Thorax 1991;46:39-42 39 Cysteine and glutathione concentrations in plasma treatment and bronchoalveolar lavage fluid after Thorax: first published as 10.1136/thx.46.1.39 on 1 January 1991. Downloaded from with N-acetylcysteine Myrtle M E Bridgeman, Mark Marsden, William MacNee, David C Flenley,* Andrew P Ryle Abstract protects lung epithelial cells in vitro against N-acetylcysteine (600 mg/day) was given oxidant injury mediated by polymorpho- to patients by mouth for five days before nuclear leucocytes9 and dogs against pul- bronchoscopy and bronchoalveolar monary oxygen toxicity in vivo."0 Thus N- lavage to determine whether N-acetyl- acetylcysteine has antioxidant potential and its cysteine could increase the concentra- main metabolite, cysteine, a precursor in the tions of the antioxidant reduced gluta- biosynthesis of reduced glutathione, is an thione in plasma and bronchoalveolar extremely important intracellular and extra- lavage fluid. Bronchoalveolar lavage was cellular antioxidant.'1 12 performed 1-3 hours (group 2, n = 9) The aim of this study was to determine if and 16-20 hours (group 3, n = 10) after N-acetylcysteine given by mouth could the last dose of N-acetylcysteine and the increase the concentrations of the antioxidants values were compared with those in a cysteine and reduced gluytathione in plasma control group receiving no N-acetyl- and in bronchoalveolar lavage fluid in man. cysteine (group 1, n = 8). N-acetyl- cysteine was not detected in plasma or lavage fluid. Plasma concentrations of Methods cysteine, the main metabolite of N- All patients were undergoing routine diagnos- acetylcysteine and a precursor of tic bronchoscopy for the investigation ofa lung reduced glutathione, were greater in the tumour. Three groups ofpatients were studied. groups receiving treatment (groups 2 Group 1 (n = 10) acted as a control group and and 3) than in group 1. Cysteine concen- received no N-acetylcysteine. Groups 2 and 3 trations in lavage fluid were similar in (both n = 12) received N-acetylcysteine in a the three groups. Concentrations of single daily dose of 600 mg for five days. reduced glutathione were greater in both Bronchoalveolar lavage was performed either http://thorax.bmj.com/ plasma and lavage fluid in group 2 than 1-3 hours (group 2) or 16-20 hours (group 3) in group 1. These data suggest that N- after the last dose of N-acetylcysteine. The acetylcysteine given by mouth is rapidly patients were randomly assigned to the groups. deacetylated to cysteine, with resulting One patient in each group had chronic obstruc- increases in the concentrations of cys- tive pulmonary disease and none had pulmon- teine in plasma and of reduced gluta- ary fibrosis. Informed consent was given by all thione in plasma and the airways, which the patients and permission was obtained from thus temporarily increase the antioxi- our local ethics committee. on September 26, 2021 by guest. Protected copyright. dant capacity of the lung. SAMPLES Plasma and bronchoalveolar lavage samples An imbalance may occur in the distal air- were obtained from three groups of patients: spaces of the lung between oxidants and anti- group 1 (n = 8, six male; mean age 70 (range 35- oxidants and may have a role in the patho- 77) years), consisting of three smokers, three genesis of emphysema.'2 Oxidants, produced ex-smokers, and two non-smokers; group 2 by inflammatory cells during endogenous (n = 9, eight male; mean age 65 (range 48-73) metabolism, xenobiotic insult, or both, may years), consisting of six smokers, and three cause degradation of lung connective tissue' ex-smokers; group 3 (n = 10, eight male; and inactivate antiproteases.34 Enhancement mean age 59 (range 31-71) years), consisting of Department of of the antioxidant capacity of both blood and two smokers, six ex-smokers, and two non- Biochemistry, University of the distal airspaces may therefore prevent smokers. Edinburgh Medical oxidative damage to the lung, though the role School, Edinburgh of an oxidant-antioxidant imbalance in the Bronchoalveolar lavagefluid M M E Bridgeman remains to be performed under M Marsden pathogenesis of lung injury Fibreoptic bronchoscopy was A P Ryle proved.5 local anaesthesia (2% topical lignocaine) after Respiratory Medicine During the past 20 years the antioxidant N- premedication with diamorphine and atropine. Unit, City Hospital, acetylcysteine has been used to treat patients The bronchoscope was wedged in a segmental Edinburgh EH10 5SB with chronic obstructive pulmonary disease.6 bronchus in the lung contralateral to the W MacNee D C Flenley* Long term oral administration of N-acetyl- tumour and 30 ml aliquots of warmed sterile cysteine reduces the number of acute exacer- saline (to a total volume of 240 ml) were Reprint requests to: Dr Bridgeman bations in patients with chronic obstructive instilled and recovered. The volumes instilled Accepted 20 October 1990 disease,78 though the mechanism underlying and returned were recorded. Lavage fluid was *Died March 1989. this effect remains unclear. N-acetylcysteine immediately filtered through four sterile gauze 40 Bridgeman, Marsden, MacNee, Flenley, Ryle swabs and then centrifuged at 4°C for 10 The elution solvent A was 8-5% (v/v) aceto- minutes at 150 g to remove cells. The super- nitrile, 0-25% (v/v) acetic acid, and 0-25% natant was removed and centrifuged again at (v/v) perchloric acid, pH 3-65; and solvent B 4°C for 10 minutes at 1400 g, to produce was 75% (v/v) acetonitrile in distilled water. Thorax: first published as 10.1136/thx.46.1.39 on 1 January 1991. Downloaded from completely cell free fluid. Isocractic conditions (100% A) were main- tained for 14 minutes, then changed to 100% B Blood samples for 10 minutes and thereafter immediately Venous blood samples (5 ml) were drawn into returned to 100% A for re-equilibration for six lithium heparin at the time of bronchoalveolar minutes. The flow rate was maintained at 1 ml/ lavage and centrifuged at 4°C for 10 minutes at min. 800 g to obtain plasma. Standard solutions of cysteine, reduced glutathione, and N-acetylcysteine were pre- ASSAYS pared daily with phosphate buffered saline (pH Thiol determinations 7 4) in the same way as the test solutions. The Concentrations of cysteine, reduced glutath- limit of the detection was 5 pmol on column. ione and free N-acetylcysteine were The coefficient ofvariation ofthe assay was 5% measured.'3 Immediately after centrifugation for repeated analyses of standard solutions (n the sample of plasma or lavage fluid (100 dl) = 20). was mixed with 8 mM monobromobimane (Thiolyte, Calbiochem) in 50 mM N-ethyl- Other assays morpholine, pH 8-0 (100 pl), and phosphate Albumin and ac,-proteinase inhibitor con- buffered saline, pH 7-4 (10 p1). Mono- centrations were determined with an enzyme bromobimane is relatively insoluble in aqueous linked immunosorbent assay (ELISA) with solutions and was predissolved in a small unconjugated (Cappel Laboratories, Dyna- amount of acetonitrile. The sample was stored tech) and peroxidase conjugated (Dakopatts) in the dark at room temperature for five antisera to human albumin and a,-proteinase minutes and then acidified to stop the reaction inhibitor. 14 The coefficient ofvariation for both by the addition of 100% (w/v) trichloroacetic assays was 5%. Catalase activity was deter- acid. The precipitated protein was removed by mined spectrophotometrically. 11'7 Tests for centrifugation for 10 minutes with a microfuge haemolysis were performed on plasma samples (Microcentaur, MSE) at high speed. Aliquots with diagnostics kit No 525 (Sigma). Lactate of the supernatants (100 pl) were applied to a dehydrogenase activity was determined by high pressure liquid chromatography column. an ultraviolet kit method (Boehringer- Mannheim). Chromatography The chromatographic separation of the thiol- Statistics monobromobimane derivatives was achieved Analyses were performed on duplicates and http://thorax.bmj.com/ using a Waters (Milford, Massachusetts) mean values were obtained. Mean values of Nova-Pak steel column (3-9 x 150 mm) packed different groups were compared by the with 4 pm octadodecyl silica reversed phase non-parametric Wilcoxon rank sum test for material. The analytical column was protected unpaired data. by a small Waters Guard-Pak precolumn packed with the same material. The chroma- tographic system consisted of two Model 410 Results pumps, an automated gradient controller, a N-acetylcysteine was not detected in plasma or on September 26, 2021 by guest. Protected copyright. Waters intelligent sampler processor model lavage fluid. 710 (automatic injection system), and a data module (M730) for peak integration. A fluores- PLASMA FINDINGS cence detector, model 420, was used for peak Plasma cysteine concentrations were higher detection (all from Waters). in groups 2 and 3 than in control patients (p < 0-05: table). Reduced glutathione concen- trations differed significantly only between group 2 and group 1 (table). Percentage recovery and albumin concentrations ofbronchoalveolar lavagefluid, cysteine and reducedglutathione concentrations in plasma and lavage fluid, and lavage fluid: plasma cysteine andglutathione ratios in three groups ofpatients (mean (SEM) LAVAGE FLUID FINDINGS values) Because of the uncertain, and probably vari- able, dilution of the epithelial lining fluid that Group I Group 2 Group 3 occurs during bronchoalveolar lavage, concen- % recovery of lavage fluid 59-0 (3-3) 51-1 (4 0) 48-9 (7-1) trations of the thiols in lavage fluid were also Lavage fluid albumin (mg/i) 27-3 (5-0) 28-5 (5-2) 24-2 (2-8) expressed in relation to albumin concentra- tions. Cysteine:albumin ratios in broncho- CYSTEINE alveolar fluid did not differ In lavage fluid (pmol/1) 0 1 (01)* 0-3 (0 1)* 0 1 lavage significantly (0-1)* from those of the control group in either group In plasma (pmol/1) 3 5 (0 3) 8-1 (1-0) 5-3 (0-5) 2 or group 3, though they tended to be higher in Lavage fluid: plasmaratio 0040A04 0-02 group 2 (fig 1).