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: m.griffiths@imperial.ac.uk

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

[12].

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

1. Park HS, Kim SR, Lee YC. Impact of oxidative stress on lung diseases. Respirology 2009;

14: 27-38.

2. Suter PM, Domenighetti G, Schaller MD, Laverriere MC, Ritz R, Perret C. N-

acetylcysteine enhances recovery from acute lung injury in man. A randomized, double-blind,

placebo-controlled clinical study. Chest 1994; 105: 190-4.

3. Jepsen S, Herlevsen P, Knudsen P, Bud MI, Klausen NO. Antioxidant treatment with N-

acetylcysteine during adult respiratory distress syndrome: a prospective, randomized,

placebo-controlled study. Crit Care Med 1992; 20: 918-23.

4. Domenighetti G, Suter PM, Schaller MD, Ritz R, Perret C. Treatment with N-

acetylcysteine during acute respiratory distress syndrome: a randomized, double-blind,

placebo-controlled clinical study. J Crit Care 1997; 12: 177-82.

5. Bernard GR, Wheeler AP, Arons MM, Morris PE, Paz HL, Russell JA, Wright PE. A trial

of antioxidants N-acetylcysteine and procysteine in ARDS. The Antioxidant in ARDS Study

Group. Chest 1997; 112: 164-72.

6. Decramer M, Rutten-van Molken M, Dekhuijzen PN, Troosters T, van Herwaarden C,

Pellegrino R, van Schayck CP, Olivieri D, Del Donno M, De Backer W, Lankhorst I, Ardia

A. Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease

(Bronchitis Randomized on NAC Cost-Utility Study, BRONCUS): a randomised placebo-

controlled trial. Lancet 2005; 365: 1552-60.

7. Zheng JP, Wen FQ, Bai CX, Wan HY, Kang J, Chen P, Yao WZ, Ma LJ, Li X, Raiteri L,

Sardina M, Gao Y, Wang BS, Zhong NS; PANTHEON study group. Twice-daily N-

acetylcysteine 600mg for exacerbations of chronic obstructive pulmonary disease

(PANTHEON): a randomised double-blind placebo-controlled trial. Lancet Respir Med 2014;

2: 187-94.

8. Idiopathic Pulmonary Fibrosis Clinical Research Network. Randomized Trial of

Acetylcysteine in Idiopathic Pulmonary Fibrosis. N Engl J Med 2014; 370: 2093-2101.

9. Kutlu CA, Williams EA, Evans TW, Pastorino U, Goldstraw P. Acute lung injury and

acute respiratory distress syndrome after pulmonary resection. Ann Thorac Surg 2000; 69:

376-80.

10. Tang SS, Redmond K, Griffiths M, Ladas G, Goldstraw P, Dusmet M. The mortality from

acute respiratory distress syndrome after pulmonary resection is reducing: a 10-year single

institutional experience. Eur J Cardiothorac Surg 2008; 34: 898-902.

11. Proudfoot A, McAuley DF, Griffiths M, Hind M. Human models of acute lung injury.

Disease Models and Mechanisms 2011; 4: 145-53.

12. Moloney ED, Mumby SE, Gajdocsi R, Cranshaw JH, Kharitonov SA, Quinlan GJ,

Griffiths MJ. Exhaled breath condensate detects markers of pulmonary inflammation after

cardiothoracic surgery. Am J Respir Crit Care Med 2004; 169: 64-9.

13. Williams EA, Quinlan GJ, Goldstraw P, Gothard JW, Evans TW. Postoperative lung

injury and oxidative damage in patients undergoing pulmonary resection. Eur Respir J 1998;

11: 1028-34.

14. Misthos P, Katsaragakis S, Milingos N, Kakaris S, Sepsas E, Athanassiadi K, Theodorou

D, Skottis I. Postresectional pulmonary oxidative stress in lung cancer patients. The role of

one-lung ventilation. Eur J Cardiothorac Surg 2005; 27: 379-82.

15. Misthos P, Katsaragakis S, Theodorou D, Milingos N, Skottis I. The degree of oxidative

stress is associated with major adverse effects after lung resection: a prospective study. Eur J

Cardiothorac Surg 2006; 29: 591-5.

16. Bastin AJ, Sato H, Davidson SJ, Quinlan GJ, Griffiths MJ. Biomarkers of lung injury

after one-lung ventilation for lung resection. Respirology 2011; 16: 138-45.

17. Michelet P, D'Journo XB, Roch A, Doddoli C, Marin V, Papazian L, Decamps I, Bregeon

F, Thomas P, Auffray JP. Protective ventilation influences systemic inflammation after

esophagectomy: a randomized controlled study. Anesthesiology 2006; 105: 911-9.

18. Craig SR, Leaver HA, Yap PL, Pugh GC, Walker WS. Acute phase responses following

minimal access and conventional thoracic surgery. Eur J Cardiothorac Surg 2001; 20: 455-

63.

19. Morrow JD, Chen Y, Brame CJ, Yang J, Sanchez SC, Xu J, Zackert WE, Awad JA,

Roberts LJ. The isoprostanes: unique prostaglandin-like products of free-radical-initiated

lipid peroxidation. Drug Metabolism Reviews 1999; 31: 117-39.

20. Quinlan GJ, Evans TW, Gutteridge JM. Oxidative damage to plasma proteins in adult

respiratory distress syndrome. Free Radic Res 1994; 20: 289-98.

21. Jalan R, Schnurr K, Mookerjee RP, Sen S, Cheshire L, Hodges S, Muravsky V, Williams

R, Matthes G, Davies NA. Alterations in the functional capacity of albumin in patients with

decompensated cirrhosis is associated with increased mortality. Hepatology 2009; 50: 555-

64.

22. ARDSnetwork. Ventilation with lower tidal volumes as compared with traditional tidal

volumes for acute lung injury and the acute respiratory distress syndrome. The Acute

Respiratory Distress Syndrome Network. N Engl J Med 2000; 342: 1301-8.

23. Serrano-Mollar A, Closa D, Prats N, Blesa S, Martinez-Losa M, Cortijo J, Estrela JM,

Morcillo EJ, Bulbena O. In vivo antioxidant treatment protects against bleomycin-induced

lung damage in rats. British journal of pharmacology 2003; 138: 1037-48.

24. Geudens N, Van De Wauwer C, Neyrinck AP, Timmermans L, Vanhooren HM,

Vanaudenaerde BM, Verleden GM, Verbeken E, Lerut T, Van Raemdonck DE. N-acetyl

cysteine pre-treatment attenuates inflammatory changes in the warm ischemic murine lung. J

Heart Lung Transplant 2007; 26: 1326-32.

25. Toklu HZ, Tunali Akbay T, Velioglu-Ogunc A, Ercan F, Gedik N, Keyer-Uysal M, Sener

G. Silymarin, the Antioxidant Component of Silybum marianum, Prevents Sepsis-Induced

Acute Lung and Brain Injury. J Surg Res 2008; 145: 214-22.

26. Syrkina O, Jafari B, Hales CA, Quinn DA. Oxidant stress mediates inflammation and

apoptosis in ventilator-induced lung injury. Respirology 2008; 13: 333-40.

27. Yim AP, Wan S, Lee TW, Arifi AA. VATS lobectomy reduces cytokine responses

compared with conventional surgery. Ann Thorac Surg 2000; 70: 243-7.

28. Jafari B, Ouyang B, Li LF, Hales CA, Quinn DA. Intracellular glutathione in stretch-

induced cytokine release from alveolar type-2 like cells. Respirology 2004; 9: 43-53.

29. Ortolani O, Conti A, De Gaudio AR, Moraldi E, Cantini Q, Novelli G. The effect of

glutathione and N-acetylcysteine on lipoperoxidative damage in patients with early septic

shock. Am J Respir Crit Care Med 2000; 161: 1907-11.

30. Spies CD, Reinhart K, Witt I, Meier-Hellmann A, Hannemann L, Bredle DL, Schaffartzik

W. Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock

patients: results from a prospective, randomized, double-blind study. Crit Care Med 1994;

22: 1738-46.

31. Szakmany T, Marton S, Molnar Z. Lack of effect of prophylactic N-acetylcysteine on

postoperative organ dysfunction following major abdominal tumour surgery: a randomized,

placebo-controlled, double-blinded clinical trial. Anaesthesia and Intensive Care 2003; 31:

267-71.

32. Molnar Z, MacKinnon KL, Shearer E, Lowe D, Watson ID. The effect of N-

acetylcysteine on total serum anti-oxidant potential and urinary albumin excretion in

critically ill patients. Intensive Care Med 1998; 24: 230-5.

33. Molnar Z, Shearer E, Lowe D. N-Acetylcysteine treatment to prevent the progression of

multisystem organ failure: a prospective, randomized, placebo-controlled study. Crit Care

Med 1999; 27: 1100-4.

34. Electronic Medicines Compendium. Summary of product characteristics for

acetylcysteine. https://www.medicines.org.uk/emc/medicine/29451. Accessed 1st June 2015.

35. Prescott LF, Donovan JW, Jarvie DR and Proudfoot AT. The disposition and kinetics of

intravenous N-acetylcysteine in patients with paracetamol overdosage. Eur J Clin Pharmacol

1989; 37: 501-6.

36. Gessner C, Hammerschmidt S, Kuhn H, Seyfarth HJ, Sack U, Engelmann L, Schauer J,

Wirtz H. Exhaled breath condensate acidification in acute lung injury. Respir Med 2003; 97:

1188-94.

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

Characteristic/parameter NAC groupn=23

Placebo groupn=24

p value

Age, years 68.0 (10.1) 63.8 (11.5) 0.19Gender (male/female), n 15/8 15/9Predicted body weight, kg 64.8 (9.3) 66.0 (10.8) 0.69Smoking history

Status (current/ex/never), nPack years

5/15/334 (22)

9/11/439 (31) 0.50

Comorbidities, nCOPDIschaemic heart diseaseHypertensionDiabetes

7352

4261

SpirometryFEV1, litres FEV1 % predictedFVC, litresFVC % predicted Ratio FEV1:FVC, %

2.23 (0.97)81 (23)

3.48 (1.17)100 (23)63 (13)

2.56 (0.81)90 (23)

3.60 (1.01)102 (20)72 (13)

0.210.150.720.820.03

Pre-operative statin therapy, nPre-operative beta-agonist therapy, nPre-operative NSAID therapy, n

663

647

Previous thoracic surgery, n 1 5Recent chemotherapy (<3 months), n 1 3Lung resection

Pneumonectomy/lobectomy/less, nRight/left, nPrimary/secondary/other, n

3/14/613/1018/5/0

0/20/412/1218/3/3

Duration OLV, minutes 134 (42) 137 (59) 0.85Parameters during OLV

Tidal volume, ml/kg PBWPeak airway pressure, cmH2OPlateau airway pressure, cmH2OPEEP, cmH2O

6.9 (1.6)24.9 (5.0)22.3 (4.3)3.3 (1.6)

7.1 (1.2)25.0 (5.1)23.3 (4.2)2.7 (1.9)

0.520.980.410.27

Blood products transfused (Y/N), nTotal packed red cell units transfused, n

4/1918

6/1825

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 pressureData 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) 0.053Placebo group (n=19) 6.73 (0.24) 6.67 (0.33) 0.312NAC 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).