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September - December 2002: 
Volume 15, Issue 3

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Measurement of serum total antioxidant status in patients with community acquired pneumonia: correlation with the severity of the disease
Abstract
Oxidant/antioxidant imbalance has been reported in various respiratory diseases including pneumonia. The purposes of this study were the measurement of total antioxidant status in the serum of patients with community acquired pneumonia (CAP) and the investigation of the probable relationship between TAS and the severity of the disease. Thirty patients (22 males, 8 females, mean age 48±21 years) were studied. Clinical, laboratory and radiographic data obtained on admission as well as on the 7th day of the hospital stay were recorded. At the same time points, serum TAS was also determined using a colorimetric method at 600 nm. TAS values on admission (TAS1) were significantly lower compared to TAS values obtained on the 7th day of the hospital stay (TAS2) (0.84±0.13 mmol/L vs 1.00±0.17 mmol/L, respectively, P=.0001). There was a positive association between TAS1/TAS2 ratio and the change in the severity score (r=.50, P=.007). TAS1/TAS2 ratio was inversely related to PaO2 on admission (r=.47, P=.008). Furthermore, the change in TAS was positively related to white blood cell count on admission (r=.399, P=.029) and significantly greater in patients at risk for CAP (P=.01) as well as in patients with Gram(-) pneumonia (P=.032). Another interesting finding is that TAS1/TAS2 ratio was significantly higher in patients with bilateral pneumonia (P=.023). In conclusion, low TAS values in patients with CAP suggest the presence of oxidative stress. The decrease in TAS values may be useful in estimating the severity of CAP and in determining the appropriateness of antioxidant administration in selected cases. Pneumon 2002, 15(3):295-304.
Full text

INTRODUCTION

Reactive oxygen species (ROS) have been implicated in a number of respiratory diseases, including acute respiratory distress syndrome (ARDS), bronchopulmonary dysplasia, emphysema, pneumoconiosis, oxygen toxicity, bleomycin toxicity, cystic fibrosis, bronchial asthma and more.1 ROS are products of the normal aerobic metabolism; the task of their neutralization is performed by a series of antioxidants, including enzymes such as superoxide dismutase, catalase, glutathione peroxidase, as well as a variety of reducing substances, such as albumin, uric acid, lactoferrin, b-caroten, vitamins C and E etc.2,3 Oxidants/antioxidants imbalance, due to either increased production of ROS or depletion of antioxidants, has a strong impact on several cellular functions and pathophysiological mechanisms.4 Oxidative load may be estimated by either direct detection of ROS, measurements of antioxidants or detection of ROS-induced damage products (e.g. lipid peroxidation products).5 Stimulated leukocytes (polymorphonuclear neutrophils and macrophages), which are believed to play a major role in the pathophysiology of ARDS and other inflammatory diseases of the lung, have been recognized as a significant source of endogenous ROS.6‑8 These cells immigrate to the lungs and release a variety of toxic substances, including ROS, through a process known as "oxidative burst". Although the release of these substances is a defense mechanism, increasing evidence suggests that such agents may also have detrimental effects.

Pneumonia is an inflammatory process of the respiratory system characterized by immigration and accumulation of macrophages and polymorphonuclear leukocytes in the pulmonary parenchyma.10 Presumably, this may lead to an increased load of ROS resulting in oxidants/antioxidants imbalance. In recent years, many efforts have been directed towards measuring total antioxidant activity in biologic fluids, thus eliminating the need for determining individual components of the complex antioxidant defense system.11 The purposes of this study are to determine total antioxidant status (TAS) in the serum of patients with community acquired pneumonia using an appropriate method, and to investigate the possible relationship between TAS and the severity and prognosis of the disease, as determined on the basis of clinical and laboratory data. 

Material - Methods

Thirty patients (22 males, 8 females), age range 48±21 years, admitted to a Pulmonary Medicine Department with community acquired pneumonia, were studied. Diagnosis was based on the presence of fever and cough with mucopurulent or purulent expectoration, accompanied by either leukocytosis or normal white blood cell count in peripheral blood, and a new pulmonary infiltrate on chest x-ray.

Age, smoking history and risk factors for pneumonia as defined by the ATS guidelines for the management of community acquired pneumonia, were recorded on admission.12 In compliance with the above mentioned guidelines, severity indicators, such as respiratory rate, degree of hypoxemia, extent of parenchymal involvement on chest x-ray, the presence of shock, the need for vasoconstrictive agents, and reduced urine output, were further recorded for each individual patient. Venous blood was obtained for complete blood count and determination of biochemical parameters, whereas arterial blood was also obtained for the determination of oxygen partial pressure (PaO2) while breathing room air. Gram-stained smear of sputum, sputum and/or blood culture and pleural fluid culture (in cases with pleural effusion) were conducted for the detection of the responsible pathogen. As regards radiographic findings, the uni- or bilateral location of the infiltrates, the presence or absence of lobar consolidation and/or pleural effusion were recorded. Clinical and laboratory data were then used to calculate a total severity score for each patient, following the scoring system proposed by Fine et al for classifying community acquired pneumonia cases according to the severity of the disease.13  

The above described laboratory and radiographic tests were repeated on the 7th day of the hospital stay and a new severity score was calculated. Separate venous blood samples were drawn on days 1 and 7 for the measurement of serum total antioxidant status.

Patients with known diabetes or blood glucose levels >150 mg/dL were not included in the study, in order to avoid falsely increased TAS values due to autoxidation of glucose. Patients that had received agents that inhibit ROS production (e.g. acetylsalicylic acid, non-steroidal anti-inflammatory agents, trimetazidine or nimesulide) in the previous month were also excluded.

Patients with severe hypoxemia received supplemental oxygen therapy with an oxygen concentration that did not exceed 40% (to avoid hyperoxia-induced ROS production). All patients received antimicrobial mono- or combination therapy, including amoxycillin+clavulanic acid, macrolides, 2nd or 3rd generation cephalosporins alone or combined with aminoglycosides. 

Total antioxidant status was measured using a kit manufactured by Randox Ltd., Crumlin, Co. Antrim, UK. The principle of the method is based on the reaction of ABTS (2,2'-azinobis-3-ethyl-behothiazoline-6-sulphonate) with a peroxidase (metamyoglobulin) that generates the free ABTS+fraction. That fraction has a green-blue color that absorbs light at 600 nm. In the presence of antioxidants, the generation of the colored product is inhibited in proportion to the antioxidants' concentration. The serum TAS was measured using the Dimension DU PONT-DADE analyzer at 600 nm and the reagents were calibrated with the standards included in the kit. 

Statistical Analysis

Statistical analysis was performed using the SPSS statistical package, Version 9.0. Mean TAS values on admission and on day 7 were compared using paired t-test. The relationship between the change in TAS (ΔTAS) from day 1 to day 7 and the white blood cell count on admission or the presence of risk factors, as well as the relationship between the TAS1/TAS2 ratio and the PaO2 on admission or the change in the severity score (ΔScore) were examined by calculating the Pearson correlation coefficient. Unpaired t-test was used for the comparisons between the ΔTAS values in patients with or without risk factors for pneumonia, and in patients with Gram(+) or Gram(-) pneumonia, as well as for the comparison between the TAS1/TAS2 ratios in patients with unilateral or bilateral pneumonia. A p value of less than .05 was considered indicative of statistical significance.

 

 

Results

Patients

All patients had a good outcome and none had to be admitted to an intensive care unit. Nineteen were smokers; eleven had risk factors for pneumonia (heart failure in 5, chronic obstructive pulmonary disease in 4, immunosuppression in 2 patients) and twenty patients had leukocytosis (>20,000/μL) on admission. Fourteen patients developed a complication during their hospitalization (parapneumonic effusion in 10, empyema in 3, and arthritis in 1 patient). Transthoracic drainage was performed in two of the three patients with empyema, whereas the third patient was transferred to a thoracic surgery department and underwent open thoracotomy. 

Bilateral infiltrates on chest x-ray were recorded only in five patients; lobar consolidation was present in a total of eleven patients. Etiologic diagnosis was established in 12 (40%) patients; there were five cases of Str. pneumoniae infection and the remaining cases were caused by Gram(-) pathogens (Haemophilus influenzae, Moraxella, Klebsiella, Acinetobacter). 

Total antioxidant status (TAS)

Total antioxidant status (TAS) on admission (TAS1= .84±.13 mmol/L) was significantly lower than the TAS value obtained on day 7 of hospital stay (TAS2= 1.00±.17 mmol/L) (p=.0001) (Figures 1 and 2). TAS value on admission was not related to age, smoking habit, high fever on admission, duration of fever during hospitalization, absolute count or percentage of polymorphonuclear leukocytes in peripheral blood, erythrocyte sedimentation rate, or the development of complications. Nevertheless, the following correlations were observed: i) TAS1/TAS2 ratio is positively associated with the difference in severity score between days 0 and 7 (r=.501, p=.007) (Figure 3); ii) TAS1/TAS2 ratio is inversely related to PaO2 on admission (r=.47, p=.008) (Figure 4); iii) the change in TAS (ΔTAS) is positively related to white blood cell count on admission (r=.399, p=.029) (Figure 5);
iv) ΔTAS is significantly greater in patients at risk for CAP as compared to patients without risk factors for CAP (.11±.095 vs .32±.032, respectively, p=.01) (Figure 6); v) ΔTAS is also significantly greater in patients with Gram(-) pneumonia (.34±.41 mmol/L) as compared to patients with Gram(+) pneumonia (.14±.10 mmol/L) (p=.032) (Figure 7); vi) TAS1/TAS2 ratio is significantly higher in patients with bilateral pneumonia (p=.023) (Figure 8).

 

Discussion

In the present study we have shown that total antioxidant activity in the serum of patients with community-acquired pneumonia is low at the initial stages of the disease, indicative of increased oxidative load, and increases after a 7-day hospital stay under appropriate antimicrobial therapy. That increase is accompanied by clinical and laboratory improvement. Total antioxidant status has also been shown to be associated with the presence of risk factors for developing pneumonia, the type of pathogen, as well as significant clinical and laboratory variables that determine disease severity and prognosis. 

The presence of increased oxidative load in the serum of pneumonia patients has been recorded by Umeki et al, who demonstrated an increase in the production of superoxide anion combined with reduced antioxidant activity, as determined by the reduced activity of superoxide dismutase. These changes were more prominent in immunocompromised patients, suggesting that the reduced activity of superoxide dismutase could be implicated in the impairment of the defense mechanisms in those patients.14 Braun et al reported increased production of reactive oxygen species by neutrophils in patients with pneumonia.15 In particular, ROS production by neutrophils in venous blood was found to be greater than that by arterial blood neutrophils, suggesting that, in patients with pneunonia, activated neutrophils emigrate from the systemic to the pulmonary circulation.

In two studies, Nowak et al found increased levels of lipid peroxidation products in the serum of pneumonia patients, suggestive of increased oxidative load generated as result of the activity of ROS on cell membrane lipids.16,17 

Our results are in agreement with those of previous studies in that increased ROS production may be involved in the impairment of the antioxidant mechanisms. Furthermore, it is interesting that the lipid peroxidation products are reported to be correlated with the radiographic findings and the white blood cell count.17 In the present study we reached a similar conclusion as regards the total antioxidant status. Nevertheless, neither the increase in lipid peroxidation products nor low TAS were found to be related to age or smoking, although evidence suggests that cigarette smoke is a significant source of oxidants.18 

Oxidant load in pneumonia and the role of phagocytes in oxidant/antioxidant imbalance has been investigated in various biological fluids as well. ROS production in bronchoalveolar lavage, as determined by chemiluminescence assay, was increased, even in immunocompromised patients.19 Neutrophil recruitment and activation was held responsible for this finding, since there was a positive association with neutrophil percentage and myeloperoxidase levels; this enzyme plays a key role in the induction of the "oxidative burst" and antimicrobial defense.19,8 Increased concentrations of nitric oxide products associated with neutrophil count and high levels of myeloperoxidase and elastase as indicators of neutrophil activation, were found in bronchoalveolar lavage of intubated patients with bronchopneumonia.20 Hydrogen peroxide (H2O2) in expired breath condensate of patients with either ARDS or acute respiratory failure due to pneumonia, was also found to be increased.21    

The key role of both neutrophils and macrophages in defense mechanisms, especially in pneumonia, has long been recognized.22 Their antimicrobial effects are carried out via activation of the cell membrane enzyme NADPH-oxidase, which reacts with oxygen and gradually forms superoxide anion (O-2 ) and hydrogen peroxide (H2O2). Further, the azurophilic granules of neutrophils release myeloperoxidase, which in turn catalyzes the formation of hypochlorous acid (HOCL) and hydroxyl radical (OH-).8,22-24 Although neutrophils have a significant contribution to ROS production, an association with blood neutrophil absolute count or percentage could not be established in our study nor in the study by Nowak et al; there was a positive relationship with total white blood cell count, though. This may be explained by the fact that ROS are also produced by other cell types in peripheral blood as well as in pulmonary parenchyma, such as alveolar macrophages. However, apart from macrophages, certain pathogens also seem to contribute to the increased ROS production seen in pneumonia. Animal studies have shown that Streptococcus pneumoniae causes alveolar epithelial cell damage not only by releasing toxic agents such as pneumolysin, but also by H2O2 production, which is thought to participate in the cell damage that characterizes pneumococcal pneumonia.25 Furthermore, several lines of evidence suggest that certain pathogens, including S. pneumoniae, interfere with the neutrophil-induced oxidative response, thus having an inhibitory effect, which may explain the increased toxicity of certain serotypes.26,27  

Oxidant/antioxidant imbalance is seen not only in bacterial but in viral pneumonia as well. Increased oxidative activity in the lungs has been found in experimental models of influenza-virus induced pneumonia.28,29 In contrast, patients seropositive for human immunodeficiency virus (HIV+) showed reduced H2O2 production by alveolar macrophages, which is aggravated in the event of opportunistic infections, such as P. carinii pneumonia.30 

There is increasing evidence that ROS are pathogenetically implicated in a variety of disease processes, thus providing a rationale for using antioxidants in the management of these conditions. Research has been inconclusive regarding this therapeutic possibility, since a number of problems and considerations have arisen.31 Although antioxidants have been found to be effective in animal studies, clinical trials are reporting controversial results. One possible reason is that the ideal antioxidant should meet certain criteria: absence of toxic effects, specific targeting of its action to ROS production sites, neutralization of a significant amount of oxidants, and effectiveness in both the extracellular and intracellular compartments.32 

As far as respiratory diseases are concerned, efforts to enhance antioxidant defense mechanisms have included attempts to increase the activity of intracellular antioxidant enzymes, administration of non-enzyme antioxidants (e.g. vitamin E), as well as administration of drugs with antioxidant properties.33 High doses of vitamin C given in animals with pneumococcal pneumonia boosted local antimicrobial activity, but were not shown to have any significant effect on the total defense mechanism.34 In contrast, the administration of vitamin E was beneficial reducing surfactant lipid peroxidation by ROS produced by a variety of bacteria.35 In addition, vitamin E in combination with glutathione reduced the oxidative damage caused in fibroblasts infected by Mycoplasma pneumoniae.36 Administration of artificial surfactant in experimental animals with bleomycin-induced neutrophilic alveolitis resulted in a reduction of ROS production by neutrophils, possibly by promoting neutrophil apoptosis.37 Influenza virus-induced lung injury is limited by increased levels of extracellular superoxide dismutase in the airways.38 Administration of N-acetylcysteine in experimental animals with lung injury following inhalation of endotoxin reduced neutrophil count in bronchoalveolar lavage, but did not have a significant effect on ROS production.39 Lactoferrin has been shown to inhibit the binding of lipopolysaccharides on neutrophil membranes, thus reducing neutrophil activation and ROS production.40  

In the present study, total antioxidant status in the serum of patients with community-acquired pneumonia was found to be reduced. Furthermore, a correlation was observed with clinical and laboratory findings that determine the severity and prognosis of the disease. Consequently, serum total antioxidant status may be indicative of the severity of the disease or even have prognostic value in those patients. Nevertheless, more studies on the oxidant/antioxidant balance in the serum and other biological fluids of pneumonia patients are required to fully clarify the role of ROS in the disease process. There is also a need for clinical trials to investigate the effectiveness and value of exogenous antioxidants as adjuvants to the standard treatment of pneumonia with antimicrobial agents.


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References