The effect of short-term, high-dose oral N-acetylcysteine treatment on oxidative stress markers in cystic fibrosis patients with chronic P. aeruginosa infection — A pilot study

Open ArchivePublished:October 22, 2014DOI:https://doi.org/10.1016/j.jcf.2014.09.015

      Abstract

      Background

      Patients with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa lung infection have increased oxidative stress as a result of an imbalance between the production of reactive oxygen species caused by inflammation and their inactivation by the impaired antioxidant systems. Supplementation with anti-oxidants is potentially beneficial for CF patients.

      Methods

      The effect of 4 weeks of oral N-acetylcysteine (NAC) treatment (2400 mg/day divided into two doses) on biochemical parameters of oxidative stress was investigated in an open-label, controlled, randomized trial on 21 patients; 11 patients in the NAC group and 10 in the control group. Biochemical parameters of oxidative burden and plasma levels of antioxidants were assessed at the end of the study and compared to the baseline values in the two groups.

      Results

      A significant increase in the plasma levels of the antioxidant ascorbic acid (p = 0.037) and a significant decrease in the levels of the oxidized form of ascorbic acid (dehydroascorbate) (p = 0.004) compared to baseline were achieved after NAC treatment. No significant differences were observed in the control group. The parameters of oxidative burden did not change significantly compared to baseline in either of the groups. A better lung function was observed in the NAC treated group with a mean (SD) change compared to baseline of FEV1% predicted of 2.11 (4.6), while a decrease was observed in the control group (change −1.4 (4.6)), though not statistically significant.

      Conclusion

      Treatment with N-acetylcysteine 1200 mg × 2/day for 30 days significantly decreased the level of oxidized vitamin C and increased the level of vitamin C (primary end-points) and a not statistically significant improvement of lung function was observed in this group of patients.

      Abbreviations:

      AA (ascorbic acid), DHA (dehydroascorbic acid (oxidized form of AA)), MDA (malondialdehyde), NAC (N-acetylcysteine), ROS (reactive oxygen species), 8isoP (isoprostane), GSH (glutathione), CI (confidence interval)

      Keywords

      1. Introduction

      From early childhood, patients with cystic fibrosis (CF) have recurrent and chronic respiratory tract infections characterized by polymorphonuclear neutrophil (PMN) inflammation. Counts of PMNs in CF airway fluid have been found to be thousands of times higher than normal [
      • Hull J.
      • Vervaart P.
      • Grimwood K.
      • Phelan P.
      Pulmonary oxidative stress response in young children with cystic fibrosis.
      ,
      • Armstrong D.S.
      • Grimwood K.
      • Carlin J.B.
      • Carzino R.
      • Gutierrez J.P.
      • Hull J.
      • et al.
      Lower airway inflammation in infants and young children with cystic fibrosis.
      ]. Sputum neutrophil counts and elastase activity correlate well with clinical measures of CF lung dysfunction, such as declining forced expiratory volume in 1 s (FEV1) or forced vital capacity (FVC) [
      • Sagel S.D.
      • Sontag M.K.
      • Wagener J.S.
      • Kapsner R.K.
      • Osberg I.
      • Accurso F.J.
      Induced sputum inflammatory measures correlate with lung function in children with cystic fibrosis.
      ], which is consistent with neutrophils playing a central role in CF airway destruction. A consequence of the PMN-dominated inflammation is the release of proteases and reactive oxygen species (ROS). The neutrophils continuous interaction with bacterial products and their inability to engulf bacteria embedded in biofilms contribute to this exaggerated ROS production. Subsequently, ROS lose their physiological role in killing pathogens and turn into toxic effectors responsible for damaging the pulmonary epithelium as well as of other components of the lung parenchyma. Importantly, ROS can also modify the antioxidant homeostasis of extracellular fluids and epithelia causing the immune-inflammatory imbalance observed in the CF lung [
      • Galli F.
      • Battistoni A.
      • Gambari R.
      • Pompella A.
      • Bragonzi A.
      • Pilolli F.
      • et al.
      Oxidative stress and antioxidant therapy in cystic fibrosis.
      ].
      Besides the increased consumption of antioxidants caused by the exaggerated production of ROS [
      • Wood L.G.
      • Fitzgerald D.A.
      • Lee A.K.
      • Garg M.L.
      Improved antioxidant and fatty acid status of patients with cystic fibrosis after antioxidant supplementation is linked to improved lung function.
      ], patients with CF have an impaired absorption of dietary antioxidants in the gut [
      • Winklhofer-Roob B.M.
      Oxygen free radicals and antioxidants in cystic fibrosis: the concept of an oxidant–antioxidant imbalance.
      ,
      • Winklhofer-Roob B.M.
      • van't Hof M.A.
      • Shmerling D.H.
      Long-term oral vitamin E supplementation in cystic fibrosis patients: RRR-alpha-tocopherol compared with all-rac-alpha-tocopheryl acetate preparations.
      ,
      • Winklhofer-Roob B.M.
      • Ellemunter H.
      • Fruhwirth M.
      • Schlegel-Haueter S.E.
      • Khoschsorur G.
      • van't Hof M.A.
      • et al.
      Plasma vitamin C concentrations in patients with cystic fibrosis: evidence of associations with lung inflammation.
      ,
      • Winklhofer-Roob B.M.
      Nutritional status in cystic fibrosis: where to go from here?.
      ,
      • Brown R.K.
      • Kelly F.J.
      Evidence for increased oxidative damage in patients with cystic fibrosis.
      ] and the inability to efflux glutathione (GSH) into the extracellular milieu of the lung [
      • Hudson V.M.
      Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation.
      ]; the most abundant intracellular antioxidant.
      Thus, the high ROS production and impaired antioxidant systems explain the systemic redox imbalance observed in CF for which evidence is available in the literature [
      • Galli F.
      • Battistoni A.
      • Gambari R.
      • Pompella A.
      • Bragonzi A.
      • Pilolli F.
      • et al.
      Oxidative stress and antioxidant therapy in cystic fibrosis.
      ,
      • Roum J.H.
      • Buhl R.
      • McElvaney N.G.
      • Borok Z.
      • Crystal R.G.
      Systemic deficiency of glutathione in cystic fibrosis.
      ]. It has been shown [
      • Tirouvanziam R.
      • Conrad C.K.
      • Bottiglieri T.
      • Herzenberg L.A.
      • Moss R.B.
      High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis.
      ] that this redox imbalance affects circulating neutrophils before they migrate to CF airways, as evidenced by marked basal intracellular GSH deficiency.
      N-acetylcysteine (NAC) is a cysteine prodrug and can be considered a GSH precursor [
      • Rushworth G.F.
      • Megson I.L.
      Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits.
      ] and oral administration of NAC replenishes the cellular levels of GSH [
      • Atkuri K.R.
      • Mantovani J.J.
      • Herzenberg L.A.
      • Herzenberg L.A.
      N-acetylcysteine — a safe antidote for cysteine/glutathione deficiency.
      ]. High-dose oral NAC has been shown to increase neutrophil GSH levels, decrease airway neutrophil recruitment and reduce neutrophilic release of airway elastase in CF patients [
      • Tirouvanziam R.
      • Conrad C.K.
      • Bottiglieri T.
      • Herzenberg L.A.
      • Moss R.B.
      High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis.
      ].
      A recent Cochrane review on the use of thiol derivatives, such as NAC, did not find sufficient evidence to recommend the use of these compounds in the management of CF lung disease, but concluded that further studies were warranted [
      • Tam J.
      • Nash E.F.
      • Ratjen F.
      • Tullis E.
      • Stephenson A.
      Nebulized and oral thiol derivatives for pulmonary disease in cystic fibrosis.
      ]. Indications of a positive effect of NAC treatment on the lung function of a subgroup of CF patients have previously been published in our center [
      • Stafanger G.
      • Koch C.
      N-acetylcysteine in cystic fibrosis and Pseudomonas aeruginosa infection: clinical score, spirometry and ciliary motility.
      ].
      Recently, a placebo-controlled randomized clinical trial (70 CF patients) was conducted in the USA to study the effect of oral NAC on lung inflammation (ClinicalTrials.gov Identifier: NCT00809094). Oral NAC was administered in a dose of 2700 mg/day divided into three dosages over a period of 24 weeks and the effects on the sputum levels of human neutrophil elastase (HNE) were assessed as a primary end-point. While no statistical significant difference was found between the two groups with regard to the primary end-point, a slight improvement in the lung function FEV1% predicted (95% CI) was observed in the NAC treated group with a change of 1.05 (−26.16 to 25.73) while a significant decrease of −5.62 (−24.54 to 19.69) of the lung function was observed in the placebo-treated group. A larger randomized, placebo-controlled clinical study (153 CF patients) investigating the effect of inhaled GSH for 6 months similarly showed a significant improvement in lung function at three months, although differences between the two groups, failed to reach statistical significance after 6 months [
      • Griese M.
      • Kappler M.
      • Eismann C.
      • Ballmann M.
      • Junge S.
      • Rietschel E.
      • et al.
      Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial.
      ].
      Like GSH, ascorbic acid (AA) is an important antioxidant of the lung and GSH also plays a central role in AA recycling [
      • Winkler B.S.
      • Orselli S.M.
      • Rex T.S.
      The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.
      ]. In a guinea-pig model of oxidative stress caused by low plasma levels of AA, we have previously demonstrated, that biofilm lung infection with Pseudomonas aeruginosa is characterized by a worsening of the PMN-dominated inflammatory response in the lung [
      • Jensen P.O.
      • Lykkesfeldt J.
      • Bjarnsholt T.
      • Hougen H.P.
      • Hoiby N.
      • Ciofu O.
      Poor antioxidant status exacerbates oxidative stress and inflammatory response to Pseudomonas aeruginosa lung infection in guinea pigs.
      ]. In this animal model, the low AA levels lead to an increased oxidative burst from PMNs [
      • Jensen P.O.
      • Lykkesfeldt J.
      • Bjarnsholt T.
      • Hougen H.P.
      • Hoiby N.
      • Ciofu O.
      Poor antioxidant status exacerbates oxidative stress and inflammatory response to Pseudomonas aeruginosa lung infection in guinea pigs.
      ], indicating that an impaired antioxidant system can in turn exacerbate the inflammatory response. This raised the hypothesis that improved AA status can decrease the inflammation in the lung. One way of improving the AA status is by GSH supplementation as GSH facilitates AA recycling and homeostasis. GSH provides 2H+ and 2e which react with the oxidized form of AA (dehydroascorbic acid DHA) and maintain AA on its reduced form [
      • Winkler B.S.
      • Orselli S.M.
      • Rex T.S.
      The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.
      ]. As NAC is a source of GSH, we hypothesized that high-dose oral NAC, as a source of GSH, would increase the antioxidant capacity of the plasma and subsequently decrease the levels of oxidative burden markers. Although used by many CF patients, especially as a mucolytic agent, no data on the effect of NAC treatment on oxidative stress markers are available.
      The aim of this study was to investigate the effect of high dose, orally administered NAC on oxidative stress markers in urine (8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanosine (8-oxoGuo)) and plasma malondialdehyde (MDA) and 8-isoprostane (8-isoP) as well as on the plasma antioxidant levels (ascorbic acid (AA), dehydroascorbic acid (DHA) and alpha- and gamma-tocopherols) as primary end-points. Lung function changes were secondary endpoints of the study. This study was intended as a pilot study enabling proper power calculations necessary for number of CF patients to be included in a larger phase II clinical study in CF patients.

      2. Material and methods

      2.1 Patients

      An open-label, controlled, randomized study was conducted at the Copenhagen CF Center (Eudract CT nr.: 2007-001401-15). The protocol was reviewed and approved by the Committee on Health Research Ethics in the Capital Region of Denmark. All subjects provided written informed consent.
      Inclusion criteria were: adult CF patients (CF defined by positive [>60 mM Cl2] sweat chloride test and/or two disease-causing mutations) with chronic P. aeruginosa lung infection, at the end of a two-week intravenous antibiotic treatment. Exclusion criteria were: hypersensitivity to N-acetyl cysteine, prior lung transplantation or if on lung transplant waiting list, patients who received NAC in the last 30 days, patients with recent hemoptysis or an abnormal liver function test (ALAT) more than twice the normal range (10–70 U/L). After written consent, patients were consecutively randomized to one of the two groups (receiving NAC or control group) 1:1. Patients were included in the study over 2 years (March 2011 to August 2013).

      3. Study design

      Twenty-one CF patients with chronic P. aeruginosa lung infection were included in the study (12 males and 9 females), median age 39 years (range 25–61 years). Chronic P. aeruginosa infection was defined as the persistent presence of P. aeruginosa for at least 6 consecutive months, or less when combined with the presence of two or more P. aeruginosa precipitating antibodies. All patients were controlled on a regular monthly basis and each patient had an average of 10 sputum cultures per year. Eleven patients were ΔF508 homozygotes (6 in NAC and 5 in control group) and ten heterozygotes (4 in NAC and 6 in control group). Pulmonary function tests were performed according to international recommendations [
      • Miller M.R.
      • Hankinson J.
      • Brusasco V.
      • Burgos F.
      • Casaburi R.
      • Coates A.
      • et al.
      Standardisation of spirometry.
      ] measuring FEV1, expressed as a percentage of predicted values for sex and height using reference equations from Wang or Hankinson [
      • Wang X.
      • Dockery D.W.
      • Wypij D.
      • Fay M.E.
      • Ferris Jr., B.G.
      Pulmonary function between 6 and 18 years of age.
      ,
      • Hankinson J.L.
      • Odencrantz J.R.
      • Fedan K.B.
      Spirometric reference values from a sample of the general U.S. population.
      ].
      The lung function at baseline of the patients in the NAC group was FEV1 (% predicted) mean (95% CI) 58.36% (46.26; 70.46) and of the patients in the control group 53.7% (37.6; 69.8). The control group did not receive placebo medication and was therefore aware of the group to which they were assigned. Patients in the NAC group received oral treatment with N-acetylcysteine, tablets of 600 mg effervescent (Mucolysin ®600 produced by Sandoz A/S), 2 tablets twice a day (a total daily dose of 2400 mg) for 4 weeks. All other medication was continued, including inhalation with pulmozyme, bronchodilators with β2 agonists and colistin and per oral treatment with ciprofloxacin and azithromycin. There were no differences between the two groups in terms of additional medication, with the exception of three patients in the intervention group receiving low-dose (5 mg ×1) prednisone. With two exceptions in the control group, all patients (19/21 90%) were pancreatic insufficient and all were advised to continue daily supplementation with multivitamin (2 capsules of AquADEKs®).
      Two of the patients belonging to the NAC group did not complete the study: One due to adverse events (intestinal pain) and one due to lack of compliance. These two patients were excluded from the final analysis, thus the effect of the treatment with NAC was evaluated in 9 CF patients compared to 10 CF controls.

      3.1 Measurements of oxidative stress

      The primary efficacy end-points were changes compared to baseline in the level of oxidative stress markers, including lipid peroxidation: plasma malondialdehyde (MDA) and 8-isoprostane (8-isoP) and urinary excretion of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanosine (8-oxoGuo), as well as plasma antioxidant levels: vitamin C or ascorbic acid (AA) and oxidized vitamin C (dehydroascorbic acid (DHA), alpha- and gamma-tocopherols).
      The secondary efficacy end-points were: lung function (FEV1 and FVC) and as inflammatory parameter: oxidative burst in the PMNs.
      Blood samples and 24-hour urine samples were collected when patients entered the study (baseline values) and 4 weeks later.

      3.2 Plasma AA and DHA measurements

      Blood samples were immediately centrifuged (16,000 ×g, 1 min). One 100 μL plasma aliquot was acidified with an equal volume of 10% m-phosphoric acid containing 2 mM EDTA, briefly vortex mixed, centrifuged and the supernatant frozen at −80 °C [
      • Lykkesfeldt J.
      Ascorbate and dehydroascorbic acid as biomarkers of oxidative stress: validity of clinical data depends on vacutainer system used.
      ]. AA and total vitamin C were measured by HPLC with colorimetric detection and the fraction of DHA was calculated by subtraction using uric acid as endogenous standard as described in details elsewhere [
      • Lykkesfeldt J.
      Ascorbate and dehydroascorbic acid as reliable biomarkers of oxidative stress: analytical reproducibility and long-term stability of plasma samples subjected to acidic deproteinization.
      ].

      3.3 MDA measurements

      Plasma (100 μL) MDA analysis was accomplished by reverse-phase HPLC with fluorescence detection of the genuine MDA (thiobarbituric acid)2 adduct as described previously [
      • Lykkesfeldt J.
      Determination of malondialdehyde as dithiobarbituric acid adduct in biological samples by HPLC with fluorescence detection: comparison with ultraviolet–visible spectrophotometry.
      ]. 8-isoP was assessed as per the manufacturer's recommendation (Cat no.: 516351, Cayman Chemicals, USA). The method is based on a competitive enzyme immunoassay using 8-isoP and an isoprostane conjugate. The concentration of bound conjugate, which is developed with Ellman's reagent and quantified spectrophotometrically, is inversely proportional to the concentration of 8-isoP in the sample. Alpha- and gamma-tocopherols were analyzed by HPLC with colorimetric detection as modified from Sattler and coworkers [
      • Sattler W.
      • Mohr D.
      • Stocker R.
      Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.
      ].

      3.4 8oxodG and 8-oxodGuo measurements

      The urinary content of the oxidized nucleosides 8-oxodG and 8-oxoGuo was quantified using a modified version of an ultra performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) assay, described in detail elsewhere [
      • Henriksen T.
      • Hillestrom P.R.
      • Poulsen H.E.
      • Weimann A.
      Automated method for the direct analysis of 8-oxo-guanosine and 8-oxo-2′-deoxyguanosine in human urine using ultraperformance liquid chromatography and tandem mass spectrometry.
      ]. Briefly, the frozen urine samples were thawed, mixed and heated to 37 °C for 5 min to re-dissolve possible precipitate and centrifuged at 10,000 ×g for 5 min. All further sample preparation was performed using a Biomek 3000 robot (Beckman Coulter, CA, USA). 110 μL of each urine sample or calibration standard were mixed with 90 μL 100 mM lithium acetate buffer and 90 μL of 50 nM internal standard. The chromatographic separation was performed on an Acquity I-class UPLC system (Waters Corp., Milford, USA) using an Acquity UPLC BEH Shield RP18 column (1.7 μm, 2.1 × 100 mm; Waters Corp.) with a column temperature of 4 °C. The mass spectrometry detection was performed on a Xevo-TSQ triple quadrupole mass spectrometer (Waters Corp., Milford, USA), using electrospray ionization in the positive mode for 8-oxodG and negative ionization mode for 8-oxoGuo. Calibration standards ranged from 1 to 60 nM. As internal standards, stable isotope-labeled 8-oxodG and 8-oxoGuo, [15N5]8-oxodG and [15N5] 8-oxoGuo, were used. To confirm the presence of the analyte and the absence of false contributions from co-elution of similar compounds in the urine samples, two specific fragments of each analyte were included in the analysis. The average within-day variation (RSD, %) estimated from the method validation was 2.3% for 8-oxoGuo, and 3.8% for 8-oxodG. The average recovery was 103.7% and 104.8%, respectively. The 8-oxodG and 8-oxoGuo urinary excretion was normalized to the urinary creatinine concentration, quantified by Jaffe's reaction. The average within-day and between-day variation (RSD, %) estimated from the method validation was 2.3% and 9.0% for 8-oxoGuo, respectively, and 3.8% and 7.4% for 8-oxodG.
      8-oxodG and 8-oxoGuo were normalized against urinary creatinine concentrations.

      3.5 Oxidative burst in PMN measurements

      The respiratory burst of PMNs was estimated by a modified flow cytometric assay [
      • Jensen P.O.
      • Lykkesfeldt J.
      • Bjarnsholt T.
      • Hougen H.P.
      • Hoiby N.
      • Ciofu O.
      Poor antioxidant status exacerbates oxidative stress and inflammatory response to Pseudomonas aeruginosa lung infection in guinea pigs.
      ] for the intracellular content of H2O2 according to the fluorescence intensity from oxidized 123-dihydrorhodamine (DHR) [
      • Rothe G.
      • Oser A.
      • Valet G.
      Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes.
      ].

      3.6 Statistical analysis

      Statistical analysis was performed by GraphPad Prism 6.04.
      The Gaussian distribution of the values was tested by D'Agostino & Pearson omnibus normality test. On normally distributed values, one-tailed t-test on paired samples was used to compare the changes of the measured parameters in the end of the study compared to baseline in NAC treated and untreated (control) patients. The level of significance was 5%. The differences between the measured parameters in the two groups at baseline were tested by two-tailed unpaired t-test.

      4. Results

      At baseline, CF patients in the NAC and control groups had similar levels of oxidative burden markers and plasma antioxidant levels. Thus, no significant differences in the levels of oxidative stress markers in plasma (MDA, 8isoP) and urine (8oxodG, 8 oxodGuo) and of the plasma levels of antioxidants (ascorbate, dehydroascorbate, alpha- and gamma-tocopherols), were observed between the two groups (Table 1). Additionally, lung function of the patients was similar in the two groups at baseline (Table 1).
      Table 1Baseline levels of measured parameters mean (95% CI) in NAC treated and control groups. Unpaired t-test was used to analyze the differences between the two groups. The patients in the two groups have similar levels of the measured parameters at baseline.
      Group (number of patients)Plasma antioxidantsPlasma markers of oxidative burdenUrinary markers of oxidative burden
      Ascorbic acid (μmol/L)Oxidized ascorbic acid (% of total)Alpha-tocopherol (μmol/L)Gamma-tocopherol (μmol/L)MDA (μmol/L)8-isoP (ng/L)DNA 8oxodG (nM/mM creatinine)RNA 8oxodGuo (nM/mM creatinine)
      NAC (11)

      Mean (95% CI)
      79.77

      (58.7; 100.8)
      4.97

      (3.73; 6.21)
      29.73

      (22.48; 36.98)
      3.52

      (2.26; 4.77)
      0.30

      (0.21; 0.38)
      53.31

      (45.64; 60.99)
      1.54

      (0.99; 2.09)
      3.73

      (2.49; 4.96)
      Control (10)

      Mean (95% CI)
      76.62

      (60.82; 92.42)
      5.25

      (3.9, 6.6)
      29.70

      (21.8; 37.6)
      2.36

      (0.98; 3.72)
      0.25

      (022; 0.28)
      57.20

      (49.6; 64.7)
      1.19

      (0.87, 1.5)
      3.31

      (2.72, 3.89)
      p0.790.730.990.170.260.420.180.52
      Group (number of patients)FEV1 (% predicted)FVC (% predicted)
      NAC (11)

      Mean (95% CI)
      58.36

      (46.26; 70.46)
      94.18

      (80.81; 107.6)
      Control (10)

      Mean (95% CI)
      53.7

      (37.6; 69.8)
      90.20

      (69.83; 110.6)
      p0.600.71
      The patients in the intervention group received NAC in a mean dose of 36 mg/kg/day (max 59 mg/kg/day and min 25.8 mg/kg/day).
      After 4 weeks of NAC treatment, a significant increase in the plasma level of ascorbic acid (AA) (p = 0.037) and decrease in the level of oxidized ascorbic acid (DHA) (p = 0.004) compared to baseline were observed, while no significant changes were observed in the control group (Fig. 1 and Table 2).
      Figure thumbnail gr1
      Fig. 1Changes in the plasma levels of ascorbic acid (AA) and dehydroascorbate (DHA) at the end of the study compared to baseline in the NAC treated group (A and C) and in the control group (B and D, respectively). Significant higher plasma levels of ascorbate (p = 0.037) and lower of oxidized ascorbate (p = 0.004) compared to baseline were found in the NAC treated patients but not in the control group. Paired t-test was used to analyze the differences between the levels at the end of the study compared to baseline.
      Table 2Differences in plasma and urine markers and lung function after 4 weeks compared to baseline in the NAC treated and control group. The p value represents the significance of the difference between levels at baseline and end of the study (t-paired, one tailed).
      Group (number of patients)Plasma antioxidantsPlasma markers of oxidative burdenUrinary markers of oxidative burden
      Ascorbic acid (μmol/L)Oxidized ascorbic acid (% of total)Alpha-tocopherol (μmol/L)Gamma-tocopherol (μmol/L)MDA (μmol/L)8-isoP (ng/L)DNA 8oxodG (nM/mM creatinine)RNA 8oxodGuo (nM/mM creatinine)
      NAC (9)

      Mean (95% CI)
      16.66

      (−2.07;35.39)

      p = 0.037
      1.45

      (−2.39; −0.51)

      p = 0.004
      5.88

      (−15.66;3.88)

      p = 0.099
      1.46

      (−3.07;0.16)

      p = 0.035
      0.04

      (−0.14; 0.06)

      p = 0.200
      1.84

      (−8.28;11.96)

      p = 0.340
      0.1

      (−0.55;0.75)

      p = 0.329
      0.11

      (−1.06;0.83)

      p = 0.394
      Control (10)

      Mean (95% CI)
      7.72

      (−6.16;21.62)

      p = 0.120
      0.10

      (−1.38;1.17)

      p = 0.427
      3.96

      (−10.55;2.63)

      p = 0.100
      0.48

      (−1.88; 0.92)

      p = 0.230
      0.02

      (−0.03;0.07)

      p = 0.224
      5.73

      (−13.54;2.07)

      p = 0.065
      0.26

      (−0.61;0.09)

      p = 0.065
      0.58

      (−1.43;0.27)

      p = 0.079
      GroupFEV1 (% predicted)FVC (% predicted)
      NAC (9)

      Mean (95% CI)
      2.11 (−1.44;5.66)

      p = 0.104
      1.44 (−3.38;6.27)

      p = 0.255
      Control (10)

      Mean (95% CI)
      1.4 (−4.7; 1.9)

      p = 0.182
      0.1 (−6.03; 6.23)

      p = 0.486
      A decrease in the serum levels of alpha- and gamma-tocopherols was observed in both groups, with a significant decrease (p = 0.03) in the levels of gamma-tocopherol in the NAC treated group.
      A non-significant decrease in the oxidative burst of the PMNs was found in the end of the study compared to baseline in both groups (data not shown).
      An improvement compared to baseline in the FEV1 (% predicted) mean (95% CI) +2.11 (−1.44; 5.66) was observed in the NAC treated group while a decrease was observed in the control group mean (95% CI) (−1.4 (−4.7; 1.9)), both changes did not reach the level of significance (p > 0.05) (Table 2).

      4.1 Safety and adverse events profile

      One patient stopped treatment with NAC due to stomach pain. No other adverse events were observed.

      5. Discussions

      Oral treatment with NAC 1200 mg × 2 daily for 30 days significantly decreased the level of oxidized vitamin C and increased the level of vitamin C in patients with CF in the present study. The significant increase in the plasma levels of AA and decrease in the DHA levels under treatment with NAC are in accordance with the role played by GSH in the ascorbate homeostasis [
      • Winkler B.S.
      • Orselli S.M.
      • Rex T.S.
      The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.
      ]. In short, it is known from in vitro assays that cells take up exogenous DHA, and in the presence of GSH convert it to AA in the cytoplasm. Our data demonstrate that the redox coupling between GSH and DHA in the regeneration of AA exposed to an oxidative challenge is well functioning in CF patients, explaining the lower plasma levels of DHA and the higher AA levels compared to baseline, following NAC administration.
      NAC has been used in our study as an antioxidant agent in a mean dose that was calculated to be 36 mg/kg/day. The optimal dosage as anti-oxidant agent in CF is not known but this dosage is higher than what is usually recommended as mucolytic agent (400–1200 mg/day). Taking into account the benign side-effect profile of NAC, the dosage can probably be safely increased to 50 mg/Kg/day [
      • Dodd S.
      • Dean O.
      • Copolov D.L.
      • Malhi G.S.
      • Berk M.
      N-acetylcysteine for antioxidant therapy: pharmacology and clinical utility.
      ].
      According to in vitro data, vitamin C (AA) spares for vitamin E (tocopherol) [
      • Winkler B.S.
      • Orselli S.M.
      • Rex T.S.
      The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.
      ] and the increased plasma AA levels after NAC treatment could thus be expected to cause an increase in the alpha-tocopherol levels. However, conflicting results have been reported from in vivo studies and Burton et al. [
      • Burton G.W.
      • Wronska U.
      • Stone L.
      • Foster D.O.
      • Ingold K.U.
      Biokinetics of dietary RRR-alpha-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not “spare” vitamin E in vivo.
      ] failed to find the sparing effect in a well-controlled experiment conducted in an animal model.
      The mean AA serum levels at baseline were 79.77 μmol/L in the NAC group and 76.62 μmol/L in the control group (Table 1), both values being above 50 μmol/L, which is considered the protective level for free radical disease. The mean serum alpha-tocopherol levels at baseline were 29.73 μmol/L in the NAC group and 29.70 μmol/L in the control group (Table 1), which is within the normal ranges reported from healthy non-CF populations (24.06 μmol/L) [
      • Ford E.S.
      • Schleicher R.L.
      • Mokdad A.H.
      • Ajani U.A.
      • Liu S.
      Distribution of serum concentrations of alpha-tocopherol and gamma-tocopherol in the US population.
      ]. The low levels of alpha-tocopherol frequently reported in CF patients [
      • Farrell P.M.
      • Bieri J.G.
      • Fratantoni J.F.
      • Wood R.E.
      • di Sant'Agnese P.A.
      The occurrence and effects of human vitamin E deficiency. A study in patients with cystic fibrosis.
      ,
      • Benabdeslam H.
      • Abidi H.
      • Garcia I.
      • Bellon G.
      • Gilly R.
      • Revol A.
      Lipid peroxidation and antioxidant defenses in cystic fibrosis patients.
      ], were thus not found in our adult CF population. The mean gamma-tocopherol serum levels at baseline were 3.52 μmol/L in the NAC group and 2.36 μmol/L in the control group; notably lower than what has been reported in healthy non-CF population (4.9 μmol/L in the corresponding age group) [
      • Ford E.S.
      • Schleicher R.L.
      • Mokdad A.H.
      • Ajani U.A.
      • Liu S.
      Distribution of serum concentrations of alpha-tocopherol and gamma-tocopherol in the US population.
      ], but similar to reports from CF patients receiving AquADEKs® supplements [
      • Sagel S.D.
      • Sontag M.K.
      • Anthony M.M.
      • Emmett P.
      • Papas K.A.
      Effect of an antioxidant-rich multivitamin supplement in cystic fibrosis.
      ].
      It has been shown that vitamin E levels increase after antibiotic treatment as a result of bronchial inflammation control [
      • Lagrange-Puget M.
      • Durieu I.
      • Ecochard R.
      • bbas-Chorfa F.
      • Steghens J.P.
      • et al.
      Longitudinal study of oxidative status in 312 cystic fibrosis patients in stable state and during bronchial exacerbation.
      ]. Based on these observations, it may be speculated, that the vitamin E levels were likely to be at their highest level at baseline in our study, as all CF patients were included in the study at the end of a 14 day antibiotic treatment. The decrease in the alpha- and gamma-tocopherol levels observed in both groups after 4 weeks may thus be explained by the dynamics of vitamin E during the course of the disease.
      In addition, the effect of GSH on tocopherol levels is thought to be indirect through the sparing of vitamin E by vitamin C and it is therefore not surprising, that such effects were not observed following a relatively short study period. An explanation for the significant decrease in the plasma levels of gamma-tocopherol in the NAC treated group is not available at the present time.
      The mechanisms by which ROS cause tissue injuries are many, and among them it is important that the role played by ROS attacks on polyunsaturated fatty acids of lipid structures (membranes) and DNA [
      • Winklhofer-Roob B.M.
      Oxygen free radicals and antioxidants in cystic fibrosis: the concept of an oxidant–antioxidant imbalance.
      ,
      • Winklhofer-Roob B.M.
      • Ellemunter H.
      • Fruhwirth M.
      • Schlegel-Haueter S.E.
      • Khoschsorur G.
      • van't Hof M.A.
      • et al.
      Plasma vitamin C concentrations in patients with cystic fibrosis: evidence of associations with lung inflammation.
      ,
      • Brown R.K.
      • Kelly F.J.
      Evidence for increased oxidative damage in patients with cystic fibrosis.
      ,
      • Brown R.K.
      • Kelly F.J.
      Role of free radicals in the pathogenesis of cystic fibrosis.
      ,
      • Brown R.K.
      • McBurney A.
      • Lunec J.
      • Kelly F.J.
      Oxidative damage to DNA in patients with cystic fibrosis.
      ,
      • Winklhofer-Roob B.M.
      • Ziouzenkova O.
      • Puhl H.
      • Ellemunter H.
      • Greiner P.
      • Muller G.
      • et al.
      Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: improvement during vitamin E supplementation.
      ].
      Malondialdehyde (MDA) is an end product of the oxidation and decomposition of unsaturated fatty acids and 8-iso prostaglandin F2α (8 isoprostane) is produced by free-radical catalyzed peroxidation of arachidonic acid.
      MDA levels have been found to be increased in plasma of CF patients compared to controls in some studies [
      • Benabdeslam H.
      • Abidi H.
      • Garcia I.
      • Bellon G.
      • Gilly R.
      • Revol A.
      Lipid peroxidation and antioxidant defenses in cystic fibrosis patients.
      ,
      • Dominguez C.
      • Gartner S.
      • Linan S.
      • Cobos N.
      • Moreno A.
      Enhanced oxidative damage in cystic fibrosis patients.
      ,
      • Portal B.C.
      • Richard M.J.
      • Faure H.S.
      • Hadjian A.J.
      • Favier A.E.
      Altered antioxidant status and increased lipid peroxidation in children with cystic fibrosis.
      ]. However, this was not found in a large cohort of CF patients [
      • Lagrange-Puget M.
      • Durieu I.
      • Ecochard R.
      • bbas-Chorfa F.
      • Steghens J.P.
      • et al.
      Longitudinal study of oxidative status in 312 cystic fibrosis patients in stable state and during bronchial exacerbation.
      ] using sensitive methods for MDA measurement. It has been suggested, that due to the fast elimination from plasma, measuring lipid peroxidation in the epithelial lining fluid is more appropriate when approximating the severity of a lesion at the site of inflammation [
      • Hull J.
      • Vervaart P.
      • Grimwood K.
      • Phelan P.
      Pulmonary oxidative stress response in young children with cystic fibrosis.
      ], however this involves invasive procedures and is cumbersome. 8 isoprostane levels were found to be increased in the plasma of CF patients [
      • Wood L.G.
      • Fitzgerald D.A.
      • Gibson P.G.
      • Cooper D.M.
      • Collins C.E.
      • Garg M.L.
      Oxidative stress in cystic fibrosis: dietary and metabolic factors.
      ,
      • Collins C.E.
      • Quaggiotto P.
      • Wood L.
      • O'Loughlin E.V.
      • Henry R.L.
      • Garg M.L.
      Elevated plasma levels of F2 alpha isoprostane in cystic fibrosis.
      ]. Due to the large diversity of the measurement methods for MDA and 8 isoP among various laboratories [
      • Lykkesfeldt J.
      Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking.
      ], no comparison of the data in our study with previously published studies was performed. In our study, no decreases in the levels of lipid peroxidation markers (MDA and 8isoP) were observed after NAC administration. As the main antioxidant which prevents lipid peroxidation is tocopherol, present in cell membranes and as NAC supplementation did not cause an improvement in the tocopherol levels, it is not surprising, that a decrease in the lipid peroxidation markers was not observed.
      Urinary 8oxodG levels, as a marker of DNA oxidation, have been reported to be higher in CF patients compared to controls [
      • Brown R.K.
      • McBurney A.
      • Lunec J.
      • Kelly F.J.
      Oxidative damage to DNA in patients with cystic fibrosis.
      ]. No data on the 8oxodGuo levels, as a marker of RNA oxidation, are available in the literature, our study being the first reporting these measurements in CF patients.
      The urinary levels of DNA and RNA oxidation products (8oxodG and 8oxodGuo) did not show significant changes compared to baseline in either of the groups, though a trend towards lower urinary levels compared to baseline was observed in the control group. This is probably due to the chronic inflammation, present for several decades in these CF patients, contributing to the overall oxidative stress and plausibly overwhelming the antioxidant system, despite higher AA levels.
      Both, the small number of CF patients included in the study and the short period of treatment (4 weeks) are draw-backs of our trial, which was meant as a pilot study.
      Due to the intensive treatment of CF patients with chronic P. aeruginosa infection, intervention studies with antioxidants are difficult to perform and require large number of patients in each group in order to find significant changes in the measured parameters and require multicenter studies [
      • Griese M.
      • Kappler M.
      • Eismann C.
      • Ballmann M.
      • Junge S.
      • Rietschel E.
      • et al.
      Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial.
      ].
      Due to difficulties in recruiting patients for a satisfactory matched paired design study in our CF center, we were not able to perform suitable power calculations for future studies. To circumvent the problem of variability between individuals in the two groups, a placebo-controlled, cross-over design study could be conducted in our CF center in the future. This kind of design has been used in a previous study on the effect of NAC in CF patients [
      • Stafanger G.
      • Koch C.
      N-acetylcysteine in cystic fibrosis and Pseudomonas aeruginosa infection: clinical score, spirometry and ciliary motility.
      ]. Using changes in FEV1 during treatment from our pilot study with NAC we calculated that a sample size of 40 patients will need to enter this two-arms (NAC and placebo) crossover study. Based on the data collected in this pilot study, we suggest a treatment period of 6 months with NAC in a dose of 50 mg/kg/day.
      Since it has been shown that inflammation in CF patients starts as early as infancy [
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • Douglas T.
      • von Ungern-Sternberg B.S.
      • Garratt L.W.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ,
      • Sly P.D.
      • Brennan S.
      • Gangell C.
      • De K.N.
      • Murray C.
      • Mott L.
      • et al.
      Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening.
      ,
      • Stick S.M.
      The first 2 years of life: implications of recent findings.
      ], clinical trials aiming at correcting the oxidant/antioxidant imbalance in young children, especially in countries with newborn screening, could be considered.
      In conclusion, in accordance with our hypothesis, this pilot study demonstrated that supplementation with the GSH precursor, NAC, improved the antioxidant capacity of plasma by increasing AA levels and decreasing the DHA levels. No further effect on the vitamin E levels and markers of lipid peroxidation were observed. The tendency towards a better lung function in this pilot study which is very much underpowered and after NAC supplementation for a short period of time (4 weeks) is encouraging and calls for new multi-centered placebo-controlled trials on a large population of CF patients that are matched in accordance with sex, age and lung function.

      Conflict of interests

      We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
      We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
      We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.
      We further confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.

      Acknowledgments

      We wish to thank the 21 CF patients with chronic P. aeruginosa infection attending the Copenhagen CF Center for accepting to be included in yet another study. Additionally, we want to thank the nurses at the Adult CF Department for the help with blood sample collection. The excellent technical assistance of Trine Henriksen, Annie Bjergby Christensen, Belinda Bringtoft, Joan Frandsen and Tina Wassermann is very much appreciated.
      The Mucolysin tablets were kindly provided by Sandoz A/S.
      The Novo Nordisk Foundation supported HKJ as a clinical research stipend.

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