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Abstract

Background

The aim of this study was to analyse platelet superoxide dismutase (SOD) activities (total SOD, manganese SOD and copper zinc SOD) and copper (Cu) and zinc (Zn) concentrations during the course of community-acquired pneumonia (CAP), and to compare them between patients with normal platelet count and those who have developed reactive thrombocytosis (RT).

Methods

Platelet count, SOD activities and Cu and Zn concentrations in platelet-rich plasma were measured in patients with CAP on admission and at discharge.

Results

Post-therapeutic platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed RT. All platelet SOD activities as well as Cu concentration were significantly lower in CAP patients than in control subjects. The initial Zn concentration was greater in CAP patients compared with controls and showed a decrease at discharge. On admission, there was no difference in all SOD activities between either subgroup with normal platelet count or subgroup with RT. At discharge all SOD activities were significantly lower in patients with RT. Also, catalytic activities of those enzymes were significantly lower in both subgroups in comparison with the initial values. Post-therapeutic Cu value was lower in patients with RT in comparison with patients having normal platelet count. Zn concentration decreased significantly at discharge when compared with the initial values only in patients with RT.

Conclusion

The pattern of changes might be indicative of a certain role of platelets in antioxidant response during treatment in CAP patients.

Introduction

Oxidative stress and free radicals generated through enhanced synthesis during oxidative processes play a major role in the pathogenesis of many diseases.^1–5 Inflammatory lung diseases, particularly chronic diseases such as asthma and chronic obstructive pulmonary disease (COPD), are characterized by impaired balance between the adverse oxidative and favourable protective antioxidative processes.^6,7 In pneumonia, epithelial cells may also sustain damage during oxidative stress⁸ because of an increased concentration of reactive oxygen species (ROS) and decreased concentration of both enzymatic and non-enzymatic antioxidative compounds.⁹ Superoxide anion (O₂ ⁻) is the first reactive oxidative compound thus formed, which is transformed to hydrogen peroxide (H₂O₂) by superoxide dismutases (SODs). Pulmonary SODs are the only enzymatic system reducing superoxide to form H₂O₂.¹⁰ According to Borzi et al.,¹ patients with idiopathic pulmonary fibrosis have an elevated concentration of CuZnSOD and appear to correlate with the disease severity in these patients.

Community-acquired pneumonia (CAP) is an acute infection of pulmonary parenchyma occurring in patients not hospitalized or residing in a long-term care facility. It is a common and serious acute infectious disease, which is associated with significant morbidity and mortality.^11,12 During the course of pneumonia, inflammatory infiltrate involves the alveoli (alveolar, bacterial pneumonia) or interstitial spaces (interstitial, atypical non-bacterial pneumonia). In some patients, pneumonia may be characterized by an increase in platelet count, which is defined as the occurrence of reactive thrombocytosis (RT; platelet count >450 × 10⁹/L).¹³ There are relatively rare literature reports on RT in pneumonia patients, although some data suggest it might be a frequent event in children with pneumonia. Othman et al.¹⁴ report on thrombocytosis in 41% of children with Mycoplasma pneumoniae infection. The platelet count increase in pneumonia as well as in other conditions underlain by inflammation occurs as a result of the increased synthesis of thrombopoietin and interleukin-6.^15,16 The possible faster patient recovery in some pathological conditions has also been attributed to RT.¹⁷ Catalytic concentrations of total intracellular SOD (MnSOD and CuZnSOD isoenzymes)^18,19 or extracellular SOD (EC SOD)^20,21 have for years been determined as one of the first parameters of protective events in the course of oxidative process.^9,22

Although ROS are known to be produced in activated platelets (via glutathione cycle, arachidonic acid metabolism, phosphoinositide metabolism and xanthine oxidase),²³ there are scarce literature reports on the platelet SOD catalytic activity.^24,25

The aim of the present study was: (i) to assess the possible changes in SOD activity (total SOD, manganese [Mn]SOD and copper zinc [CuZn]SOD) in platelet-rich plasma (PRP) from pneumonia patients; (ii) to determine the possible presence and degree of association between changes in SOD activity and development of RT in these patients; and (iii) to determine zinc (Zn) and copper (Cu) concentration in platelets from these patients. Platelet count, total SOD, MnSOD, CuZnSOD, Zn and Cu concentrations in PRP were determined on hospital admission and at discharge from the hospital (i.e. upon therapy completion). The values thus obtained were compared with those measured in the control group of healthy volunteers, and expressed per platelet count.

Materials and methods

Subjects and samples

The study included 60 CAP patients (group I), mean age (x̄ ± SD) 44 ± 17 y, and 30 healthy volunteers, mean age (x̄ ± SD) 40 ± 4 y, as a control group (group II). Diabetes diagnosis was recorded in one CAP patient. Patients receiving anticoagulant therapy were excluded. Individuals suffering from gastrointestinal, renal, cardiac, lung (asthma, COPD), autoimmune or endocrine diseases and hypertension, and those taking medications such as antibiotics, calcium antagonists, diuretics, laxatives or alcohol were not included in the control group. Pneumonia patients were hospitalized at University Hospital for Infectious Diseases for severe clinical picture and had received antibiotic therapy (penicillin, azithromycin, ceftriaxone) for a mean (x̄ ± SD) of 12 ± 5 d. Patients were discharged from the hospital upon defeverscence and definitive clinical and radiological improvement. Diagnostic work-up was performed according to the standardized procedure, in line with ethical principles, 1975 Helsinki Declaration on Human Rights and 2004 Tokyo amendments. An informed consent in writing was obtained from all study subjects.

The diagnosis of CAP was based on clinical findings and chest radiography. Radiographic diagnosis required the finding of a new pulmonary infiltrate. Clinical diagnosis of CAP was supported by at least two of the following signs and symptoms: cough, chest pain, dyspnoea, auscultatory findings (rales and/or sign of consolidation), axillary temperature >38°C, or leukocytosis (white blood cell count > 10 × 10⁹/L). Microbiological tests such as blood culture, Gram staining, microscopy and culture of expectorated sputum or endotracheal aspirate were performed to define the bacterial aetiology of pneumonia, while serological tests were directed to atypical pathogens (M. pneumoniae, Chlamydia pneumoniae, Legionella pneumophila).

Microbiological testing revealed bacterial pneumonia in 53% (32/60), atypical pneumonia in 38% (23/60) and interstitial pneumonia in 8% (5/60) of study patients. Platelet count, total SOD, MnSOD, CuZnSOD, Zn and Cu concentrations were determined in PRP. According to platelet count, patients were divided into those without (subgroup A) and with (subgroup B) the developments of thrombocytosis during hospital stay.

Blood sampling

In control subjects, blood samples were collected between 07:00 h and 10:00 h and in patients between 07:00 h and 20:00 h. In patients, blood sampling was done on admission (sample 1) and at discharge from the hospital, upon completion of treatment, at a mean of 12 d of hospital admission (sample 2).

Platelet-rich plasma

Platelets were isolated upon whole blood overlaying with OptiPrep™ solution (Axon Laboratories AG, Le Mont-sur-Lausanne, Switzerland) and centrifugation in a Hettich Rotina 35 R centrifuge (Tuttlingen, Germany) at 350 G for 15 min.²⁶ Total platelet count and PRP platelet count was determined on a Coulter HmX-AL, Beckmann Coulter, SIN AE 20240 autoanalyzer. PRP was stored at −20°C; samples were submitted to a triple thawing/freezing cycle before determination of SOD catalytic activity and Zn and Cu concentrations.

Methods

Determination of SOD catalytic activity

The method used for the determination of total SOD catalytic activity and MnSOD is based on the formation of superoxide radicals from xanthine by the action of xanthine oxidase, which reacts with 2-(4-iodophenyl)3-(4-nitrophenyl)5-phenyltetrazole chloride to produce formazan red stain. SOD activity is measured as the grade of inhibition of this reaction.²⁷ MnSOD was determined upon sample incubation with 1 mmol/L KCN that inhibits CuZnSOD, and CuZnSOD activity was calculated as difference between total SOD activity and MnSOD activity.^28,29 Catalytic activity was determined on an Olympus AU400 selective autoanalyzer with RANSOD (Randox Laboratories Ltd, Ardmore, Diamond Road, Crumlin,Co. Antrim, UK) reagents. SOD catalytic activity was expressed as activity per platelet.

Determination of zinc and copper concentrations

Zn and Cu concentrations were determined by the method of atomic absorption on a Perkin Elmer Analyst 200 atomic absorption spectrophotometer (Waltham, Massachusetts, USA).³⁰

Statistics

Data were stored and prepared for statistical analysis using the Microsoft Office Excel 2000 software (Microsoft, USA). The variables with normal distribution were described by arithmetic mean (x̄) and standard deviation (SD), and those not showing normal distribution were presented by median (M) and interquartile range (IQR). Normality was tested using D′ Agostino-Pearson test for normal distribution. Paired samples Student's t-test and Wilcoxon test were used for comparison of dependent variables (for normal distribution and asymmetric distribution, respectively). Independent samples Student's t-test and Mann-Whitney test were used for independent variables (for normal distribution and asymmetric distribution, respectively). The values P < 0.05 were considered statistically significant. MedCalc (Medisoftware Mariakerke, Belgium) was used.

Results

The platelet count in CAP patients at admission was statistically significantly lower when compared with control group (217 ± 74 × 10⁹/L vs. 266 ± 56 × 10⁹/L; P < 0.005). Upon completion of therapy in CAP patients, the platelet count (434 ± 163 × 10⁹/L) was statistically significantly higher than at hospital admission (P < 0.0001). By the end of treatment, 42% (25/60) of patients developed RT. RT was found to be equal in all patients, regardless of ethiological profile of pneumonia (i.e. bacterial, atypical or interstitial pneumonia). Pooled results recorded in CAP patients, and statistical significance of results between groups, subgroups and samples are presented in Tables 1–3. Table 1 shows pooled results recorded in CAP patients irrespective of the occurrence of RT or normal platelet count throughout the course of disease. Both pretherapeutic and post-therapeutic SOD (i.e. total SOD, MnSOD and CuZnSOD) catalytic activities per platelet were statistically significantly lower in comparison with control group (Table 3).

Table 1

Superoxide dismutase (SOD) catalytic activity (total SOD, MnSOD and CuZnSOD), and copper (Cu) and zinc (Zn) concentrations in all community-acquired pneumonia (CAP) patients (group I) on admission (sample 1) and at discharge (sample 2), and in control group (group II)

Sample		Total SOD/platelet (U/plt)	Mn SOD/platelet (U/plt)	CuZn SOD/platelet (U/plt)	Cu/platelet (μmol/plt)	Zn/platelet (μmol/plt)
Group I (n = 60)
Sample 1	Median	0.60	0.44	0.16	0.08	0.39
	IQR	0.48–0.78	0.35–0.57	0.09–0.22	0.06–0.10	0.23–0.55
Sample 2	Median	0.46	0.34	0.09	0.04	0.26
	IQR	0.37–0.60	0.28–0.46	0.07–0.14	0.03–0.05	0.17–0.34
Group II (n=30)
	x̄ ± SD	–	–		0.08 ± 0.03
	Median	0.69	0.54	0.17	–	0.22
	IQR	0.61–0.82	0.46–0.65	0.12–0.22	–	0.14–0.38

All values are expressed per platelet. plt, platelet; IQR, interquartile range

Table 2

SOD catalytic activity (total SOD, MnSOD and CuZnSOD), and copper (Cu) and zinc (Zn) concentrations in CAP patients (group I, subgroups A and B) on admission (sample 1) and at discharge (sample 2)

Sample		Total SOD/platelet (U/plt)	Mn SOD/platelet (U/plt)	CuZn SOD/platelet (U/plt)	Cu / platelet (μmol/plt)	Zn/platelet (μmol/plt)
Subgroup A (n=35)
Sample 1	Median	0.62	0.44	0.18	0.09	0.47
	IQR	0.49–0.80	0.35–0.57	0.09–0.21	0.07–0.12	0.24–0.60
Sample 2	x̄ ± SD		0.42 ± 0.16		0.05 ± 0.02
	Median	0.54	–	0.12	–	0.32
	IQR	0.40–0.62	–	0.07–0.17	–	0.24–0.38
Subgroup B (n=25)
Sample 1	x̄ ± SD	–	–	–	0.09 ± 0.05	0.41 ± 0.23
	Median	0.60	0.40	0.16	–	–
	IQR	0.47–0.76	0.33–0.57	0.10–0.22	–	–
Sample 2	x ± SD	–	–	–	0.04 ± 0.02	0.28 ± 0.18
	Median	0.41	0.30	0.07	–	–
	IQR	0.34–0.49	0.27–0.39	0.06–0.10	–	–

All values are expressed per platelet. Subgroup A, patients with normal platelets count; subgroup B, patients with reactive thrombocytosis; plt, platelets; IQR, interquartile range

Table 3

Statistical significance of results between groups, subgroups and samples expressed as P values

	Total SOD/platelet	Mn SOD/platelet	CuZn SOD/platelet	Cu/platelet	Zn/platelet
I – sample 1: II	0.0167	0.0036	NS	NS	0.0023
I – sample 2: II	<0.0001	<0.0001	<0.0001	<0.0001	NS
I – sample 2: I – sample 1	0.0008	0.0180	0.0001	<0.0001	0.0006
I-A sample 1: II	NS	0.0228	NS	NS	0.0016
I-A sample 2: II	0.0001	0.0048	NS	0.0058	NS
I-B sample 1: II	0.0215	0.0001	NS	NS	NS
I-B sample 2: II	<0.0001	<0.0001	<0.0001	<0.0001	NS
I-A sample 1: I-B sample 1	NS	NS	NS	NS	NS
I-A sample 2: I-B sample 2	0.0151	0.0414	0.0205	<0.0001	0.0002
I-A sample 2: I-A sample 1	NS	NS	0.0203	<0.0001	NS
I-B sample 2: I-B sample 1	0.0009	0.0170	0.0013	<0.0001	0.0003

I – CAP patients irrespective of the occurrence of reactive thrombocytosis or normal platelet count; I-A – CAP patients with normal platelet count; I-B – patients with reactive thrombocytosis; II – control group; NS, not significant (P > 0.05); sample 1, at admission; sample 2, on discharge

In CAP patients, post-therapeutic Cu concentration per platelet was statistically significantly lower than the initial value and also significantly lower than that recorded in control group (both P < 0.0001). The initial Zn concentration per platelet was statistically significantly greater in comparison with control group (P = 0.0023) and showed a decrease at discharge (P=0.0006).

In group B patients, both pretherapeutic (239 ± 69 × 10⁹/L; P < 0.05) and post-therapeutic (591 ± 123 × 10⁹/L; P < 0.0001) platelet counts were statistically significantly greater when compared with group A patients (200 ± 69 × 10⁹/L and 321 ± 68 × 10⁹/L, respectively). Though remaining within the reference range, platelet count showed an increase with treatment in patients with normal platelet count (group A, increase index 1.6) and more than two-fold increase in patients with RT (group B, increase index 2.47). During hospital stay, platelet count showed a statistically significant increase in both patient groups (P < 0.0001).

Patient results according to the development of RT during the course of the treatment are illustrated in Table 2.

In both subgroups (i.e. subgroups A and B), both pretherapeutic and post-therapeutic values, respectively, platelet total SOD and MnSOD catalytic activities were statistically significantly lower in comparison with control group values (Table 3). In subgroup B, post-therapeutic (P < 0.0001), but not pretherapeutic CuZnSOD activity was statistically significantly lower in comparison with control group. On admission, there was no difference in any of the SOD activities between subgroup A or subgroup B patients (P > 0.05) though at discharge all SOD activities were statistically significantly lower in subgroup B in comparison with subgroup A. Also, catalytic activities of those enzymes/isoenzymes were statistically significantly lower in both subgroups in comparison with the initial values.

In both subgroups, only post-therapeutic platelet Cu concentration was statistically significantly lower in comparison with the control group (subgroup A: P = 0.0058; subgroup B: P < 0.0001). Post-therapeutic Cu value was statistically significantly lower in subgroup B in comparison with subgroup A patients (P < 0.0001). Cu was statistically significantly lower at discharge when compared with the initial values in both subgroups (both P < 0.0001). On admission, platelet Zn concentration was statistically significantly greater in subgroup A in comparison with control group. At discharge, Zn concentration in subgroup B patients was significantly lower (P = 0.0002) than in subgroup A patients. Zn concentration was statistically significantly lower at discharge when compared with the initial values only in subgroup B (P < 0.0003).

All changes were more pronounced in patients with RT.

Discussion

Changes in platelet SOD activities as well as Cu and Zn concentration (as CuZn SOD isoenzyme cofactors) might be indicative of an active role of platelets in antioxidant response during treatment in CAP patients. Increased production of ROS in patients with CAP causes increased consumption of antioxidant compounds, reduced expression of SODs and consecutive decrease of their catalytic activities, especially at the end of treatment. All changes were more pronounced in patients with RT. It can be presumed that platelet SOD pool as a first line of defence is exhausted during the course of pneumonia. We have already shown that RT occurred in patients with a severe form of pneumonia.¹³ Although the aetiology of pneumonia varied in our patients, there was no statistically significant aetiology-related difference in the results of study parameters.

Our previous study investigating the platelet catalytic activity in CAP patients indicated that patients had a higher initial gamma-glutamyl transpeptidase (GGT) catalytic activity in platelets when compared with healthy subjects, which was significantly reduced at the end of treatment in PRP of patients with RT when compared with patients having normal platelet count.³¹ This suggests a potential role of platelets in supporting the oxidative/antioxidative balance during CAP.

ROS are products of normal aerobic metabolism and are inactivated by various antioxidative mechanisms including enzymes such as SOD, catalase, glutathione peroxidase and other non-enzymatic compounds, such as glutathione, albumin, uric acid, lactoferrin, β-carotene and vitamins C and E.³² ROS synthesis is enhanced in most pulmonary diseases, such as adult respiratory distress syndrome, bronchopulmonary dysplasia, emphysema, hyperoxia, cystic fibrosis, bronchial asthma and pneumonia. The origin of oxidants varies with each individual case.³³ The oxidant/antioxidant imbalance causes cellular damage and pathophysiological impairment. In pneumonia patients, pronounced oxidative stress may reduce the antioxidant concentration and activity in both adults³⁴ and children.⁹ MnSOD, mitochondrial isoenzyme, is a predominant pulmonary SOD isoenzyme.¹⁰ Pulmonary MnSOD is mostly active in bronchial epithelium, alveolar macrophages and alveolar epithelium, type II pneumocytes in particular, whereas the activity of cytosolic CuZnSOD is predominantly found in bronchial epithelium.³⁵ According to Bowler et al.,²¹ extracellular SOD plays a major role in attenuating inflammatory response in the lungs, to an even greater extent than the proinflammatory cytokine release from phagocytes. There are scarce literature data on the SOD catalytic activity in platelets. SOD has been reported to prolong the platelet functional ability in the circulation, including inflammation.³⁶ Dietrich-Muszalska et al.²⁴ demonstrated the reduction in platelet SOD activity in schizophrenia patients, whereas Dwivedi et al.²⁵ showed the same in patients with myocardial infarction. In asthmatics, a reduced catalytic activity of SOD when compared with healthy controls was demonstrated not only in the airways but also in monocytes and neutrophils.³⁷ Thus, the reduced catalytic activity especially of MnSOD found in PRP of our patients when compared with control subjects was consistent with literature data.

In addition to the catalytic activity of MnSOD, our study in CAP patients also included determination of the catalytic activity of CuZnSOD in PRP. Borzi et al.¹ report an increased activity of CuZnSOD in patients with idiopathic pulmonary fibrosis, which correlated with the disease severity. Kurosawa et al.³⁸ demonstrated a significantly higher CuZnSOD activity in the platelets from asthmatic children in comparison with control group. In contrast to these reports, in our study the post-therapeutic catalytic concentration of CuZnSOD was statistically significantly lower when compared with control group.

We used PRP in our study, which may not be the most appropriate sample type, for the measurement of Cu and Zn concentration in particular, because the Cu and Zn concentrations measured in PRP cannot be attributed exclusively to their platelet concentrations but also to their plasma concentrations. Yet, we did choose PRP for the determination of SOD catalytic activity because of the platelet fragility³⁹ and absence of measurable SOD activity in the plasma if determined by the Randox method (data from package insertion). In plasma, SOD could be measured using ELISA.⁴⁰ Other authors also used PRP for studying platelet function, considering that PRP implies a physiological setting superior to buffer-suspended platelets.^41,42

In the present study, Cu and Zn concentrations differed between the patients with and without RT. Interestingly, in patients with normal platelet count, the initial Zn concentrations was higher than those in the control group. We found no data on platelet Cu and Zn concentrations and their role in inflammation in the available literature, and our results may be the first ones pointing to certain dynamics of changes in Cu and Zn platelet concentrations in CAP patients. Since Cu and Zn are cofactors for the catalytic activity of CuZnSOD, changes in their concentrations could be result of dynamic changes of CuZnSOD activity. There is only one study of Chan et al.⁴³ which notes that plasma Zn deficiency leads to increased oxidative stress and decreased antioxidant activity in platelet. Plasma zinc could be reduced during pneumonia in elderly.⁴⁴ Serum Cu concentrations were found to be inversely associated total antioxidative status in chronic inflammation.⁴⁵ Decreased concentrations of Zn and Cu after anti-inflammatory therapy are also found.⁴⁶ Moreover, in some studies trace element supplementation was used to enhance the antioxidant capacity in patients after major burns accompanied severe pneumonia.⁴⁷

The role of these trace elements in pneumonia patients needs to be investigated.

Based on the results obtained in the present and previous studies suggesting the possible association of antioxidative processes and occurrence of RT in CAP patients,³³ platelets are postulated also to be involved in the antioxidative response in pneumonia patients. Many questions remain open and should be tested. These include: (i) if platelet numbers in some patients increase but platelet SOD activities decrease, what happens to total platelet SOD activity per volume of blood; (ii) what physiological significance do these changes have; (iii) how long does the reparation of SOD pool last? Additional studies with long-term follow-up should be focused on the platelet role in inflammation and interaction of other antioxidative compounds with SOD isoenzymes in the development and management of inflammatory processes.

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Superoxide dismutase,copper and zinc concentrations in platelet-rich plasma of pneumonia patients

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Subjects and samples

Blood sampling

Platelet-rich plasma

Methods

Determination of SOD catalytic activity

Determination of zinc and copper concentrations

Statistics

Results

Discussion

References