Online data supplement. Matthias Griese, Jan Ramakers, Angela Krasselt, Vitaliy Starosta, Silke van

Transcription

1 Online data supplement Improvement of Alveolar Glutathione and Lung Function but Not Oxidative State in Cystic Fibrosis Matthias Griese, Jan Ramakers, Angela Krasselt, Vitaliy Starosta, Silke van Koningsbruggen, Rainald Fischer, Felix Ratjen, Bernhard Müllinger, Rudolf M. Huber, Konrad Maier, Ernst Rietschel, and Gerhard Scheuch

2 Additional details on Methods and Results Subjects and study design 21 adolescents or adults with CF participated in the study (detailed clinical data in Tab. E1 below). All medications were kept constant 4 weeks before and during the study period. All subjects were in a clinically stable condition. In particular, they did not have symptoms like increased frequency, duration, and intensity of cough, increased or new onset of sputum production, change in sputum appearance, new onset or increased hemoptysis, increased shortness of breath and decreased exercise tolerance, decrease in overall well-being, increased fatigue, weakness, fever, poor appetite. In addition there were no physical signs like increased work of breathing, increased respiratory rate; new onset or increased crackles on chest examination; increased air trapping; fever or weight loss. Also there was no decrease in FEV 1 of 10% or greater compared with their baseline in the preceding 6 months and no decrease in SaO 2. Initially the intrathoracic deposition achieved with the nebulizer used, was assessed in 6 CF patients. Intrapulmonary deposition Intrathoracic lung deposition was determined with 99m Tc-labelled Fe 3 O 4 aerosol particles (d ae = 3.1 µm)(e1). A standardized inhalation was performed by means of an AKITA inhalation device (INAMED, Gauting, Germany). The AKITA device used in this study is a unit that connects to a regular Pari LCplus nebulizer and directs the nebulization via regulated generation of pressure and flow to the nebulizer and by this also the breathing pattern, i.e. duration and breathing depth. The pre-set breathing pattern used in this study was achieved by an individualized inhalation volume of 75% of the patient s inspiratory capacity, which was 31

3 calculated from of the difference of vital capacity (IVC) and expiratory reserve volume (ERV). The flow rate was fixed to 200 ml/s during in- and exhalation without endinspiratory breath hold time. After end of the inhalation procedure regional deposition and peripheral lung deposition were determined (E2). The deposition efficiency was expressed as % of the emitted dose and was defined as the fraction of the aerosol particles that had left the nebulizer and were deposited in the lungs. The device was programmed via an individual SmartCard and all inhalation maneuvers, the respective time points and durations were recorded for later analysis, i.e. compliance with the inhalation schedule. The individual results for compliance are given in Table E1. Assessment of the deposition of glutathione by bronchoalveolar lavages The second part of the study assessed deposition of glutathione in 17 CF patients with mild to moderate lung disease defined as a FEV 1 > 45% of predicted. All patients were clinically stable for at least 6 weeks. Patients were not eligible for this study, if they had allergic bronchopulmonary aspergillosis or were using N-acethylcysteine as a mucolytic agent. The intrapulmonary changes in glutathione concentrations were measured by BAL (see Tab. E1, supplementary material, for details on the subjects). Three to seven days later, inhalation with glutathione was started at 3 times daily doses of 300 or 450 mg for 14 days. 4 subjects received doses of 300 mg; BAL was performed 1 h after the last inhalation. 13 subjects received 450 mg glutathione t.i.d., of which nine were assessed by BAL 1 h and four 12 h after the last inhalation. The study was approved by the ethics committee of the participating centers and informed consent from the patients or parents and young adults was obtained before the study. Estimation of the amount of glutathione to be nebulized before the start of the study 32

4 Roum et al. (1999) had found in patients with cystic fibrosis basal glutathione-epithelial lining fluid level of 56 ± 12 µm (n = 7; Mean ± SE)(E3). After inhalation of 2 x 600 mg glutathione per day for 3 days, the mean glutathione level in BAL after 60 min was 174 ± 46 µm; p < 0.05, i.e. an increase of about 120 µm. In patients with COPD we had previously found a deposition rate of about 80% (unpublished). This deposition rate was about 4-fold better than the usual rate of about 20 %, that we assumed for the nebulization in the study of Roum et al. We assumed and also proved (see results from the deposition study) that such a high rate can also be achieved in CF patients. We further assumed a hourly reduction of glutathione by about 50 %, necessitating dosing at least three times a day. The goal set were peak level after 1 hour of at least about 350 µm, i.e. equivalent to an increase by about 300 µm. This range was derived from the normal controls of the Roum group who had level of about 278 ± 21 µm (E4), Cantin et al. (1987) and Behr et al. (1995) had found level of about 400 ± 21 µm and 568 ± 53 µm in healthy controls (E5,E6). Thus with 300 mg of glutathione (this is half of a single dosage of Roum et al, but the same daily dose, i.e. using 3 x 300 mg here and 2 x 600 mg by Roum et al, resulting both in 1200 mg/day), and with a 4 fold better deposition we expected at least twice the level of the Roum et al study (i.e. about 240 µm). Taking into consideration that we dosed 3 times a day and that we had the same daily dosage, however not for only 3, but 14 days, an optimistic estimate was a 4 fold increase of that seen by Roum et al, i.e. about 480 µm. Therefore the 300 mg per dosage were our starting point and we planned to study 4 subjects with this dosage first. In case that our estimates turned out to be too pessimistic, we planned to step down to 150 mg/inhalation. In case we did not find the estimated increases and 33

5 thus could deliver more glutathione, we would increase the dosage to 450 mg/inhalation. We thought that this would be an upper amount of glutathione, as judged from osmolarity, amount of fluid to be nebulized and time necessary for that. Inhalation of the study drug Reduced glutathione sodium salt for intravenous application (Biomedica Foscana, Ferentino, Italy) was reconstituted at a concentration of 200 or 300 mg/ml, resulting in a tonicity of 1301 mosm/l or 1952 mosm/l), and a ph at 200 mg/ml of 7.0 ± 0.05 and at 300 mg/ml of 7.1 ± 0.05 (n = 5). The ph was stable over time at room temperature in an open vial for up to 30 min. The solution was filled into the reservoir of a Pari LC Star nebulizer (Pari, Starnberg, Germany) driven by the AKITA inhalation device. The total emitted volume was set to 1.5 ml which was delivered in an individualized number of breaths according to the patients vital capacity, as described above. This volume was selected to limit the inhalation time to 20 min or less. 2.5 ml of drug were filled in the reservoir and the amount left in the nebulizer after delivering the predefined amount to the lungs, was in the order of 1 ml. Because the AKITA inhalation device controls the amount of drug that has left the nebulizer and controls the inhalation flow rate and the inhalation volume of the patient, in all patients a similar amount of the drug has been deposited. The glutathione solution to be nebulized and the aerosol, after recovery within a cascade impactor, were analyzed for reduced glutathione and oxidized glutathione. Before nebulization reduced glutathione was 95.5 ± 0.5 % of total, and after nebulization 96.4 ± 0.4 % of total (n=4). This demonstrated that there was no oxidation from nebulization of the solution. Bronchoalveolar lavage, sample preparation and biochemical analysis 34

6 Flexible fiberoptic bronchoscopy and bronchoalveolar lavage were performed at baseline and 14 days after start of the inhalation of glutathione. Bronchoalveolar lavage was performed via a flexible bronchoscope with an outer diameter of 4.9 mm diameter. Topical anesthesia was achieved via inhalation with 2-4 ml of a 4 % lidocaine solution prior to BAL and 3-5 ml of 2 % lidocaine as needed while introducing the bronchoscope into the airways. Patients were sedated with midazolam ( mg/kg body weight) for the bronchoscopic procedure. The bronchoscope was wedged in the middle lobe or one of its segments in all patients. BAL was performed with 4 ml/kg body weight normal saline warmed to body temperature in 4 aliquots of 1 ml/kg bw, up to a maximum of 50 ml per aliquot. All manipulations of the samples were done immediately and on ice. The first aliquot of the recovered BAL fluid was treated separately; all other samples were pooled for analysis. The recovery of BAL fluid from the initial BAL and from the second BAL 14 days after start of the glutathione inhalations, did not differ significantly and were 43 ± 4 % and 49 ± 4 % of the instilled volume. After recovery of the BAL fluid, it was immediately put on ice, filtered through gauze, centrifuged at 500g for 10 min at +4 C in a pre-cooled centrifuge and a pre-cooled rotor, and ice cold 10 % trichloracetic acid was added to equal volume aliquots of the supernatant, mixed, snapfrozen by liquid nitrogen, and stored at -70 C for analysis. In pre-experiments we had assessed the reproducibility and stability of samples prepared this way with storage time. Once deeply frozen, storage time was always minimized (< 5 days) and the material appeared to be relatively stable in its acidic environment and frozen at 70 C. This is similar to the experience of other authors who acidified the samples and in contrast to the native material. In BAL from adult patients with suppurative lung diseases (n=6), undergoing diagnostic lavages we observed with time a small but not significant decrease of both the concentration of 35

7 reduced and oxidized glutathione by repeated measures ANOVA. This was for reduced glutathione from 100 % to 92.2 ± 2.5 % after 1 day and to 79.4 ± 5.5 after 40 days. The actual concentrations in this set of experiments were 2097 ± 601 µmol/ml immediately, 1999 ± 642 µmol/ml after 1 day frozen at 70 and 1925 ± 687 µmol/ml after 40 days of frozen storage (P = 0.708). For oxidized and reduced glutathione together the loss was from 100 to 97.9 ± 1.1% after 1 day and to ± 1.4 % after 40 days. Here the actual concentrations were 2799 ± 676 µmol/ml immediately, 2746 ± 665 µmol/m after 1 day frozen at 70 and 2811 ± 649 µmol/ml after days of frozen storage (P= 0.113). Thus by using exactly the same conditions of sample preparation and storage the analytical procedures met the clinical and analytical purposes of this study. To address the question if changes in glutathione may occur before the cells were separated from the lavage fluid at 4 C, we investigated the stability of the lavage which was immediately treated with trichloracetic acid, i.e. before the cells were removed. This resulted in little but not much higher values and more scatter of the data, possibly from damage to the cells and liberation of glutathione into the supernatant from the trichloracetic acid treatment. The corresponding numbers from immediate analysis of the samples, i.e. trichloracetic acid added to the BAL without prior separation of the cells by centrifugation gave 2691 ± 499 µmol/ml for reduced glutathione (+ 137 % compared to the above values) and 3584 ± 532 µmol/ml for reduced and oxidized glutathione (+ 115 % compared to the above values). Again, with storage time, there were also no differences between immediate analysis after removal of the cells and analysis after an additional day of storage at 70 C. This supported the 36

8 view that not much, if any glutathione was lost during the 10 to 15 min period of centrifugation directly after the recovery of the BAL fluid. However that period cannot be skipped in studies looking at lavages for technical reasons, i.e. to avoid the release of intracellular components into the BAL fluid by the trichloracetic acid treatment. All studies performed so far have centrifuged the BAL for cells, before treating the supernatant and storing the glutathione. An aliquot of the native BAL was used for total cell count and viability by trypan blue exclusion. From the cells collected by centrifugation cytospin preparations were made, stained according to May-Grünwald-Giemsa and by counting at least 600 cells in each subject, a differential cellular distribution was obtained. Reduced and total glutathione were measured within 5 days by RP-HPLC (E7). Total protein was determined as described (E8), the fraction of carbonyls assessed by a sensitive slot blot assay (E9). Aliquots of the samples containing 2.5 µg of protein were slot-blotted onto PVDF membranes. These were sequentially treated with 2,4-dinitrophenylhydrasine, a primary antibody specific for the 2,4-dinitrophenole group, and a peroxidase-labeled second antibody for detection. The level of the immunostaining was quantitated by densitometry. Oxidized bovine serum albumin was used as a standard. The protein thiols were measured using a colorimetric assay kit (t-6060) from Molecular probes, Eugene, OR, USA. Briefly, the protein thiols present in 50 µg of protein, were determined based on the reduction of a disulfide-inhibited derivative of papain by protein thiols or inorganic sulfides, which release stoichiometrically the active enzyme. The activity of the enzyme is then measured using the chromogenic papain substrate, N-benzoyl- L-arginine, p-nitroanilide (L-BAPNA). The L-cysteine at known concentrations from 0 to 1.5 µm serves as a standard in the reaction, and Ellman's reagent is used for accurately determining the actual thiol concentration of L-cysteine standard solutions. Cystamine, when added to the 37

9 reaction, permits the detection of poorly accessible thiols on proteins or thiols that have high pka values. Cystamine, a disulfide, undergoes an exchange reaction with protein thiols, yielding 2- mercaptoethylamine (cysteamine), which then releases active papain. The lipid peroxides were measured by the peroxoquant quantitative peroxide assay kit from Pierce Biotechnology Incorp., Rockford, IL, USA, according to the manufacturer. Urea was determined by the urease technique (E10). The assay range was from 2.5 to 40 mg/dl. Statistical analysis Individual data and means ± standard error of the mean (SE) for n independent determinations are given. Comparisons were made by the one-sided t-test, i.e. we tested for an increase in glutathione concentrations. Multiple regression analysis, Pearson correlation analysis and linear regression were performed. P < 0.05 was set as level of significance and exact P values are reported (E11). 38

10 Reference List E1. Moller W., W. Barth, M. Kohlhaufl, K. Haussinger, W. Stahlhofen, and J. Heyder Human alveolar long-term clearance of ferromagnetic iron oxide microparticles in healthy and diseased subjects. Exp.Lung Res. 27: E2. Brand, P., I. Friemel, T. Meyer, H. Schulz, J. Heyder, and K. Häußinger Total deposition of therapeutic particles during spontaneous and controlled inhalations. J Pharm Sci in press. E3. Roum, J. H., Z. Borok, N. G. McElvaney, G. J. Grimes, A. D. Bokser, R. Buhl, and R. G. Crystal Glutathione aerosol suppresses lung epithelial surface inflammatory cellderived oxidants in cystic fibrosis. J Appl Physiol. 87: E4. Roum, J., R. Buhl, N. McElvaney, Z. Borok, and R. G. Crystal Systemic deficiency of glutathione in cystic fibrosis. J.Appl.Physiol. 75: E5. Cantin, A. M., S. L. North, R. C. Hubbard, and Crystal R.G Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol. 63: E6. Behr, J., B. Degenkolb, K. Maier, B. Braun, T. Beinert, F. Krombach, C. Vogelmeier, and G. Fruhmann Increased oxidation of extracellular glutathione by bronchoalveolar inflammatory cells in diffuse fibrosing alveolitis. Eur Respir J 8: E7. Kuhn, K. S., A. I. Krasselt, and P. Fuerst Glutathione and glutathione metabolites in small tissue samples and mucosal biopsies. Clinical Chemistry 46:

11 E8. Griese, M., N. Maderlechner, P. Ahrens, and R. Kitz Surfactant proteins A and D in children with pulmonary disease due to gastroesophageal reflux. Am J Resp Crit Care Med 165: E9. Robinson C.E., A.Keshavatzian, D.S.Pasco, T.O.Frommel, D.H.Winship, and E.W.Holmes Determination of protein carbonyl groups by immunoblotting. Anal.Biochem. 266: E10. Hilliard, J. B., M. W. Konstan, and P. B. Davis Inflammatory mediators in cf patients. Methods Mol Med 70: E11. Motulsky, H Intuitive biostatistics Oxford University Press, New York. 40

12 Legend to the figures Figure E1: Scintigraphic images (Diacam, Siemens, Munich, Germany) were acquired over a 10 min period (anterior, supine) 20 to 60 min after the end of the inhalation. The numbers refer to the subjects in Tables E1 and E2. Figure E2: Peak expiratory flow in the morning and evening before the inhalation of the glutathione aerosol. The best of three efforts was recorded by the patients in a diary. Values given are means ± SEM. Linear regression was calculated, the slopes (morning: ± 0.4, evening: ± 0.3) were significantly different from zero, the P values, goodness of fit and the corresponding lines are indicated in the figures. Figure E3: Multivariate correlation analysis between the improvement of FEV 1 during the study and the clinical other variables listed in Table 1. Except for a weak negative correlation between the change in FEV 1 with treatment and the starting FEV 1, i.e. the lower the starting FEV1 was, the larger was the improvement, no other correlations were found. 41

13 Table E1. Clinical characteristics of the study subjects at baseline and compliance with the study medication during the glutathione inhalation study Subject Age Sex CFTR- FEV 1 FVC MEF 25 RV/TLC Compliance (% of (years) Mutation (% pred.) (% pred.) (% pred.) (% pred.) total dose) F df508/ df ND M df508/ ND 80.2 unknown M df508/ df M df508/ df F df508/ df * F df508/ df * M df508/ df * M R347P/ Q493x F df508/ df F df508/ df M df508/

14 unknown F df508/ unknown M df508/ df * M df508/ unknown M df508/ ND 40.6 unknown F df508/ unknown F R553/ unknown F df508/ df NA F df508/ NA unknown * M df508/ df NA 43

15 * M Unknown NA Means ± SEM *Patients 34.9 ± ± ± ± ± 8.0 NA participating in the deposition study Patients 24.2 ± ± ± ± ± ± 3.3 participating in the glutathione inhalation study (Pts 1-17) Subjects 18 to 20 did not participate in the glutathione-inhalation study and were excluded after the initial bronchoalveolar lavage. All 3 were scheduled to inhale the 450 mg dose and were randomised for the 12 h time point. Subjects 6, 7, 8, 20 participated also in the deposition study, subject 21 merely in the deposition study. NA, not applicable; ND, not done. 44

16 Table E2. Deposition of radio labelled 3 µm particles in 6 subjects with cystic fibrosis Subject Intrathoracic deposition (% of emitted aerosol) Intrathoracic deposition, peripheral (% of emitted aerosol) Intrathoracic deposition, central (% of emitted aerosol) Extrathoracic Deposition (% of emitted aerosol) Exhaled dose (% of emitted aerosol) 24 hr retention (% of intrathoracic deposition) Mean ± SEM 85.5 ± ± ± ± ± ± 3 45

17 Table E3 Bronchoalveolar concentrations of reduced glutathione, oxidised glutathione and total glutathione in patients with cystic fibrosis BAL before inhalation of glutathione BAL after 14 days of 3 times daily inhalation of glutathione Subject Reduced Oxidised Total % of Reduced Oxidised Total % of glutathione glutathione glutathione reduced glutathione glutathione glutathione reduced (µmol/l) (µmol/l) (µmol/l) glutathione (µmol/l) (µmol/l) (µmol/l) glutathione Inhaled dose 300 mg, lavage 1 hour after end of inhalation Mean ± SEM 4.01 ± ± ± ± ± ± ± ± 5 P Inhaled dose 450 mg, lavage 1 hour after end of inhalation *

18 * * Mean ± SEM 3.83 ± ± ± ± ± ± ± ± 6 P < Inhaled dose 450 mg, lavage 12 hours after end of inhalation * Mean ± SEM 1.37 ± ± ± ± ± ± ± ± 9 P * indicates the subjects also participating in the deposition study. 47

19 Table E4. Tolerance of the GSH-inhalations in 17 patients with CF, as assessed from the notes of a diary Symptom Number of subjects no complaints 7 cough 9 continous 4 only days bad taste 5 headache 2 running nose 2 48

20 Figure E Scintigram from patient 14 was very similar to those presented here, but was unfortunately lost after the end of the study

21 Figure E2 Peak expiratory flow (l/min) Morning r=0.584, P= Peak expiratory flow (l/min) Evening r=0.681, P= Time inhaling glutathione (days) 50

22 Figure E3 Starting FEV 1 (% predicted) 125 r = P = Change in FEV 1 during the study 51