1 Department of Anaesthesiology and Intensive Care, Hotel-Dieu Hospital, 1 Place de lhôpital, F-69288 Lyon cedex 02, France. 2 Department of Biochemistry, Edouard Herriot Hospital, Lyon, France*Corresponding author
Accepted for publication: February 18, 2002
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Abstract |
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Methods. We studied plasma and bronchoalveolar lavage (BAL) samples for markers of oxidative stress, taken from patients with VAP. Seventy-eight patients likely to have VAP and 10 patients who were not suspected of having VAP were studied prospectively. A diagnosis of VAP was based on a positive quantitative mini-lavage culture of 103 colony-forming units per ml. Blood and BAL samples were collected and analysed for thiobarbituric acid reactant substances (TBARS) and antioxidant activity.
Results. Plasma and alveolar TBARS increased significantly in patients who developed VAP compared with those who did not, by 43% and 259% respectively. Red cells and alveolar glutathione peroxidase concentrations (antioxidant activity) were lower in those with VAP compared with the non-VAP group, by 43% and 68% respectively. No significant differences were found for serum superoxide dismutase activity. Significant differences were found for alveolar glutathione peroxidase concentrations over time in the VAP group. No significant differences between survivors and non-survivors were found in the blood or BAL assays, in patients with VAP.
Conclusions. VAP is associated with evidence of early oxidative stress in the alveolar fluid and blood.
Br J Anaesth 2002; 89: 2316
Keywords: complications, oxidative stress: complications, nosocomial pneumonia; lung, mini-bronchoalveolar lavage
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Introduction |
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Improved knowledge of the disease process may improve the treatment of VAP. Inflammatory mediators and free radicals are released in generalized inflammatory reactions involving the activation of leucocytes.3 Airway inflammation has a central role in the disease process in asthma, adult respiratory distress syndrome and chronic obstructive pulmonary disease (COPD) and is associated with increased markers of oxidative stress in fluid from bronchoalveolar lavage (BAL) and in the blood.47
To our knowledge, no data exist on oxidative stress in VAP, so we measured markers of oxidative stress in BAL fluid and serum from patients at risk of VAP.
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Methods |
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Sampling technique
We did 88 mini-BAL procedures using a blind catheter technique (CombicathTM, Plastimed, St Leu La Foret, France). During the lavage, 100% oxygen was given and patients were sedated and paralysed. Topical anaesthesia was not used, and the circulation and oxygen saturation were monitored during the procedure. Tracheal sputum was aspirated and collected before introducing the protected catheter. The catheter was inserted using a previously described technique.8 After blind insertion into a bronchus, the inner catheter was advanced until resistance was encountered and 20 ml sterile saline was injected. Fluid was then aspirated by hand into the infusion syringe. When at least 5 ml fluid had been retrieved, the entire catheter was removed. The sampling procedure lasted less than 3 min and was always done by the same physician. Meanwhile, blood samples were collected. Samples of the alveolar fluid were sent for bacteriological analysis (Gram staining and quantitative culture). Other samples were centrifuged (1500 rpm, 10 min) and the supernatant was stored immediately at 80°C for later measurement of oxidant and antioxidant activity. Repeat samples were taken for measurement of oxidant and antioxidant activity 3 and 7 days after the first samples.
Bacteriological analysis
The same technician processed all the bacteriological samples.
Microscopic examination
Aliquots (0.2 ml) from the original suspension were dropped into a cytospin and centrifuged at 300 g for 10 min. Slides were Gram stained and were examined at high magnification (x100). Between 10 and 50 fields were inspected for microorganisms and classified according to morphology.
Quantitative cultures
The fluid was diluted to obtain concentrations of 101, 103 and 105. The samples were then plated onto Petri dishes containing Colombia agar, chocolate agar, trypticase soy, or McConkey and Sabouraud agar. Bacterial colonies were counted and identified using conventional techniques.
Oxidative stress analysis
The same technician did all the measurements. Malondialdehyde (MDA), glutathione peroxidase activity (GPX) and superoxide dismutase (SOD) concentrations were measured. MDA is currently estimated by measurement of thiobarbituric acid reactant substances (TBARS). TBARS were measured in plasma by a fluorescence method.9 GPX catalyses the oxidation of glutathione (GSH) by cumene hydroperoxide. In the presence of GSH reductase and NADPH, the oxidized GSH is reduced and NADPH is oxidized to NADP. GPX activity was measured by the decrease in NADPH absorbance at 340 nm.10 SOD activity was measured by the auto-oxidation of pyrogallol according to the method described by Marklund and colleagues.11 One unit of SOD activity is defined as the amount of the enzyme required to inhibit the rate of pyrogallol autooxidation by 50%. GPX and SOD activity results are expressed as unit per gram of haemoglobin in blood (units g Hb1) and as unit per litre in BAL (units litre1).
The tests had the following characteristics. TBARS: intra-assay coefficient of variation (CV), 5%; interassay CV, 5%; linearity, 0.58 µmol litre1, detection limit, 0.06 µmol litre1; SOD assay: intra-assay CV, 6%; interassay CV, 7%; linearity, the SOD activity is measured by the degree of inhibition to form a redformazan dye, sample should give a degree of inhibition within 3060% of the sample diluent rate; detection limit, 30%; GPX assay: intra-assay CV, 6%; interassay CV, 8%; linearity, 900 units litre1; detection limit, 20 units litre1.
Diagnostic categories
The diagnosis of pneumonia was based on a positive mini-lavage quantitative culture (cutoff 103 colony-forming units per ml). VAP was excluded if the following were present: negative or non-significant growth on culture of BAL and full recovery without antimicrobial therapy, or diagnosis of another disease of the chest that could account for the abnormal chest radiograph. The definition of bacterial infection was based on the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference Criteria and was used to categorize patients into those with sepsis, severe sepsis and septic shock.12
Statistical analysis
Patients were classified into one of three categories: those with VAP (VAP group), those without VAP (non-VAP group) and those with no infectious disease (postoperative group). Values are expressed as mean and 95% confidence interval. Statview 5.0TM (SAS Institute Inc., Cary, North Carolina, USA) was used for data analysis. Data were compared using a chi-square test or a one-way analysis of variance, as appropriate. A two-way analysis of variance for repeated measures was used to compare differences in alveolar and blood TBARS, SOD and GPX (significance: P<0.05). Correction for multiple comparisons was made using the Bonferroni method.
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Results |
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Alveolar and serum TBARS concentrations were greater in the VAP patients compared with the non-VAP and the postoperative groups (Fig. 1A). Alveolar and serum GPX concentrations were lower in the VAP group compared with the non-VAP and postoperative groups (Fig. 1B). No significant differences were found in serum SOD activity between the VAP group and the non-VAP group (Fig. 2). SOD was not detected in alveolar lavage fluid. In patients with VAP, there were no differences between the non-survivors and the survivors with respect to alveolar and blood TBARS, GPX and SOD concentrations (data not shown). No significant differences were found between the postoperative group and the non-VAP group for alveolar and blood TBARS measurements and red cell GPX concentrations (Fig. 1A and B). Alveolar GPX and serum SOD concentrations were significantly lower in the non-VAP group compared with the postoperative group (Figs 1B and 2). There were no changes with time for the alveolar and plasma TBARS (Fig. 3), serum SOD (Fig. 4) and red cell GPX concentrations (Fig. 5B), whereas significant changes with time were found in serial alveolar GPX measurements in the VAP group (Fig. 5A).
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Discussion |
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Limitations of the study
Direct measurement of specific markers of oxidative injury resulting from excessive free radical activity can be made by electron spin resonance, but this cannot be currently applied to the study of tissues. In our study, we measured oxidative stress indirectly by measuring free radical activity in biological fluids, as increased lipid peroxidation. Although these markers suggest that oxidative stress has occurred, they do not indicate that this is necessarily involved in the pathogenesis of VAP.
Antimicrobial therapy was started after examining the bacterial cultures (i.e. Gram staining). This could affect the measurement of oxidative stress on days 3 and 7, and explain our inability to detect significant changes in the serial concentrations of plasma TBARS, serum SOD and red cell GPX.
Free radicals such as superoxide and hydroxyl radicals are constantly produced by normal aerobic metabolism.13 Oxidative stress results from an imbalance between radical-generating and radical-scavenging systems, leading to cell membrane impairment or DNA damage.13 TBARS indicates lipid peroxidation whereas SOD and GPX are important antioxidant defences, involved in the clearance of superoxide and hydrogen peroxide (H2O2) to prevent damage.13 SOD converts superoxide to H2O2 and oxygen; the former is then catabolized by catalase and GPX. Our findings suggest that local and systemic oxidative stress occurs in VAP.
One cause of oxidative stress associated with VAP is increased inflammation. Monton and colleagues studied 20 patients with severe VAP and found greater inflammation, shown by higher concentrations of cytokines in serum and BAL.14 Tissue necrosis factor and interleukin (IL)-6 levels were greater systemically than in the lung. In alveolar fluid, IL-6 was the only cytokine that increased. In vitro oxidants cause the release of inflammatory mediators such as IL-1, IL-8 and nitric oxide from macrophage cell lines and alveolar and bronchial epithelial cells. These events are associated with increased expression of the genes for these inflammatory mediators and increased nuclear binding or activation of nuclear factor kappa B, an oxidant mediator.15 Thus, the release of reactive oxygen species could, in theory, enhance the greater level of inflammatory mediators expressed by higher concentrations of local and circulating cytokines.
Alveolar GPX and serum SOD concentrations were significantly lower in the non-VAP group than in the postoperative group. One possible explanation could come from the mechanical ventilation. Kotani and colleagues studied the aggregation of alveolar macrophages in BAL fluid of 41 mechanically ventilated patients.16 They found larger aggregates and lower viability of macrophages during mechanical ventilation. Systemic SOD activity was not different in the VAP group compared with the non-VAP group. However, the adaptive antioxidant response of SOD is not accompanied by GPX enzyme upregulation. Boya and colleagues studied antioxidant status and oxidative stress in peripheral blood mononuclear cells from 49 patients with chronic hepatitis C.17 They found higher concentrations of SOD with reduced GSH activity, whereas oxidative stress was present, expressed by higher concentrations of TBARS. We could not measure alveolar concentrations of SOD because of technical difficulties. In our laboratory, SOD activity is routinely measured in erythrocytes by the method described above, but this procedure could not be applied to the BAL samples.
Overproduction of reactive oxygen species is one of the putative mechanisms in generalized (i.e. sepsis, transplantation, ischemic-reperfusion injury, burns) or local (i.e. asthma, COPD, chronic hepatitis, nephrotic syndrome) inflammation. Our results show that patients with pneumonia caused by mechanical ventilation have more systemic and local oxidative stress than patients in whom infection is not found. Based on these results, we speculate that the administration of antioxidants such as vitamin E or N-acetyl-L-cysteine might be of interest.
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References |
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