Abstract
OBJECTIVES
Neutrophil elastase has been reported to play an important role in acute lung injury, which is a major cause of postoperative mortality after pulmonary resection. Neutrophil elastase released in the lungs reaches the peripheral circulation via the pulmonary veins. This study was performed to compare neutrophil elastase activity in pulmonary venous blood (collected during lobectomy) with that in the peripheral blood, and to determine the perioperative changes of neutrophil elastase activity.
METHODS
In 34 patients undergoing lobectomy with mediastinal lymph node dissection, the leucocyte count, neutrophil count, neutrophil elastase activity and levels of tumour necrosis factor-α, interleukin-6 and interleukin-8 were measured in the pulmonary venous blood and peripheral arterial blood before and after surgery. Then, these parameters were compared between before and after surgery with peripheral and pulmonary blood.
RESULTS
Neutrophil elastase activity was found to be significantly higher in pulmonary venous blood at the completion of surgery than at the start (during thoracotomy), while the neutrophil elastase activity of peripheral arterial blood showed no significant change between the start and completion of surgery.
CONCLUSIONS
In conclusion, measurement of neutrophil elastase activity in pulmonary venous blood revealed changes associated with lobectomy.
Keywords: Lung cancer, Surgery, Neutrophil elastase activity, Inflammatory cytokine, Acute lung injury, Postoperative complications
INTRODUCTION
In Japan, the mortality rate of patients undergoing surgical resection of lung cancer is about 1% [1], a low rate that may be ascribable to both preoperative cardiopulmonary function testing and advances in perioperative management. We have obtained a favourable outcome in our patients by selecting a safe pulmonary resection procedure through a battery of preoperative cardiopulmonary function tests [2, 3]. Nonetheless, it remains impossible to completely avoid death after lung cancer surgery. Acute lung injury and acute respiratory distress syndrome, including acute exacerbation of idiopathic interstitial pneumonia, are responsible for about 40% of such deaths [1]. Although the mechanisms underlying postoperative acute lung injury or respiratory distress are not clearly understood, several reports have indicated that fatal multiple organ failure is associated with a marked increase in inflammatory cytokines and/or neutrophil elastase activity in patients with acute lung injury due to severe sepsis or resection of oesophageal cancer [4, 5]. Despite this, it was reported that patients who developed pulmonary injury after undergoing lobectomy for lung cancer showed no changes of neutrophil elastase activity in the peripheral arterial blood [6]. Serum neutrophil elastase activity has been investigated in blood from the left atrium during pulmonary resection [7], but there have been no reports about neutrophil elastase activity in blood from the pulmonary vein of the residual lung, which is the organ most severely affected by this highly invasive surgical procedure. The present study was performed to compare neutrophil elastase activity between pulmonary venous blood (collected during lobectomy) and peripheral blood, and to determine the perioperative changes of neutrophil elastase activity.
MATERIALS AND METHODS
This study included patients with a confirmed diagnosis of lung cancer preoperatively who underwent lobectomy with mediastinal lymph node dissection between January 2007 and December 2009 at the Surgical Department of Omuta National Hospital. Selection of the surgical procedure for lobectomy was done after performing a battery of preoperative cardiopulmonary function tests in accordance with the Institutional Criteria for Objective Determination of Pulmonary Resection Procedures [3]. Patients who were found to have severe adhesions during thoracotomy and those in whom a diagnosis of lung cancer could not be established preoperatively were excluded because rapid collection of pulmonary venous blood during thoracotomy was impossible. Patients who did not require mediastinal lymph node dissection were also excluded because of the difference in invasiveness between procedures with and without dissection. However, patients who underwent bilobectomy (right middle and lower lobectomy) were included because the pulmonary resection volume was similar to that for left upper lobectomy. All patients received anaesthesia by systemic one-lung ventilation combined with epidural anaesthesia. Nitrous oxide and sevoflurane were used as the inhalational anaesthetics. The oxygen concentration was maintained at 50% during surgery. All smokers stopped smoking for at least 4 weeks before surgery. Operations were performed by four thoracic surgeons, and one board-certified thoracic surgeon was involved in all procedures as either the chief operator or assistant in order to maintain uniform quality. Thoracotomy was usually done by the muscle-sparing antero-axial method, but posterolateral thoracotomy was employed in patients with large tumours and those with a possible need for angioplasty or bronchoplasty (no such patients in this series). Postoperative pain was managed by epidural analgesia with continuous injection of 1% mepivacain hydrochloride until the chest tube was removed. Patients were monitored in the intensive care unit for at least 24 h postoperatively.
Pulmonary venous blood samples were collected from the pulmonary vein at the start and completion of surgery (i.e. during thoracotomy and before closing the chest incision). Peripheral arterial blood samples were also collected at the start and completion of surgery via a catheter inserted into the radial artery. At the start of surgery, pulmonary venous blood was collected from the lobe undergoing resection, while blood was collected from the pulmonary vein of the residual ipsilateral lobe at the end of surgery. Immediately after collection, each blood sample was centrifuged (3000 rpm, 10 min) to separate plasma, which was stored at −34°C. Plasma samples were analysed to determine the leucocyte count, neutrophil count and neutrophil elastase activity as well as the levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8).
Neutrophil elastase activity was determined with the specific synthetic substrate Suc-Ala-Pro-ValpNA [8] by the method of Yoshimura et al. [9]. IL-6 and TNF-α were measured with a Quanti Glo Human IL-6 Immunoassay 2nd Generation kit and Quanti Glo Human TNF-α Chemiluminescent Immunoassay kit, respectively (R&D Systems, Minneapolis, MN, USA). IL-8 was measured with an enzyme immunoassay (IL-8 EASIA kit; Invitrogen, Minneapolis, MN, USA). All tests were performed by Mitsubishi Chemical Medience Inc. (Tokyo, Japan).
This study was approved by the Institutional Review Board (IRB) of Omuta National Hospital (Nos 18-9 and 20-2). Patients were fully informed about this study and all gave consent.
Statistical analysis
Results are expressed as the mean ± standard deviation. Statistical analysis was performed by the paired t-test with SPSS software for Windows (version 13.0J) (SPSS, Chicago, IL, USA). Differences were considered significant at P < 0.05.
RESULTS
A total of 123 patients underwent resection of primary lung cancer between January 2007 and December 2009, with 89 patients having lobectomy plus mediastinal lymph node dissection. All of the required blood samples were obtained from 34 patients (38.2%), who were included in the present study. There were 23 men (67.6%) and 11 women (32.4%) aged from 48 to 84 years (mean: 69.4 ± 9.4 years). Twenty-five patients (73.5%) were smokers and 9 patients (26.5%) were non-smokers. Fifty-five patients were excluded from the study even though they underwent lobectomy with mediastinal lymph node dissection, with the reason for exclusion being failure to confirm the diagnosis of lung cancer preoperatively in 32 patients. One patient each was excluded because of inappropriate storage of the plasma sample, preoperative chemotherapy, double cancer and severe adhesions encountered during thoracotomy. Fourteen patients were excluded according to the decision of the attending surgeon and five patients were excluded because the IRB discontinued the study before evaluation was completed. None of the patients had interstitial pneumonia preoperatively based on contrast-enhanced high-resolution computed tomography of the chest. The surgical procedure was lobectomy in 30 patients and bi-lobectomy in 4 patients, with mediastinal lymph node dissection in all 34 patients. Muscle-sparing antero-axial thoracotomy was performed in 31 patients. Posterolateral thoracotomy was selected for three patients, including two with lymph node involvement detected preoperatively (angioplasty or bronchoplasty was thought to be necessary) and one with a large tumour. The mean operating time was 253 ± 41 min (range: 188–350 min) and the mean intraoperative blood loss was 154 ± 130 g (range: 45–621 g). No patient received perioperative blood transfusion. The pathologic diagnosis was adenocarcinoma in 26 patients, squamous cell carcinoma in 5 patients, adenosquamous cell carcinoma in 2 patients and large cell carcinoma in 1 patient. Lung cancer was stage I in 26 patients, stage II in 4 patients and stage III in 4 patients (Table 1).
Table 1:
Clinical characteristics of the patients
| Gender (M/F) | 23/11 |
| Age (years) | 69.4 ± 9.4 |
| Smoking history (yes/no) | 25/9 |
| Operating time (min) | 253 ± 41 |
| Blood loss (g) | 154 ± 130 |
| Tumour histology | |
| Adenocarcinoma | 26 |
| Squamous cell carcinoma | 5 |
| Adenosquamous cell carcinoma | 2 |
| Large cell carcinoma | 1 |
| Pathologic stage | |
| I | 26 |
| II | 4 |
| III | 4 |
Changes of the leucocyte count and neutrophil count
The leucocyte count showed significant elevation in both pulmonary venous blood and peripheral arterial blood after surgery compared with the levels during thoracotomy (Table 2). Similarly, the neutrophil count showed significant elevation in both pulmonary venous and peripheral arterial blood after surgery.
Table 2:
Changes of leucocyte and neutrophil
| Start of surgery | Finish of surgery | P-value | |
|---|---|---|---|
| Leucocyte in pulmonary venous blood (per mm2) | 5515 ± 1253 | 11921 ± 3049 | <0.001 |
| Leucocyte in peripheral arterial blood (per mm2) | 5818 ± 1256 | 12532 ± 3011 | <0.001 |
| Neutrophil in pulmonary venous blood (per mm2) | 3294 ± 1075 | 9851 ± 2614 | <0.001 |
| Neutrophil in peripheral arterial blood (per mm2) | 3443 ± 1161 | 10165 ± 2583 | <0.001 |
Changes of neutrophil elastase activity
Neutrophil elastase activity in pulmonary venous blood was 0.96 ± 1.16×10 nmol/ml/24 h at the start of surgery versus 1.68 ± 2.48×10 nmol/ml/24 h on completion of surgery, showing significant elevation (P = 0.019). In contrast, neutrophil elastase activity in peripheral arterial blood was 0.92 ± 1.22×10 nmol/ml/24 h at the start of surgery and 1.29 ± 1.28×10 nmol/ml/24 h on completion, showing no significant change (Table 3 and Fig. 1).
Table 3:
Changes of neutrophil elastase activity and inflammatory cytokines
| Start of surgery | Finish of surgery | P-value | |
|---|---|---|---|
| Neutrophil elastase activity in pulmonary venous blood (×10 nmol/ml/24 h) | 0.96 ± 1.16 | 1.68 ± 2.48 | 0.019 |
| Neutrophil elastase activity in peripheral arterial blood (×10 nmol/ml/24 h) | 0.92 ± 1.22 | 1.29 ± 1.28 | 0.133 |
| TNF-α in pulmonary venous blood (pg/ml) | 0.89 ± 0.41 | 0.82 ± 0.36 | 0.165 |
| TNF-α in peripheral arterial blood (pg/ml) | 0.85 ± 0.42 | 0.81 ± 0.38 | 0.525 |
| IL-6 in pulmonary venous blood (pg/ml) | 4.19 ± 6.29 | 248 ± 116 | <0.001 |
| IL-6 in peripheral arterial blood (pg/ml) | 4.11 ± 7.73 | 177 ± 104 | <0.001 |
| IL-8 in pulmonary venous blood (pg/ml) | 9.22 ± 3.47 | 14.3 ± 7.21 | <0.001 |
| IL-8 in peripheral arterial blood (pg/ml) | 9.72 ± 3.87 | 13.8 ± 8.36 | 0.001 |
TNF-α: tumour necrosis factor-α; IL-6: interleukin-6; IL-8: interleukin-8.
Figure 1:
Neutrophil elastase activity in pulmonary venous blood was showing significant elevation between the start of surgery and completion of the surgical procedure. In contrast, neutrophil elastase activity in peripheral arterial blood was showing no significant change.
Changes of inflammatory cytokines
IL-6 and IL-8 levels showed significant elevation in both pulmonary venous blood and peripheral arterial blood after surgery compared with during thoracotomy. In contrast, TNF-α levels showed no significant change in pulmonary venous blood or peripheral arterial blood after surgery (Table 3).
DISCUSSION
Neutrophil elastase is a proteolytic enzyme with a role in host defences and intercellular signalling [10–12]. High neutrophil elastase activity is detected in the peripheral blood of patients with acute lung injury and neutrophil elastase plays a key role in this condition [5, 6]. Therefore, if treatment is started immediately after detecting elevation of neutrophil elastase activity, this may prevent subsequent acute lung injury by inhibition of intercellular signalling.
The present study investigated changes of the leucocyte count, neutrophil count and IL-6, IL-8 and TNF-α levels during resection of lung cancer. These inflammatory cytokines are involved in intercellular signalling and are thought to participate in postoperative acute lung injury by inducing migration of neutrophils into the pulmonary tissues. Sakamoto et al. [13] reported that peripheral blood levels of IL-6 and IL-8 increased during surgery for oesophageal cancer, peaked at least 6 h postoperatively and then gradually declined. In the present study, there was marked elevation of the leucocyte count, neutrophil count and IL-6 and IL-8 levels at the completion of lobectomy compared with the start of surgery, and these changes showed no differences between pulmonary venous blood and peripheral arterial blood. Therefore, it seems to be possible to monitor changes of IL-6 and IL-8 by measurement of peripheral blood samples and investigating these cytokines in pulmonary venous blood does not provide additional information.
In contrast, this study revealed that neutrophil elastase activity in pulmonary venous blood was 0.96 ± 1.16×10 nmol/ml/24 h at the start of lobectomy and 1.68 ± 2.48×10 nmol/ml/24 h at the completion of surgery, showing significant elevation. However, despite the significant postoperative increase in the peripheral blood neutrophil count, the neutrophil elastase activity of peripheral arterial blood was 0.92 ± 1.22×10 nmol/ml/24 h at the start of lobectomy and 1.29 ± 1.28×10 nmol/ml/24 h on completion of surgery, showing no significant change. Thus, elevation of neutrophil elastase activity in the lungs could only be detected by measurement of pulmonary venous blood. Accordingly, measurement of neutrophil elastase in pulmonary venous blood is more useful for monitoring changes of neutrophil elastase activity associated with lobectomy than measurement of peripheral blood.
Waller et al. [7] measured the serum neutrophil elastase level in blood samples collected from the left atrium during pulmonary resection, and reported no change of neutrophil elastase associated with surgery. In the present study, we collected blood samples from the pulmonary vein, which is closer to the site of lobectomy than the left atrium. Another difference is that Waller et al. [7] measured the serum level of neutrophil elastase, while we measured neutrophil elastase activity, which is more directly related to acute lung injury than the serum level [14]. Thus, our findings suggested that changes of neutrophil elastase associated with lobectomy could be revealed by measurement of this enzyme's activity but not by measuring its serum level.
The present study had several limitations. First, neutrophil elastase activity increases rapidly in blood samples at room temperature. To minimize this increase, all blood samples were placed in ice-cold water (0°C) immediately after collection and during transport to the laboratory, and then were immediately centrifuged to separate plasma that was cryopreserved for storage. Secondly, analysis of neutrophil elastase activity is a time-consuming procedure, so that data may not be available when needed. Thirdly, the elevation of neutrophil elastase activity associated with lobectomy was slight compared with that usually observed in the setting of acute lung injury, suggesting that neutrophil elastase is influenced by other factors in patients with lung injury.
In conclusion, changes of neutrophil elastase activity associated with lobectomy can be assessed by measurement of the activity in pulmonary venous blood collected intraoperatively. Accordingly, intraoperative measurement of neutrophil elastase activity in pulmonary venous blood could be useful for future studies to identify effective drugs for the prevention of acute lung injury.
Conflict of interest: none declared.
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