate and the perfusate volume used (300 mL), a PMN count of 2,000/µL is required to release 1,000 nmol/min of hypochlorite. This cell count represents a level in the range rather low to normal. Much higher PMN counts are found during inflammatory states. Considering these calculations, the infusion of the hypochlorite dosages used (500 to 2,000 nmol/min) is regarded as an appropriate dosage. Together with a flow rate of 100 mL/min, this dosage results in perfusate hypochlorite concentrations between 5 µmol/L and 20 µmol/L.

Hypochlorite causes oxidative modification of free functional groups (preferentially thiol and thioether groups).26 27 The same low hypochlorite concentrations that are used in the present study have been reported to cause oxidation of plasma membrane sulfhydryls and disturbances of various plasma membrane functions in a cell culture study.42 This study demonstrated a relation between alterations of the oxidative state of the cell membrane and disturbances of its function. In line with these results, the hypochlorite-induced alterations in PAP and vascular permeability are accompanied by a decrease in tissue rGSH concentration in our isolated organ model as well. The hypochlorite-induced decrease in tissue rGSH is dose dependent and is closely correlated with the maximum alterations of PAP and Kf,c. Whether rGSH depletion plays a causative role in hypochlorite-induced lung injury or is only an indicator of vascular injury cannot be concluded on the basis of this correlation. Decreased total glutathione and rGSH levels in the alveolar fluid are found in patients with ARDS.22 Experimental studies43 44 as well as clinical trials45 46 in patients with ARDS have, however, demonstrated that replacement of rGSH by use of N-acetylcysteine reduces oxidative stress-induced lung injury. These findings support the concept that depletion of rGSH is an important mechanism contributing to lung injury in ARDS.

Glutathione is the most prevalent cellular nonprotein thiol that occurs ubiquitous in eucaryotic cells.47 It plays a crucial role in protecting tissue against oxidizing conditions and in maintaining intracellular redox balance.48 The lung is exposed physiologically to a large oxidative burden due to its barrier function against the atmosphere and due to the oxidative activity of the alveolar macrophages. In addition to the intracellular glutathione pool, the pulmonary epithelial cells are protected by epithelial lining fluid that contains glutathione in a higher concentration than plasma to maintain the extracellular redox balance.49 Glutathione is synthesized in the liver and released into the lining fluid from type II cells.47 More than 96 to 99% of the total glutathione is reduced under physiologic conditions.47 As there is no difference in the rGSH level between the c-0 and the c-105 groups, and as the rGSH resembles 96 to 99% of total glutathione,47 a significant decrease in total glutathione may be ruled out under control conditions. It is not possible to ascertain whether the decrease in rGSH level in the hypochlorite groups is due to oxidation of rGSH to oxidized glutathione (GSSG), to a decrease in total glutathione, or to both mechanisms on the basis of the data obtained in this study. Independently of the mechanism, the decrease in rGSH tissue concentration indicates impaired antioxidative defense. The rGSH concentration is measured in frozen lung tissue and represents the intracellular and extracellular rGSH. Therefore, this study does not provide information about differences between intracellular and extracellular changes in rGSH concentration. As hypochlorite is infused into the perfusate, the oxidative injury may first affect endothelial cells and later involve the pulmonary epithelial cells. Finally, the epithelial lining fluid may be affected either due to disturbed redox balance in type II cells or due to direct oxidation.

In our study, the hypochlorite-induced rGSH depletion is dose dependent. The tissue rGSH content in the 1,000 nmol/min HOCl group is lower than in the 500 nmol/min HOCl group. No significant difference, however, is found between the 1,000 nmol/min group and the 2,000 nmol/min HOCl group. This observation may be explained by the following considerations: when the increase in lung weight due to edema formation is > 50 g, the experiments are terminated. Edema formation is caused by an increase in vascular permeability. As the increase in vascular permeability, ie, in Kf,c, is closely correlated with rGSH depletion, the experiments are terminated when the rGSH depletion reaches a defined level of approximately 50 nmol/mg protein. In the present study, premature termination due to edema formation occurs in the 1,000 nmol/min HOCl group and 2,000-nmol/min HOCl group after a mean recording time of 78.8 ± 6.1 min and 41.2 ± 3.3 min, ie, after total hypochlorite doses of approximately 78.8 µmol/L and 82.4 µmol/L, respectively. Obviously, this hypochlorite dosage causes a certain rGSH depletion (to approximately 50 nmol/mg protein) that is accompanied by severe edema formation resulting in termination of the experiments. This hypochlorite dose and therefore this rGSH depletion is not reached in the 500 nmol/min HOCl group after the regular recording time of 105 min.

Hypochlorite may induce depletion in tissue rGSH via the following mechanisms: (1) both hypochlorite-derived and hypochlorite-derived chloramines cause oxidative modification of sulfhydryl groups,27 resulting in the formation of GSSG; and (2) the maintenance of physiologic redox balance with restoration of rGSH from GSSG by the reduced nicotinamide adenine dinucleotide-dependent enzyme, glutathione reductase, and the reduced nicotinamide adenine dinucleotide providing glucose-6-phosphate dehydrogenase is an energy-dependent process47 that may be affected by hypochlorite.42

The observed rGSH depletion to approximately 35% of the baseline value represents a severe disturbance of the redox balance. The impaired antioxidative defense may fail to maintain the oxidative integrity of functionally relevant sulfhydryl and methionyl groups of several enzymes for cellular energy or calcium metabolism. This way, rGSH depletion may precede and cause alterations of energy and calcium-dependent functions, such as increased vascular tone or increased endothelial permeability. These considerations and the correlation between rGSH depletion and parameters of lung injury may allow us to conclude that hypochlorite-induced rGSH depletion plays an important role in hypochlorite-induced lung injury.

In line with other studies16 17 18 19 20 21 22 of oxidative stress-induced lung injury, our study shows a dose-dependent, hypochlorite-induced increase in LPO products in lung tissue. In contrast to these studies, it uses a single oxidant, hypochlorite. Reactive oxygen species such as hydroxyl radical, alkoxyl radicals, and the hydroperoxy radical, but not the nonradical oxidants hydrogen peroxide and hypochlorite, are able to abstract the first hydrogen atom from a methylene group, which is required to initiate LPO.50 Although some studies do not find hypochlorite-mediated LPO,32 51 there are data and considerations that may explain hypochlorite-induced LPO. Hypochlorite may give rise to the hydroxyl radical by an iron-dependent reaction.52 Although iron or any other transition metal are not a constituent of the perfusate, they occur in the intracellular compartment. Furthermore, hypochlorite-induced LPO has been shown to occur independently of hydroxyl radical formation.35 Organic hydroperoxides, which are found in vivo, have been shown to interact with hypochlorite and form free radicals that are initiators of LPO.36

In line with these data, our study demonstrates, for the first time, hypochlorite-induced LPO in a more complex system such as an isolated organ preparation. It might be expected that LPO of the cell membrane would evoke a nonspecific increase in membrane permeability and nonspecific cell damage. However, this may be ruled out, as release of LDH into the perfusate is not observed. There is no correlation between LPO product accumulation and lung injury. This may be because of several reasons: (1) the measurement of malonaldehyde as a LPO product represents only a single parameter of LPO and does not rule out correlations between other indicators of LPO and lung injury; and (2) this study determines whole-lung malonaldehyde content. It is possible that LPO represents an important mechanism only in a distinct compartment, which is small in comparison with the whole lung. This may lead to a potential correlation being missed. The results of our study verify a previous study53 using a model of oxidative stress-induced lung injury in sheep that also did not find a correlation between lung injury and LPO.

In the present study, hypochlorite-induced oxidative lung injury is accompanied by tissue rGSH depletion and by accumulation of LPO products. LPO, which requires the interaction between hypochlorite and transition metals or organic hydroperoxides, may, of course, occur due to hypochlorite administration. However, the highly reactive hypochlorite-induced oxidative modification of free functional groups, especially of thiols, which correlates with parameters of lung injury, may represent a major mechanism in neutrophil-derived oxidative stress-induced acute lung injury.


   Footnotes
 
Abbreviations: c-0 = control experiment terminated after 30-min steady-state period; c-105 = control experiment terminated after continuous buffer administration during the whole recording time of 105 min; GSSG = oxidized glutathione; Kf,c = capillary filtration coefficient; Kf,cmax = maximum capillary filtration coefficient; LDH = lactate dehydrogenase; LPO = lipid peroxidation; PAP = pulmonary artery pressure; PAPmax = maximum pulmonary artery pressure; {Delta}PAPmax = maximum increase in pulmonary artery pressure; PMN = polymorphonuclear leukocytes; PVP = pulmonary venous pressure; rGSH = reduced glutathione

Received for publication November 30, 2000. Accepted for publication August 9, 2001.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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