Systemic Inflammation and Oxidative Stress Post-Lung Resection: Effect of Pre-Treatment

Systemic inflammation and oxidative stress post-lung resection: effect of pre-treatment

with N-acetylcysteine

Anthony J Bastin1, Nathan Davies2, Eric Lim3, Greg J Quinlan4,5, Mark J Griffiths1,4,6

1 Adult Intensive Care Unit, Royal Brompton Hospital, London, United Kingdom

2 Liver Failure Group, UCL Institute for Liver and Digestive Health, Royal Free Hospital

Campus, University College of London (UCL), Pond Street, London, United Kingdom.

3 Academic Division of Thoracic Surgery, Royal Brompton & Harefield NHS Foundation

Trust, London, UK National Heart and Lung Division, Imperial College, London, UK.

4 National Institute for Health Research Respiratory Biomedical Research Unit at the Royal

Brompton & Harefield NHS Foundation Trust and Imperial College, London, United

Kingdom

5 Vascular Biology, National Heart and Lung Institute, Imperial College London, United

Kingdom

6 Leukocyte Biology, National Heart and Lung Institute, Imperial College London, United

Kingdom

Correspondence:

Dr Mark J Griffiths

Royal Brompton Hospital Campus

Sydney Street, London SW3 6NP, United Kingdom

Email: [email protected]

Summary at a Glance

In a human model of lung injury and oxidative stress, pre-operative administration of the anti-oxidant N-acetylcysteine augmented plasma thiols, but did not attenuate post-operative systemic inflammation or oxidative stress associated with lung resection. ABSTRACT

Background and objective

N-acetylcysteine has been used to treat a variety of lung diseases, where is it thought to have an anti-oxidant effect. In a randomised placebo-controlled double-blind study, the effect of

N-acetylcysteine on systemic inflammation and oxidative damage was examined in patients undergoing lung resection, a human model of acute lung injury.

Methods

Eligible adults were randomised to receive pre-operative infusion of N-acetylcysteine

(240mg/kg over 12h) or placebo. Plasma thiols, interleukin-6, 8-isoprostane, ischaemia- modified albumin, red blood cell glutathione and exhaled breath condensate pH were measured pre- and post-operatively as markers of local and systemic inflammation and oxidative stress.

Results

Patients undergoing lung resection and one-lung ventilation exhibited significant post- operative inflammation and oxidative damage. Post-operative plasma thiol concentration was significantly higher in the N-acetylcysteine-treated group. However, there was no significant difference in any of the measured biomarkers of inflammation or oxidative damage, or in clinical outcomes, between N-acetylcysteine and placebo groups.

Conclusions

Pre-operative administration of N-acetylcysteine did not attenuate post-operative systemic or pulmonary inflammation or oxidative damage after lung resection. Clinical trial registration: NCT00655928 at ClinicalTrials.gov

Key words: Lung injury, thoracic surgery, inflammation, critical care medicine, lung cancer

Short title: N-acetylcysteine pre-lung resection INTRODUCTION

Oxidative stress contributes to inflammation in patients with acute and chronic lung diseases, including acute lung injury (ALI), chronic obstructive lung disease (COPD) and idiopathic pulmonary fibrosis (IPF) [1]. The anti-oxidant N-acetylcysteine (NAC) has been widely investigated in laboratory and clinical studies. NAC is a pro-drug of cysteine, the availability of which is the rate-limiting step in the formation of glutathione (GSH), a component of lung antioxidant defence. NAC also ‘directly’ scavenges reactive species, by virtue of its thiol group. Treatment with NAC was associated with improvements in oxygenation and lung injury score in ‘early’ lung injury [2] but not in established ALI [3-5]. In COPD, a large multicentre study showed no significant effect of NAC on pulmonary function or exacerbations [6], but did reduce exacerbation rate in moderate to severe disease [7]. In IPF,

NAC offered no significant benefit in preservation of lung function versus placebo [8].

The incidence of ALI is 1-3% following lobectomy and up to 11% following pneumonectomy, with a mortality of 40-70% [9, 10]. Patients undergoing lung resection and one-lung ventilation (OLV) develop excessive oxidative stress through several mechanisms including ventilator induced lung injury (VILI), ischaemia-reperfusion injury, and surgical trauma, which are also among factors implicated in the development of ALI. Lung resection and OLV can be viewed as a human model that reflects pathophysiology of ALI common to other aetiologies [11, 12]. In clinical studies, oxidative stress-related damage to proteins and lipids was associated with lung re-expansion following OLV [13, 14] and with morbidity after lung resection and OLV [15]. Furthermore, the rise in plasma interleukin (IL)-6, a marker of systemic inflammation after lung resection [16], was attenuated by a protective ventilatory strategy [17] and less invasive surgery [18]. Modulating the degree of oxidative stress, and preventing oxidative damage to biomolecules and associated inflammation with anti-oxidant intervention, might therefore be more effective than treating established disease.

We hypothesised that pre-operative administration of NAC would attenuate the post- operative rise in plasma concentration of biomarkers of oxidative damage and systemic inflammation in patients undergoing lung resection and OLV.

METHODS

Adults undergoing elective lung resection for cancer at the Royal Brompton Hospital,

London, UK were eligible. Patients taking glucocorticoids, NAC or carbocysteine within the preceding month, with a history of asthma, unable to receive the standardised anaesthetic approach, or enrolled in another interventional study were excluded. Written informed consent was required. The study was approved by the St Mary’s Research Ethics Committee,

London, UK (National Research Ethics Service).

Operative management was carried out as previously described [16], further details are in the

Supplementary Appendix 1.

Patients were randomised 1:1 in blocks of 10 to receive NAC (Pliva Pharma, Hampshire, UK:

240mg/kg in 1000mL 0.9% saline) or placebo (1000mL 0.9% saline) intravenously over 12h commencing the evening prior to surgery in a double-blind fashion.

Sample collection and biomarker assays

Blood was collected before (T1) and after (T1a) administration of study drug, at the end of

OLV (T2), 1 hour (T3) and 4-6 hours (T4) after the end of OLV, and on post-operative days 1

(T5) and 2 (T6). Samples were collected on ice and immediately centrifuged. The anti- oxidant butylated hydroxytoluene (20μM final concentration; BDH Chemicals Ltd, Poole,

UK) was added to samples in which markers of oxidative stress were assessed [19]. Plasma thiols were measured immediately after centrifugation, remaining plasma was stored at

-80°C.

Plasma IL-6 was measured in duplicate using ELISA (Hycult Biotechnology, Uden,

Netherlands). 8-isoprostane was measured using gas chromatography and mass spectroscopy

(GC-MS) at T1 and T3, the time of maximal oxidative stress in a previous study [14]. Plasma thiol concentration was determined using Ellmann’s assay [20]. Thiols were expressed as a ratio to albumin concentration [13]. Red cell GSH was measured in the first 26 patients

(Calbiochem, Merck Chemicals, UK). Ischaemia modified albumin (IMA) was measured using minor modifications to a previously described method, results were expressed as the ratio of IMA to albumin (IMAR) [21]. Exhaled breath condensate (EBC: 0.5 to 1mL) was collected in a polypropylene tube with a one-way valve (Rtube, Respiratory Research,

Charlottesville, VA) and pH was measured (UB-10 pH meter, Denver Instrument, Colorado,

USA) after argon de-aeration [16]. Paired preoperative and end-OLV EBC pH measurements were available for 37 patients (19 placebo group and 18 NAC group) owing to clinical constraints, mainly the inability to sample at end-OLV due to return to two-lung ventilation for clinical reasons. Further assay details are given in the Supplementary Appendix 1.

Data collection

Peri-operative ventilatory parameters were recorded using a computerised clinical information system (CareVue, Phillips Medical Systems, Reigate, UK) at 5-minute intervals.

Data were reported as averaged values during OLV. Tidal volume was expressed as mL/kg of predicted body weight [22]. Clinical data recorded are listed in the Supplementary Table S1. Study design

The study was randomised double-blind placebo-controlled. The primary endpoint was post- operative plasma IL-6. Increases in plasma IL-6 after lung resection are attenuated by a protective ventilatory strategy [17], where a 50% reduction in IL-6 at end-OLV and 18h post- operatively was seen. NAC pre-treatment in murine models of lung injury reduced pulmonary and systemic IL-6 concentration [23-26]. Based on previous data [16], a minimum of 23 patients was required in each group to detect a 50% reduction in post-operative plasma IL-6 with 80% power and a significance level of 5%. Secondary endpoints were post-operative 8- isoprostane, IMAR and EBC pH.

Statistical analysis

Data were analysed using GraphPad Prism version 4.02 (GraphPad Software, San Diego,

USA). Data normality was assessed using the Kolmogorov-Smirnov test. Non-normally distributed data are presented as median (inter-quartile range), and normally-distributed data as mean (standard deviation). Baseline characteristics between groups were compared using a two-tailed t-test. When comparing data from two groups at multiple time points, the influence of clustering (grouping) of patient data was taken into account by estimating the results with

Generalised Estimating Equations (GEE) with exchangeable correlation structures, performed using Stata 9.1 (StataCorp, College Station, TX, USA). Where the GEE suggested a difference between groups, t-tests were performed to investigate differences at each time point. We sought to investigate associations between duration of OLV and plasma biomarkers using Spearman correlations of duration of OLV and peak biomarker levels. A p- value of less than 0.05 was considered statistically significant for all tests. RESULTS

Ninety patients were assessed; 38 were excluded and 5 did not receive the allocated intervention. Forty-seven patients (24 in the placebo group and 23 in the NAC group) were included in the final analysis (Figure 1). More patients in the placebo group had undergone previous thoracic surgery, and FEV1:FVC was lower in the NAC group. Three pneumonectomies were performed, all in the NAC group. Baseline characteristics and intraoperative parameters were otherwise similar in both groups (Table 1).

Plasma IL-6 concentration was undetectable in both groups at T1, and peaked at T3 [111(72-

162)pg/ml and 126(80-180)pg/ml in placebo and NAC groups respectively] (Figure 2). There were no significant differences between placebo and NAC groups. There was a significant correlation between peak post-operative IL-6 concentration and OLV duration [r=0.37, p=0.011 (Spearman), n=47], consistent with the hypothesis that OLV-induced lung inflammation contributed to systemic IL-6 levels.

EBC pH was lower at end-OLV than pre-operatively [6.61(0.34) vs 6.72(0.29), p=0.053

(paired t-test), n=37, Table 2]. There was no significant difference in EBC pH pre-operatively or at end-OLV between placebo and NAC groups (Table 2).

Plasma thiol concentration in the placebo group decreased significantly from a baseline of

355(59)μM to 250(52)μM at T3 (Figure 3A). In the NAC group it increased from a baseline of 365(44)μM to 413(71)μM at T1a, and remained significantly higher than in the placebo group until T4 (Figure 3A). Plasma albumin concentration decreased between T1 and T1a from 42.7(3.1)g/l to 35.3(3.8)g/l and from 42.7(3.3)g/l to 35.8(2.6)g/l in the NAC and placebo groups respectively, due to the effects of dilution following administration of 1 litre 0.9% saline. Plasma albumin concentration decreased further during the operation between

T1a and T2, and remained constant thereafter. There were no significant differences in plasma albumin concentration between the placebo and NAC groups. The ratio of plasma thiol/albumin increased following administration of NAC, and remained significantly higher than the placebo group until T4, whereas the thiol/albumin ratio remained constant in the placebo group (Figure 3B).

There were no significant differences in red cell GSH between NAC and placebo groups

(n=13 in each group) (Figure 3C).

Plasma 8-isoprostane increased significantly between T1 and T3 in the NAC group from

355(264-507)pg/ml at T1 to 574(461-990)pg/ml at T3, n=17, p=0.0004 (Wilcoxon matched pairs signed rank test), and in the placebo group from 292(242-484)pg/ml at T1 to 495(417-

747)pg/ml at T3, n=20, p=0.0002 (Wilcoxon matched pairs signed rank test) (Figure 4).

There was no significant difference at T3 between NAC and placebo groups (p=0.43, Mann-

Whitney test). There was no significant correlation between plasma 8-isoprostane level at T3 and OLV duration [r=0.06, p=0.71 (Spearman), n=37].

IMAR increased significantly in both groups, peaking at T4 (Figure 5). There were no significant differences between placebo and NAC groups. Furthermore, there was no significant effect of NAC when considering only patients with a longer duration (>120 minutes) of OLV (n=14 in placebo group, n=13 in NAC group). The peak post-operative

IMAR correlated with the OLV duration [r=0.42, p=0.0036 (Spearman), n=47].

Clinical outcomes were similar between the 2 groups (Supplementary Appendix 2-results and

Table S1). One mild adverse reaction (NAC group) was treated with oral antihistamines.

After an interruption of 1 hour, the infusion was restarted at half rate, and subsequently increased to full rate without worsening of symptoms. DISCUSSION

In a human model of systemic and lung inflammation and oxidative stress, pre-operative administration of NAC significantly augmented plasma thiol concentration for at least 4-6 hours after surgery. However, NAC did not modulate post-operative systemic inflammation as measured by plasma IL-6 concentration (primary end-point), post-operative oxidative damage as measured by plasma 8-isoprostane, IMAR, pulmonary inflammation as measured by EBC pH, or clinical outcomes versus placebo.

Biomarkers of inflammation and oxidative damage in plasma and EBC

The increase in plasma IL-6 after lung resection and OLV is consistent with previous reports

[18, 27]. Similarly, the increase in plasma 8-isoprostane, indicating significant post-operative oxidative stress, is consistent with previous studies, in which plasma protein carbonyls [13] and malondialdehyde (MDA) [14], alternative markers of oxidative damage, were measured.

Several in vitro and in vivo studies provide a biological rationale for studying the clinical effects of NAC on inflammation and oxidative stress; stretch-induced release of IL-6 and isoprostanes from alveolar type 2 cells was inhibited by NAC, through an increase in intracellular glutathione [28]. Pre-treatment with NAC in an animal model of VILI prevented a rise in bronchoalveolar lavage (BAL) cytokines and serum isoprostanes [26]. Modest effects of NAC on biomarkers of oxidative damage to lipids in patients with sepsis have been demonstrated [29]. However, despite these and other cellular and animal models suggesting a benefit of NAC in a variety of settings, clinical studies of NAC in patients with sepsis [30], after high risk major surgery [31], and in mixed critically ill patients [32] have failed to demonstrate significant clinical benefit, and may even be harmful if administered late [33]. In most experimental models, NAC was administered immediately before or coinciding with the insult. In clinical studies in patients with ALI, NAC was administered at doses ranging from 40mg/kg/day to 150mg/kg loading followed by 20mg/kg/hour, for 3-10 days [2-5]. This may have resulted in greater anti-oxidant augmentation than in our study, where the 12h infusion of NAC 240mg/kg was completed 2-6 hours before surgery. The 12h schedule was chosen to ensure study drug administration pre-surgery whilst maximising safety (by administering NAC at a lower concentration than that thought to cause side-effects, and at a similar total dose to the UK licensed indication for paracetamol poisoning). NAC half-life following intravenous administration is 2-6 hours [34, 35] suggesting that it may not be available in its ‘free’ form at the start of surgery. Although plasma thiol concentration was augmented immediately pre-operatively, the peak ‘anti-oxidant’ effect may have been earlier.

Administering NAC during and after surgery may have been more effective. Red cell glutathione was similar in both groups, which may at least partly explain the lack of effect of

NAC. The correlation between OLV duration and IL-6 was relatively weak; factors other than OLV are likely to contribute to inflammation after lung resection, including surgical trauma, and possibly hyperperfusion of the operated lung, and ischaemia-reperfusion.

Ventilation during OLV was relatively ‘protective’ (Table 1), and OLV duration may be more reflective of other factors (e.g. duration of surgery, hyperperfusion) rather than injurious ventilation. The relative importance of these factors, and their potential for modulation by NAC, is not known.

The reduction in EBC pH comparing pre-operative with end-OLV is consistent with previous findings [16]. Lower EBC pH may reflect lung inflammation [36], suggesting the potential for modulation with antioxidants via changes in inflammation. However, EBC pH was not affected by NAC in this study. We hypothesised that IMAR would increase after lung resection and OLV, due to ischaemia and oxidative stress in the non-ventilated lung, and that pre-treatment with NAC would attenuate the increase in IMAR. Since ischaemia of the non-ventilated lung is thought to be important in the pathogenesis of ALI after lung resection, we investigated the relationship between lung ischaemia (duration of OLV) and IMAR, and whether this was modifiable by

NAC. IMAR was elevated at the end of OLV compared to preoperatively, and there was a correlation between duration of OLV and peak post-operative IMAR, consistent with the hypothesis that IMAR increased due to ischaemia in the non-ventilated lung. However, was no significant in IMAR difference between placebo and NAC-treated patients. It is likely that the study was underpowered to show an effect of NAC in patients with greater ischaemic damage.

Study limitations

We did not sample the pulmonary compartment for markers of inflammation or oxidative damage bronchoscopically due to reliance on OLV during surgery. This may have led to underestimation of both pulmonary inflammation, and the effect of anti-oxidant supplementation. Previous studies of VILI in peri-operative patients suggest that the lungs may exhibit inflammation measurable in BAL without evidence of systemic inflammation

The insult associated with lung resection and OLV is less severe than in animal models of lung injury or in established ARDS. The latter are associated with marked physiological derangement and tissue damage, sustained inflammation, and significant oxidative stress, and the opportunity for successful intervention is greater in these circumstances. This study was powered to the end-point of a 50% reduction in post-operative plasma IL-6. Although this end-point was achieved using protective ventilation in a similar cohort [17], it was perhaps ambitious to expect such a reduction with NAC pre-treatment. However, powering a study on clinical end-points such as ALI incidence or mortality would require recruitment of infeasible numbers of patients.

In summary, pre-operative administration of NAC increased plasma thiol concentration, but did not influence plasma IL-6 or 8-isoprostane concentrations. Further work is needed to evaluate biomarkers of inflammation and oxidative damage in patients at risk of ALI, but the role of NAC pre-treatment in patients undergoing lung resection for this goal appears limited.

Acknowledgements

The authors wish to thank the study pharmacists (Vibha Teli, Nancy Jones and Jermaine

Wright, Royal Brompton Hospital), and the Departments of Thoracic Surgery and

Anaesthesia for their assistance with this study, and Mr Winston Banya for reviewing statistical methods. Dr Bastin was supported by a Royal College of Physicians Dunhill

Medical Trust Joint Research Fellowship. Otherwise, this work was funded and supported by the British Lung Foundation and the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. REFERENCES

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1188-94. Table 1. Baseline characteristics and intraoperative parameters for patients receiving placebo or N-acetylcysteine prior to lung resection.

Characteristic/parameter NAC group Placebo group p value n=23 n=24 Age, years 68.0 (10.1) 63.8 (11.5) 0.19 Gender (male/female), n 15/8 15/9 Predicted body weight, kg 64.8 (9.3) 66.0 (10.8) 0.69 Smoking history Status (current/ex/never), n 5/15/3 9/11/4 Pack years 34 (22) 39 (31) 0.50 Comorbidities, n COPD 7 4 Ischaemic heart disease 3 2 Hypertension 5 6 Diabetes 2 1 Spirometry FEV1, litres 2.23 (0.97) 2.56 (0.81) 0.21 FEV1 % predicted 81 (23) 90 (23) 0.15 FVC, litres 3.48 (1.17) 3.60 (1.01) 0.72 FVC % predicted 100 (23) 102 (20) 0.82 Ratio FEV1:FVC, % 63 (13) 72 (13) 0.03 Pre-operative statin therapy, n 6 6 Pre-operative beta-agonist therapy, n 6 4 Pre-operative NSAID therapy, n 3 7 Previous thoracic surgery, n 1 5 Recent chemotherapy (<3 months), n 1 3 Lung resection Pneumonectomy/lobectomy/less, n 3/14/6 0/20/4 Right/left, n 13/10 12/12 Primary/secondary/other, n 18/5/0 18/3/3 Duration OLV, minutes 134 (42) 137 (59) 0.85 Parameters during OLV Tidal volume, ml/kg PBW 6.9 (1.6) 7.1 (1.2) 0.52 Peak airway pressure, cmH2O 24.9 (5.0) 25.0 (5.1) 0.98 Plateau airway pressure, cmH2O 22.3 (4.3) 23.3 (4.2) 0.41 PEEP, cmH2O 3.3 (1.6) 2.7 (1.9) 0.27 Blood products transfused (Y/N), n 4/19 6/18 Total packed red cell units transfused, n 18 25 Epidural catheter inserted, n 8 8

NAC, N-acetylcysteine; COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; NSAID, non-steroidal anti- inflammatory drug; OLV, one-lung ventilation; PBW, predicted body weight; PEEP, positive end expiratory pressure Data are expressed as number (n) or mean (standard deviation). Groups were compared using a two-tailed t-test. Table 2. Exhaled breath condensate pH in patients undergoing lung resection

EBC pH Pre-operative End OLV P value* All patients (n=37) 6.72 (0.29) 6.61 (0.34) 0.053 Placebo group (n=19) 6.73 (0.24) 6.67 (0.33) 0.312 NAC group (n=18) 6.70 (0.35) 6.55 (0.35) 0.105

Data are pre-operatively and at the end of one-lung ventilation (OLV) in patients randomised to receive placebo or N-acetylcysteine (NAC) pre-operatively. Data are expressed as mean (standard deviation). *Data were compared using a paired t-test. Figure legends

Figure 1. CONSORT diagram depicting recruitment of participants

Figure 2. Plasma interleukin-6 concentration in patients undergoing lung resection. Placebo (open circles, n=24) and NAC (black circles, n=23). Data are expressed as mean (SD). P=ns for between-group effect (Generalised Estimating Equation).

Figure 3a). Plasma thiol concentration in patients undergoing lung resection and one-lung ventilation. Data are expressed as mean (SD). Open circles represent placebo and black circles represent NAC group. P<0.0001 for between-group effect (Generalised Estimating Equation). ***p<0.0001, **p<0.001, *p=0.05 (t-test comparing NAC and placebo groups at specified time point)

Figure 3b). Plasma thiol:albumin ratio in patients undergoing lung resection and one-lung ventilation. Data are expressed as mean (SD). Open circles represent placebo and black circles represent NAC group. P<0.0001 for between-group effect (Generalised Estimating Equation). ***p<0.0001, **p<0.001 (t-test comparing NAC and placebo groups at specified time point)

Figure 3c). Red cell glutathione concentration in patients undergoing lung resection.

Data are expressed as mean (SD). Open circles represent placebo (n=13) and black circles represent NAC group (n=13). P=ns for between group effect (Generalised Estimating Equation).

Figure 4. Plasma 8-isoprostane in patients undergoing lung resection Plasma 8-isoprostane increased significantly between T1 and T3: NAC group (black bars) from 355 (264-507) pg/ml at T1 to 574 (461-990) pg/ml at T3, n=17, p=0.0004 (Wilcoxon matched pairs signed rank test); placebo group (grey bars) from 292 (242-484) pg/ml at T1 to 495 (417-747) pg/ml at T3, n=20, p=0.0002 (Wilcoxon matched pairs signed rank test). Data are expressed as median (IQR). There was no significant difference in 8-isoprostane levels at T3 between NAC and placebo groups (p=0.43, Mann-Whitney test).

Figure 5. Ischaemia modified albumin ratio (IMAR) in patients undergoing lung resection Patients were pre-treated with NAC (n=23, closed circles) or placebo (n=24, open circles). Data are expressed as mean (SD). P=ns for between-group effect (Generalised Estimating Equation).