Effects of Aminoguanidine, an Inhibitor of Inducible Nitric Oxide Synthase, on Nitric Oxide Production and Its Metabolites in Healthy Control Subjects, Healthy Smokers, and COPD Patients

Original Research COPD

Effects of Aminoguanidine, an Inhibitor of Inducible Nitric Oxide Synthase, on Nitric Oxide Production and Its Metabolites in Healthy Control Subjects, Healthy Smokers, and COPO Patients* Caterina Brindicci, MD, PhD; Kazuhiro Ito, PhD; Olga Torre, MD; Peter J. Barnes, DM, DSc; and Sergei A. Kharitonov, MD, PhD

Background: Nitric oxide (NO) is produced by resident and inflammatory cells in the respiratory tract by the enzyme NO synthase (NOS), which exists in three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS. NO production is increased in patients with COPD, and the production of NO under oxidative stress conditions generates reactive nitrogen species that may amplify the inflammatory response in COPD. Methods: To examine the role of increased NO in COPD, we administered a relatively selective iNOS inhibitor, aminoguanidine, by nebulization in a double-blind, placebo-controlled study in COPD patients, healthy smokers, and healthy nonsmoking subjects. We investigated whether aminoguanidine had any effect on exhaled NO produced in the central lung (flux of NO from the airways UNO] and peripheral lungs (concentration of NO in peripheral lung [CALv], on NO metabolites (nitrite [N0 2 -]lnitrate [N03-], peroxinitrite [ONOO-], nitrotyrosine), and on a marker of oxidative stress (8-isoprostane) in exhaled breath condensate (EBC) and in sputum. Results: Aminoguanidine administration resulted in a significant reduction in JNO compared with administration of the saline solution control in healthy subjects, smokers, and COPD patients. CALV in smokers and in COPD patients was not completely inhibited 1 h after aminoguanidine inhalation, in marked contrast to previous results in asthma. Moreover, ONOO- and N0 2 -/N03levels were also increased in EBC and in sputum of smokers and COPD and were not completely inhibited following aminoguanidine inhalation. 8-lsoprostane levels were also increased in smokers and in COPD patients but were not reduced after aminoguanidine inhalation. Conclusions: These results suggest that the constitutive NOS isoform as well as iNOS might be involved in NO release and contribute to the high CALV and ONOO- production in patients with COPD. Trial registration: Clinicaltrials.gov Identifier: NCTOO180635. (CHEST 2009; 135:353-367) Key words: nitric oxide; nitric oxide metabolites; nitric oxide svnthase inhibitor; nitrosative stress; peripheral inflammation Abbreviations: CALV = concentration of NO in peripheral lung; CI = confidence interval; EBC = exhaled breath condensate; iNOS = inducible nitric oxide synthase; JNO = flux of nitric oxide from the airways, nNOS = neuronal nitric oxide synthase; NO = nitric oxide; N0 2 - = nitrite; N0 3 - = nitrate; NOS = nitric oxide synthase; O 2 - = superoxide anions; ONOO~- = peroxynitrite; ppb = parts per billion; RNS = reactive nitrogen species

It

has been postulated that elevated bronchial nitric oxide (NO) [flux of NO oxide from the airways ONO)] levels in asthma are due to an upregulation of inducible nitric oxide synthase (iNOS), I and we have previously shown that a relatively selective iNOS inhibitor, aminoguanidine, markedly

www.chestjournal.org

reduces JNO in asthmatic patients." In patients with COPD, increased levels of exhaled NO have been detected-", however, it is not clear whether this is due to iNOS up-regulation. Although iNOS has been the principal nitric oxide synthase (NOS) isoform considered as a therapeutic CHEST /135/2/ FEBRUARY, 2009

353

target, the role of the constitutive NOS isoforms in airways disease is hecoming increasingly important. The paradigm of constitutive NOS and iNOS isoforms has been modified from its original conception: although neuronal NOS (nNOS) and endothelial NOS are constitutively expressed, it is now clear that their activity can be regulated by various factors." The importance of constitutive NOS and iNOS isoforms as enzymatic sources of the NO in exhaled breath remains matter of debate. Indeed, interpretation of studies of NOS inhibition is limited by the lack of selectivity of the agent used. However, a number of selective inhibitors have been recentlv developed and used as pharmacologic tools ..5 Th~ most widely used have been NG-monomethylL-arginine,fi N omega-nitro-L-arginine and its methyl ester prodrug NG-nitro-L-arginine methyl ester.i" and aminoguanidine.v " ! A selective iNOS inhibitor (SC-.51) considerably reduced exhaled NO levels in asthmatic patients.I? and another selective and potent iNOS inhibitor (GW274 1.50) has also been tested in asthma.F' Since exhaled NO arises from hath airway and alveolar regions, we investigated the effects of aminoguanidine on JNO and concentration of NO in peripheral lung (CALV) in COPO patients compared to healthy smokers and nonsmokers. Under physiologic conditions, NO is unstable, reacting with oxygen to form oxides of nitrogen, nitrite (N0 2 -) and nitrate (NO.3 -), and with superoxide anion (0 2 -) to form peroxynitrite (ONOO-). ONOO- also reacts with tyrosine residues in protein to form nitrotyrosine derivatives. 14 The effect of aminoguanidine on NO products in exhaled breath condensate (EBC) and in sputum was also investigated together with the effects on 8-isoprostane, a stable marker of oxidative stress, in the same subject population. MATERIALS AND METHODS Suhjects

Study subject demographics are shown in Tahle 1. Ten patients had COPD diagnost'd according to the Global Initiative for *From the Section of Airway Disease, National Heart and Lung Institute, Imperial College, London, UK. Financial support was provided by the National Heart and Lung Institute. Imperial College, London, UK. The authors declare no conflicts of interest related to this studv. Manuscript received April 10. 20013; revision accepted August 5, 20013. !.l-p]1roduction ofthis article is pr.ohibited without written permission from the American Collegt' of Chest Physicians (www.chestjoumaL orglmisc/reprints.shtml) . Correspondence to: Sergei A. Kharitonoc, MD, PhD, Section of Airiuu] Disease, National Heart and Lung Institute, Imperial College London, Docchouse St, London S\\13 6Lr, UK; e-mail: s.kha ritonoctioimpcrial.ac.uk: 001: lO.1378/chest.08-0964 354

Chronic Obstructive Lung Disease guidelines.'; They all had chronic cough, sputum production, dyspnea, a smoking history (> 10 pack-years), postbronchodilator FEV, <1)0% of predicted and FEV /FVC < 70% of predicted, and negative bronchodilator reversibility test results. Healthy smokers (n = 10) had no history of respiratory disease, had normal spirometry results, and had a smoking history> 10 pack-vrs, Ten healthy nonatopic nonsmokers with negative skin-prick test results to house dust mite iDennatoptiaooide« pteronusstnus), cat hair, grass pollen, and Aspergillus jrllnigatus, with normal lung function were also enrolled. Exclusion criteria included a respiratory tract infection or COPD exacerbation in the preceding 6 weeks. The study was approved by the Ethics Committee of the Royal Brompton Hospital, and all the subjects gave written consent. Study Design

This was a double-blind, randomized, placebo-controlled, twoperiod, cross-over study. The study design is described in the data supplement. Methods Measurement of NO Exhaled at Multiple Exhalation Flau:s

NO at multiple exhalation flows was measured, and the flow-independent NO parameters confined to the two compartments, the conducting airways (JNO) and the peripheral airways (CALV), were calculate-d as previously described."·'h.17 See also the data supplement for a description of the measurements performed, Lung Function Tests

FEV" FVC, and FEV/FVC were measured with a dry spirometer (Vitalograph Ltd; Buckingham, UK), and the best of three maneuvers was expressed as an absolute value (in liters) and as a percentage of the predicted value. For each subject, a second FVC maneuver was performed 20 min after inhaling 400 fLg of albuterol by metered-dose inhaler. TIlt' reversibility test result with bronchodilator was considered positive when an increase in FEV, 2: 12% and 2: 200 mL over baseline was found.!" EBC

Subjects were asked to breath tidallv through a mouthpiece connected to the condenser (EcoScreen; Jaeger; \Vurzburg, Germany) while wearing a nose clip for a period of 10 min. EBC samples were collecn-cl and stored at - I)O°C in a 2-mL, sterile centrifuge tubes. Sputum Induction and Processing

Sputum was induced and sputum samples processed as previously descnbed.!''-" See also the data supplement for a detailed description of the procedures. ONOO- Measurements ONOO- was detected using a modification of the method by \Vang and [oseph'" and Ito et al"" based on oxidation of 2',7' -dichlorofluon-scin bv ONOO- in EBC Set' also the data supplement for a dl'tail~d description of the measurements performed. Original Research

Table I-Subject Demographics* Variables

Subjects, No.

ICS Use, No.

Male/Female Conder, No.

Age, yr

Smoking History, Pack/yr

Healthy control subjects Healthy smokers COPD

10 10 10

NA NA 6

6/4 5/5 6/4

57.2:!:: 6 59.7:!:: 5 62.4:!:: 3

36:!:: 12 41:!:: 10

*Data are presented as mean z SD unless otherwise stated. NA

=

NA

not applicable.

8-Isoprostane Measurements

considered significant. Statistical software (Prism 4; Graph Pad Software; San Diego, CAl was used for all statistical tests.

8-Isoprostane in EBC and sputum supernatant was quantified by a competitive enzyme immunoassay (Cayman Europe; Boldon, UK) as previously described. 2.1 .24

RESULTS

Effects of Aminoguanidine/Placebo on Lung Function Test Results and BP

N0 2-/NO,J-

N0 2 - /NO.) - released in EBC and sputum supernatant were measured (Nitrate/Nitrite Colorimetric Assay Kit; AXXORA Ltd; Nottingham, UK) as previously descrtbed.s"

Lung function and BP remained unchanged after aminoguanidine and placebo inhalation at any time point in all subjects (Tables 2, 3, respectively). No adverse effects were observed.

Nitroturosine Measurements Nitrotyrosine in EBC and sputum supernatant was measured with a specific enzyme immunoassay (Cayman Chemical; Ann Arbor, MI) as previously described."

Effects of Aminoguanidine/Placebo on Different Biomarkers The effects of aminoguanidine and placebo on different biomarkers in healthy volunteers, healthy smokers, and COPD patients are summarized in Tables 4-6 and Tables 7-9, respectively, and in Figures mentioned below.

Statistical Analysis No formal calculation of the sample size was performed because the aim of this study was exploratory and no hypotheses were tested. Data are presented as mean z SEM or median (range) and medians with 95% confidence intervals (CIs). Nonparametric tests were applied because the distribution of these variables was not known and there were insufficient data for normal distribution analysis. Differences between the variables in the three groups were analyzed by Mann-Whitney tests. Wilcoxon matched-pairs test was used to compare the variables at different time points. Correlations between variables were sought using Spearman rank correlation test. A p value <0.05 was

Effects of Aminoguanidine/Placebo on JNO and CALV

JNO values before aminoguanidine inhalation in healthy control subjects were higher compared with

Table 2-Effects of Aminoguanidine Inhalation in the Studied Groups* Aminoguanidine Variables Healthy control subjects FEV b % predicted FVC, % predicted FEV,/FVC, % Systolic/diastolic BP, mm Hg Smokers FEV" % predicted FVC, % predicted FEV,/FVC, % Systolic/diastolic BP, mm Hg COPD FEV" % predicted FVC, % predicted FEV,/FVC, % Systolic/diastolic BP, mm Hg

Baseline

30 min

60 min

120 min

24 h

100.8 :!:: 3.3 100.9 :!:: 2.6 82.69:!:: 1.7 140/80

101.3 :!:: 3.1 99:!:: 3.6 83.1 :!:: 1.2 140/80

99.2:!:: 1.6 98:!:: 4 84 :!:: 1.2 145/90

98.3:!:: 3 100.3 :!:: 2.3 81 :!:: 2.1 130/80

lOLl :!:: 1.6 98.3:!:: 2 83.1 :!:: 2.1 130/80

92.7:!:: 2.5 96.7 :!:: 2.8 78.8:!:: 1.2

95:!:: 4 98:!:: 3 78:!:: 1.6

93 :!:: 2.4 99 :!:: 2.4 79:!:: 3 100nO

95:!:: 5 98:!:: 1.4 . 75.2:!:: 2

93 :!:: 1.6 94:!:: 1.5 7.9:!:: 1 115/80

56:!:: 2.5 67.6 :!:: 4.1 61.4 :!:: 1.6

57:!:: 2.3 68 :!:: 3.4 62.6:!:: 2.5

55:!:: 2 70.2:!:: 3.5 63.3 :!:: 3.1

130nO

125nO

120nO

106nO

120n8

58.2 :!:: 5.4 70.7:!:: 4.7 64.4:!:: 3.7 127/80

57:!:: 5 68:!:: 4.2 63.5:!:: 2.3 130/80

110/80

*Data are presented as mean z SEM unless otherwise indicated. www.chestjournal.org

CHEST /135/2/ FEBRUARY, 2009

355

Table 3-Effects of Placebo Inhalation in the Studied Groups* Plaeebo Variables Healthy control subjects FEV], % predicted FVC, % predicted FEV/FVC,% Systolic/diastolic Bp, nun Hg Smokers FEV], 'If predicted FVC, % predicted FEV/FVC,% Systolic/diastolic ur. nun fig COpD FEV]. % predicted FVC, % predicted FEV/FVC,% Systolic-diastolic Bl', nun Hg

so

Baseline

:30 min

97.1 2: :3.0 })9.6 2: .3.0 S2.:3 2: 1.6 1:3.5/S2

WI 2: 2.1i 98.72: l.7 S4 2: 1.7 130/80

})6.2 2: 1.6 101 2: :3.4 S:3.6 2: :3.6 1:30/S2

})IoU 2: 2.6 S7.22: 2.1

}):3.S 2: :3.0 })6.2 2: 2.}) 7S..5 2: 1.1 110/74

})5 2: 4 })7 2: 3.6 77.1 2: 2..5 120nS

62.}) 2: .5.6 77.72:2.7 66.2 2: .5.9 13.5n.5

632:2 76.2 2: 2.2 64.3 2: .3.1 1351S0

min

120 min

24 h

100 2: 2..5

1.30/S0

WI 2: :3 100.2 2: 2.2 S4 2: 2.1 1.30/S0

}):3 2: 2.4 })6.2 2: 2..5 76.:3 2: 1.4 120/S0

% 2: :3.2 })41 2: 2.2 77.1 2: I.S 110/S0

})4 2: :3.:3 })6 2: 1.:3 7S.1 2: 1.:3 llSnS

6:3.1 2: 2.3 74.32: 1.7 62.62: :3.1 1:30/S0

63 2: 1.6 76.2 2: 1.2 65.1 2: 2.1 130/S0

64 2: :3 75.S 2: 2.1 67.1 2: 1.2 120lS0

*Data an- presented as mean 2: SEM uuless otherwise indicated.

healthy smokers and COPD patients (787.4 ± 58.2 pUs, 358.2 ± 60.1 pUs, and 514.1 ± 60.6 pUs, respectively; p < 0.05). Aminoguanidine caused a significant (p < 0.05) reduction in JNO levels in healthy control subjects and healthy smokers, and remained low for 2 h and returned to baseline value within 24 h. In COPD patients, JNO levels remained low also the day after aminoguanidine inhalation (Fig 1, top, A). CALV values were higher in COPD patients compared with healthy control subjects (3.6 ± 01 parts per billion [ppb] vs 1..5 ± 0.1 ppb; p < 0.0001) and not Significantly different compared with healthy smokers (3.1 ± 0.3 ppb). CALV levels were significantly reduced (p < 0.0.5) by aminoguanidine in healthy smokers 60 min after inhalation and remained low for 2 h before returning to baseline value. CALV values in COPD patients were reduced 30 min after aminoguanidine inhalation (p < 0.005) and remained low (p < 0.05) the following day (Fig 2, top, A). No differences were seen in JNO and CALV values after placebo inhalation in any group (Fig 1, bottom, B, and Fig 2, bottom, B, respectively). A negative correlation was found in all subjects in both visits at baseline between CALV and FEV, percentage of predicted: p < 0.0001, r = - 0.7; FVC percentage of predicted: p < 0.005, r = - 0..5; and FEV /FVC percentage of predicted: p < 0.00.5, r = - 0.6. Effects

ofAnunoguanuline/Placebo Oil

ONOO~

ONOO- levels before aminoguanidine inhalation were Significantly higher in COPD patients compared to healthy control subjects and healthy smokers (295.1 ± ,52.7 nmol, 37..3 ± 9.8 nmol, and 87.4 ± 356

1.3.8 nmol, respectively; p < 0.0001). Aminoguanidine did not reduce ONOO- values in healthy control subjects but significantly decreased ONOOlevels in smokers (.33.3 ± 7 nmol, p < 0.05) and COPD patients (99.9 ± 34.4 nmol, p < 0.(001) [Fig 3, top, A]. No differences were seen in 0 N 00 - after placebo inhalation in any group (Fig 3, bottom, B). There was a negative correlation in all subjects at baseline between ON 00 - and FEV 1 percentage of predicted: P < 0.0001, r = - 0.8; FVC percentage of predicted: p < 0.00,5, r = - 0.6; and FEV/FVC percentage of predicted: p = <0.0001, r = - 0.8. CALV and ONOO- correlated positively in all subjects in both visits at baseline (before aminoguanidine: p < 0.0001, r = 0.7; before placebo: P < 0.0001, r = 0.7) and after placebo inhalation (p < 0.0001, r = 0.7), whereas no correlation was found after aminoguanidine inhalation (Fig 4). Effects ofAmillogllallidillelP[acebo 8-1sop rostane

011

8-1soprostalle in EBC

8-Isoprostane values before aminoguanidine inhalation were higher in COPD patients (43.7 ± 2.8 pglmL) compared with healthy smokers (24.9 ± 2.2 pglmL; P < 0.05) and healthy control subjects (11..5 ± 0.9 pglmL; p < 0.00(1). Aminoguanidine did not reduce 8-isoprostane levels in healthy control subjects, neither in smokers nor in COPD patients at any time point (Fig 5, top, A). No differences were seen in 8-isoprostane levels after placebo in the study groups (Fig 5, bottom, B). A negative correlation was found at baseline in all subjects between 8-isoprostane and FEV, percentage of predicted: P < 0.0001, r = - 0.8; FVC perOriginal Research

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78H ::': 58.2 1.50::': 0.10 37.3::': 9.8 11.5::': 0.9 17.7::': 2.1 7.2 ::': 0.8

(740.0; 645-930) (1.4; 1.2-1.8) (34.1; 13.1-61.5) (11.1; 9.4-13.7) (16.0; 12.9-22.4) (7; 5.2-9.1)

Baseline

30 min

358.2 ::': 60.1 (348.9; 211-505) 200.0 ::': 30..5 (185.5; 125-275) 3.10 ::': 0.3 (2.9; 2.3-3.8) 2.40 ::': 0.3 (2.8; 1.6-3.2) 87.4 ::': 13.8 (100.8; 53.5-121.3) 24.9 ::': 2.2 (23..5; 19Jh30) 24.7::': 2.2 (25.5; 19.6-29.7) 7.6 ::': 1.0 (7.5; 5.2-9.9)

Baseline

*Data are presented as mean::': SEM (median; 95% CI).

CALV,ppb ONOO', nmol 8-Isoprostane EBC, pglmL NO/NO:l EBC, u.mol Nitrotyrosine EBC, nglmL Sputum 8-Isoprostane sputum, pglmL NO/NO, sputum, umol Nitrotyrosine sputum, nglmL

JNO, pUs

Smokers Breath

Variables

640.2 ::': 94.6 (670.0; 408-870 1.47::': 0.09 (1.4; 1.2-1.7)

30 min

15.0::': 1.9 (15.5; 10.5-19.4) 438.2 ::': 24.7 (456; 382.3-494) 119.2 ::': 10.2 (117; 96-142.3)

523.3::': 79.3 (450.0; 329-714) 1.48::': 0.10 (1.4; 1.1-1.8) 28.3::': 5.7 (31.1; 14.2-42.4) 10.9::': 1.3 (9.4; 7.9-13.9) 12.3 ::': 1.2 (11.0; 9.5-15) 7.0::': 0.8 (6.5; 5.1-8.8)

60 min

Aminoguanidine

12.5 ::': 1.3 (12.0; 9.6-15.5) 18.3::': 2.1 (16.5; 13.4-2.3.0 7.9::': 0.9 (7.5; 5.7-10)

11.6::': 1.16 (11.0; 9-14.3) 17.1::': 2.5 (14.5; 11.3-22.8) 7.5 ::': 0.9 (7; 5.3-9.6)

91.5::': 8.7 (88.5; 71.8-110 .549.3 ::': 63.2 (525.0; 406-692.5) 505.6::': 88.2 (500.0; 305.9-705)

191.3::': 26.5 (211; 126-256) 1.78::': 0.2 (1.7; 1.3-2.2) 33.3::': 7.0 (41.0; 16.0-50.5) 26.4 ::': 1.9 (26.0; 22-30.8) 19.4 ::': 1.9 (18..5; 14.9-2.3.1\) 6.3::': 0.7 (6; 4.6-7.9)

60 min

Aminoguanidine

23.5 ::': 2.3 (22.5; 18.3-28.7) 22.3::': 2.0 (22.0; 17.6-26.9) 8.5 ::': 1.0 (7.5; 6.2-10.7)

269.8::': 43.5 (234.9; 163-376) 1.84::': 0.2 (1.7; 1.4-2.2)

120 min

21\.2::': 2.4 (27.5; 22.7-33.6) 25.9 ::': 2.4 (25..5; 20.2-31..5) 9.3 ::': 1.2 (9; 6..5 -12)

35.5.1::': 64..5 (273.2; 197-513) 2.40::': 0.3 (2.6; 1.7-3.1)

24 h

799.9::': 81.2 (748.0; 601-999) 1.47::': 0.09 (1.4; 1.2-1.7)

24 h

532.7::': 81.6 (460.0; 333-733) 1.45::': 0.10 (1.4; 1.2-1.7)

120 min

Table 5-Effects of Aminoguanidine Inhalation on Studied Biomarkers at Different Time Points in Healthy Smokers*

*Data are presented as mean::': SEM (median; 95% CI).

CALV, ppb ONOO', nmol 8-Isoprostane EBC, pglmL NO/NO, EBC, p.mol Nitrotyrosine EBC, nglmL Sputum 8-Isoprostane sputum, pglmL NO/N0 3 sputum, urnol Nitrotyrosine sputum, nglmL

JNO,

Healthy control subjects Breath

Variables

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Table 4-Effects of Aminoguanidine Inhalation on Studied Biomarkers at Different Time Points in Healthy Volunteers*

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514.1 :!: 60.6 (510.8; :368-660) :3.6 :!: 0.1 (:3.7; :3.2-4) 295.1 :!: .52.7 (2:39.5; 176---414) 4:3.7:!: 2.8 (45.5; .372~50.1) :37.8 :!: :3.4(:3,5.5; .30-4.5..5) 24.6 :!: 2.2 (26.0; 19.4-29.7)

Baseline

Baseline

*Data are presented as mean:!: SEM (median; 9,5% en.

Healthy control subjects Breath 827 ..5 :!: 74.0 (7:37.1; 646-1,009) JNO, pUs 1.6:!: 0.1 (1.7; 1.4-1.9) CALV, ppb 41.:3 :!: 12.0 (:3:3.:3; 11.8-70.9) ONOO', nmol/L 1O.6:!: l.l (9.1; 8.0-1.3..3) 8-lsoprostane EBe, pglmL 16.6:!: 1.7 (17.8; 12..5 -20.7) NO/NO, EBe, u.mol 6.9 :!: 0.7 (7..5; .5.1-8.6) Nitrotyrosine EBe, nglmL Sputum 8-lsoprostane sputum, pglmL NO/NO;l sputum, p.mol Nitrotyrosine sputum, nglmL

Variables

:318.4 :!: :38.9 (282..5; 2:30-4(6) 1.9 :!: 0.2 (2; 1.4-2.4)

:30 min

177.0:!: 14.1 (192; 145-208.9) 678.9 :!: 98.8 (549.5; 455-902..5) 888.4 :!: 110.2 (78:3.5; 6:39.1-11:38)

290.6:!: 19.8 (287.9; 246-.'3:35) 2.0 :!: 0.2 (2; 1.4-2.6) 99.9:!: :34.4 (:3.5.7; 22.1-177.8) 46.9 :!: :3.0 (49..5; :39.:3~5.'3.8) 2:3.0 :!: 1.8 (22.5; 18.7-27.2) 21.6 :!: 2 (21.0; 17-26.1)

60 min

60 min

Placebo

1:3.8 :!: 2.0 (14..5; 9.2-18..'3) 449.1 :!: 49.0 (484.5; :3:38.2~560) 104.1 :!: 9.9 (98.0; 81.6-126..5)

797.1 :!: 65.8 (722.8; 6:36-9.58) 796.8 :!: 58.:3 (7:36.0; 654-9:39) 1.5 :!: 0.1 (1.6; 1.1-1.8) 1.4:!: 0.1 (1..5; 1.2-1.7) :37.9 :!: 10 (:3,5.0; 12.4-6.3.4) 1O.9:!: l.l (9.2; 8..3-1.3.5) 16.8 :!: 1..5 (18.0; 1:3.:3-20.2) 7.6 :!: 0.8 (7..5; 5.6-9..5)

:30 min

------

24 h

4:3.6 :!: 2.4 (4.5; :38.1-49.1) 28.5 :!: 1.9 (29; 24--:32.9) 2:3.0:!: 1.9 (2.5.0; 18.7-27.2)

24 h

--,

9.9 :!: 1.2 (8.5; 7.0-12.7) 1.5.7:!: 1.4 (16.0; 12.4-18.9) 6.0:!: 1.1 (4..5; :3.4-8.6)

1O.5:!: 1.4 (10.0; 7.:3-1.3.8) 17.:3 :!: 2.5 (15.0; 11..5-2:3) 7.1 :!: 0.4 (7.0; 6.0-8.1)

789.9:!: 50.5 (750.0; 666-914) 824.8:!: 72.6 (7:38.0; 647-1,(02) 1.:3:!: 0.1 (1.:3; 1.0-1..5) 1.:3:!: 0.1 (1.4; 1.0-1..5)

120 min

45.0:!: t.s (4:3; 40.6---49.:3) :39.4 :!: 2.5 (:38..5; :3:3.7-45.1) 25.6 :!: 2.2 (28.0; 20.,5-.'30.6)

285.4 :!: 28.0 (297..5; 222-:349) 41:3.8:!: 74.8 (:359.2; 245-58:3) 2.1 :!: 0.2 (2; 1.6-2.6) 2.8 :!: 0.1 (2.9; 2.4--:3.2)

120 min

Table 7-Effects of Placebo Inhalation on Studied Biomarkers at Different Time Points in Healthy Volunteers*

*Data arc presented as mean:!: SEM (median; 95% CI).

JNG,

pUs CALV,ppb ONOO·, nmol S-Isoprostane- EBC, pglmL NO/NO, EBe, u.mol Nitrotyrosine EBC, nglmL Sputum 8-lsoprostane sputum, pglmL NO/NO;l sputum, u.mol Nitrotyrosine sputum, nglmL

COPD Breath

Variables

Aminoguanidine

Table 6-Effects of Aminoguanidine Inhalation on Studied Biomarkers at Different Time Points in COPD*

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Baseline

*Data are presented as mean ::t SEM (median; 95% CI).

JND, pUs

95.2 ::t 9.0 (95.0; 74.8-ll5.6) 742.6 ::t 60.9 (700.0; 604.7-880.5 516.1 ::t 86.1 (491.0; 321-7ll)

379.7 ::t 59.0 (402.0; 253-524) 2.5 ::t 0.18 (2.7; 2.0-2.9) 72.5 ::t 9.4 (62.4; 49.3-95.6) 25.5 ::t 2.1 (25.2; 20.5-30.4) 34.3::t 1.2 (35.0; 31.5-37.1) 11.3 ::t 1.8 (9.0; 7.1-15.5)

60 min

30 min

(504.2; 363-790) (3.4; 2.8-3.9) (240.6; 143.2-403) (44.5; 36.1-50.8) (44.5; 40.1-51.0) (27.5; 20.8-30.3) 196.0::t 13.1 (197.5; 166-225.8) 1,124 ::t 102.5 (1,l30.0; 891.7-1,355) 1,017::t llO (911.0; 768-1,266)

576.5 ::t 94.2 3.4 ::t 0.2 273.2 ::t 57.4 43.50 ::t 3.2 45.6 ::t 2.4 25.6 ::t 2.0

60 min

Placebo

24 h

23.6 ::t 1.6 (23.0; 19.8-27.3) 26.7::t 2.1 (27.5; 21.9-31.4) 1O.8::t 1.4 (11.0; 7.5-14.0)

24 h

45.2 ::t 3.2 (47.0; 37.9--52.5) 39.0 ::t 2.7 (38.0; 32.7-45.2) 22.7 ::t 2.2 (21.0; 17.6-27.7)

43.2 ::t 3.7 (42.7; 34.8-51.6) 39.8 ::t 4.2 (39.0; 30.2-49.3) 24.2 ::t 2.3 (23.5; 18.8-29.5)

509.9 ::t 76.2 (480.0; 337-682) 503.7 ::t 59.5 (.532.8; 369-638) 3.7 ::t 0.2 (3.5; 3.0-4.4) 3.8 ::t 0.3 (3.6; 3.0-4.4)

120 min

27.1 ::t 1.8 (25.5; 22.8-31.3) 32.1 ::t 2.2 (33.0; 26.9-37.2) 12.4 ::t 2.0 (12.0; 7.8-16.9)

378.5 ::t 80.1 (396.0; 182-575) 373.8::t 62.6 (410.0; 220--527) 2.6::t 0.17 (2.9; 2.2-3.1) 3.0 ::t 0.21 (2.8;2.5-3.5)

120 min

Table 9-Effects of Placebo Inhalation on Studied Biomarkers at Different Time Points in COPD*

543.0 ::t 79.5 (532.1; 363-723) 523.2 ::t 73.9 (515.9; 356-690) 3.5 ::t 0.2 (3.7; 3.0-4.1) 3.6 ::t 0.2 (3.5; 3-4.1) CALV, ppb 261.2 ::t 52.7 (245.7; 141.9-380.4) ONOO', nmol 43.4 ::t 3.1 (46.5; 34.9-51.0) 8-lsoprostane EBC, pglmL 37.7 ::t 2.8 (38.5; 31.2-44.1) NO!N0 3 EBC, umol 26.5 ::t 2.5 (24.0; 20.6-32.3) Nitrotyrosine EBC, nglmL Sputum 8-lsoprostane sputum, pglmL NO!N0 3 sputum, u.mol Nitrotyrosine sputum, nglmL

COPD Breath

Variables

30 min

419.0::t 103.3 (411.8; 166-671) 404.7::t 96.0 (326.0; 170-ti40) 2.7 ::t 0.16 (2.6; 2.3-3.1) 2.6::t 0.13 (2.6; 2.2-2.9) 65.8 ::t 7.9 (67.4; 46.4-85.2) 24.8 ::t 1.7 (23.5; 20.9--28.7) 27.2 ::t 1.9 (27.5; 22.8-31.5) ILl ::t Ll (11.5; 8.5-13.7)

Baseline

*Data are presented as mean ::t SEM (median; 95% CI).

CALV, ppb ONOO', nmol 8-lsoprostane EBC, pglmL NO!N03 EBC, urnol Nitrotyrosine EBC, nglmL Sputum 8-lsoprostane sputum, pglmL NO!N03 sputum, umol Nitrotyrosine sputum, nglmL

JND, pUs

Smokers Breath

Variables

Placebo

Table 8-Effects of Placebo Inhalation on Studied Biomarkers at Different Time Points in Healthy Smokers*

A 1000

.

750

:::r

.S:

500

..,z 0

250

*

0

o

.

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* 60min

120min

24hrs

81000

___ healthy controls .....-smokers

750

--+-COPD

:::r

.S: 500

..,z

Healthy Smokers controls

COPO

Healthy Smokers controls

COPO

8

0

250

o o

30rrin

60min

120min

24hrs

FICURE 1. Effect of aminoguanidine (top, A) and placebo thottom, B) inhalation on central airway-derived NO (INO) in all the study groups at different time points. Data are expressed as mean:':: SEM; *p < 0.0.5, #p < 0.005 vs haseline value.

centage of predicted: p < 0.00.5, r = - 0.7; and FEV/FVC percentage of predicted: p < 0.0001, r = - 0.8. A positive correlation was found between 8-isoprostane levels and smoking history in healthy smokers and COPD patients (p < 0.0001, r = 0.9).

o o z o

FIGURE 3. ONOO- levels before (white bars) and after (black bars) aminoguanidine inhalation and before (grey bars) [top, A] and after (black bars) [botto/ll, B] placebo inhalation in all study groups. Data are ex!)ressed as mean:':: SEM *p < 0.05, **p < 0.0001 vs values )efClft' aminoguanidine inhalation.

8-Isoprostane in SPUtUIII

8-Isoprostane levels in sputum were higher in COPD patients than in healthy smokers and non-

A 3

i

.S:2 >

J

o

30min

60min 120min

24hrs

8

--- ..

..... healthy controls .....-smokers

--+-COPD

•

•

•

o o

30min

60min 120min

24hrs

2. Effect of aminoguauidine (top, A) and placebo (CALV) in all the study groups at different time points. Data are expressed as mean::':: SEM; *p < 0.05. #p < 0.005 vs baseline value. FICURE

ibott.nn, B) inhalation on peripheral NO

360

smokers (196 ± 13 pg/mL, 95.2 ± 9 pglmL, and 13.8 ± 2 pglmL, respectively; p < 0.0001). Aminoguanidine did not decrease 8-isoprostane levels in any of the groups (Fig 6, top, A). A negative correlation was observed between 8-isoprostane and FEV I percentage of predicted: p < 0.0001, r = - 0.66; FVC percentage of predicted: p < 0.0001, r = - 0.65; and FEV/FVC percentage of predicted: p < 0.0001, r = - 0.66; and a positive correlation was seen between 8-isoprostane and smoking history (p < 0.05, r = 0.69; Fig 7). Effects of AminoguanidinelPlacebo on N0 2 - INO.'] N0 2 - IN0 3 - in EBC

N0 2 - IN0 3 - levels before aminoguanidine inhalation were higher in COPD (37.8 ± 3.4 u.mol) compared with healthy smokers (24.7 ± 2.2 umol, p < 0.0.5) and healthy control subjects (17.7 ± 2.1 urnol, p < 0.0001). Aminoguanidine caused a reduction (p < 0.05) in N0 2 - IN 0.3 - levels in healthy control subjects and healthy smokers 60 min after aminoguanidine inhalation followed by an increase within 2 h. In COPD patients, aminoguanidine reduced (p < 0.0.5) N0 2 - IN0 3 - levels at 60 min Original Research

A6

r=O.7

p
••

i

I i i

o

200

,

400 600 ONOO"(nM)

800

r=O.7

•

p
• o

,

o

,

200

,

400

i

i

600

800

&Omin

120min

24hrs

FIGURE .5. Effects of aminoguanidine (top, A) and placebo (bottom, B) on 8-isoprostane levels in EBC in all study groups at different time points. Data are expressed as mean ± SEM.

ONOO"(nM) r=O.7

p
ed: p < 0.05, r = - 0.6; and FEV /FVC percentage of predicted: p < 0.05, r = - 0.6. N0 2 - IN0 3 - in Sputum

• i

o

,

,

,

,

200

400

600

800

ONOO" (nM) FIGURE 4. Correlation between CALV and ONOO- concentrations before aminoguanidine inhalation (top, A), before placebo inhalation (center, B), and 60 min after placebo inhalation (bottom, C) in all study groups.

and 120 min after inhalation, and the values returned to baseline levels within the next day (Fig 8, top, A). No differences were seen in N0 2 - IN0 3 -levels after placebo in healthy control subjects. There was an increase in N0 2 -IN03 - levels (p < 0.05) in healthy smokers and in COPD patients 60 min after placebo inhalation, and the values returned to baseline within 120 min (Fig 8, bottom, B). A negative correlation was found at baseline in all subjects between N0 2 -I N0 3 - levels and FEV I percentage of predicted: p = < 0.05, r = - 0.6; FVC percentage of predictwww.chestjournal.org

N0 2 - IN0 3 - levels were higher in healthy smokers than in nonsmokers (742.6 ± 60.9 u.mol vs 449.1 ± 49 umol, p < 0.05) and further increased in COPD patients (1,124 ± 102 urnol, p < 0.00(1). Aminoguanidine decreased N02 -IN 0: 3 levels in healthy control subjects, in healthy smokers, and in COPD patients (438.2 ± 24.7 u.mol, .549.3 ± 63.2 umol, and 678.9 ± 98.8 umol, respectively; p < 0.05) [Fig 6, center, B]. There was a negative correlation between NO 2 -IN 0:3 - levels and FEV I percentage of predicted. p < 0.005, r = - 0.5; FVC percentage of predicted: p = 0.0009, r = - 0..5; and FEV/ FVC percentage of predicted: p < 0.0.5, r = - 0.6. Effects of AminoguanidinelP[aceho on Nitrotyrosine Nitrotyrosine in EBC

Nitrotyrosine levels before aminoguanidine inhalation were higher in COPD patients (24.6 ± 2.2 nglmL; p < 0.0001) compared to healthy smokers (7.2 ± 0.8 nglmL) and healthy control subjects (8.7 ± 1.2 nglmL) [Fig 9, top, A]. No differences were seen in nitrotyrosine levels after aminoguanidine and placebo in all subjects (Fig 9, top, A, and bottom, B, respectively). A negative correlation was CHEST /135/2/ FEBRUARY. 2009

361

-

A 250

~

--t~

I to

--~

~

200

Q.

-. I I)

i

--

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200

c::

150

cu til

0

100

Q.

100

p<0.001

r=0.9

0

.!!!

50

co

0 Smokers

o

capo

1250

o

25 50 pack/years

75

FIGlJHE 7. Correlation lx-tweeu S-lsoprostan« levels in EBC and in spntnm and smoking history (pack/vears) in healthy smokers and in COPD patients.

1000 750 500 250 0

C

o

E C,

Healthy Controls

B

300

Healthy Controls

Smokers

capo

1400

104.1 ::!::: 9.9 nglmL; P < 0.000l). The levels were further higher in COPD patients (l,Oli ::!::: 110 ngl ml., P < 0.00(1) and were significantly reduced by aminoguanidine (888.4::!::: 110.2 nglmL; P < 0.0.5) [Fig 6, hottoui, Cl There was a negative correlation between nitrotyrosine levels and FEV 1 percentage of predicted: P < 0.0001, r = - 0.68; FVC percentage of predicted: P < (WOOl, r = - 0.6; and FEV/FVC percentage of predicted: P < 0.0001, r = - 0.67.

1200

DISCUSSION

~

NO, together with its products NO z - INO: 3 -, ONOO-, and nitrotyrosine, may be used to measure

~

I.. ...

:!

0 Healthy Controls

Smokers

capo

FIGl'HE 6. 1'0/). A: S-lsoprostanl' lewIs in sputum I h after placebo (white bars) and aminoguanidine (black bars) inhalation in all studv gronps. Data are expressed as mean:+: SEM. Center, B: 1\;02 - /,\;C}, - levels in sputum I h after placebo (white bars) and aminoguanidin(' (bla.-k bars) inhalation in all studv groups. Data an- c-xpn-ssed as mean z SE!v1; *p < 0.0.5 \'s placebo. Bottom. C: Nitrotvrosm« levels in sputnm 1 h after placebo (white bars) and aminoguanidiuc (black bars) inhalation in all the studv gronps. Data are expressed as mean:+: SE\1.

found at baseline in all subjects between nitrotyrosine levels and FEV I percentage of predicted: P = < 0.00,5, r = - 0.6; FVC percentage of predicted: P < 0.0001, r = - 0.7; and FEV/FVC percentage of predicted: P < 0.0,5, r = - 0.6. Nitrotu rosine ill Sputum

Nitrotyrosine levels were higher in healthy smokers than in nonsmokers (.516.1::!::: 86.1 nglmL vs 362

o

8

50

i"40 ::t CI)

30

'c

20

~

!'c

10

o

60min

120min

24hrs

*

.. o

60min

___ healthy controls --.- smokers

. 120min

--CaPD

'" 24hrs

FIGl'HE S. Effects of aminognanidine (top. A) and placebo ibattom, B) ou N0 2-/NO:,- !L·n·ls in EBC in all stndy groups at different time points. Data arc expn-ssed as meun z; SEM; *p < 0.0,5, #p < 0.00,5 vs baseline value. Original Research

A

i

B

o

!::::::::a i 60nin

120nin

i 24hrs

_____ Healthy controls --.- Smokers

-+-COPD

I

I

o

&Orrin

•

•

• 12Om1n

.. 24hrs

FIGURE 9. Effects of aminozuanidine (top, A) and placebo (hottom, B) on nitrotyrosine levels in EBC in all the study groups

at different time points. Data are expressed as rnean z; SEM.

nitrative stress, and 8-isoprostane may be used to measure oxidative stress in capo. In this study, we confirm that CALV is elevated in patients with COPU3; thus, the source of NO in capo is likely to be the peripheral lung rather than the main bronchi, in contrast to asthma, in which JNO is predominantly elevated.s? We have also shown that a single dose of aminoguanidine significantly reduced JNO levels in healthy volunteers, healthy smokers, and capo patients, and decreased CALv in healthy smokers and capo patients. The effect on JNO is not surprising because iNOS expression is regulated by both transcriptional and posttranslational mechanisms. Di Stefano et al21> demonstrated an increased expression of p65, the major subunit of nuclear factor-xh, in the bronchial epithelium of smokers with capo and normal lung function. Augusti et al29 found that nuclear factor-ids activation and iN as induction occur in skeletal muscle of capo patients. These findings suggest the involvement of iNOS in the smoking-induced airway inflammation. Our study also confirms our previous evidence- of increased CALv in peripheral lung of healthy smokers and capo patients, and shows for the first time a decreasing effect of aminoguanidine on CALV levels in these patients. Interestingly, comparing the effects of aminoguanidine in asthma and in capo, we observed a significant increase in JNO in asthma followed by a marked reduction (approximately 90%) after aminoguaniwww.chestjournal.org

dine inhalation.? in contrast to capo, in which the increased CALV decreased following aminoguanidine inhalation by only 44.5%. Alveolar macrophages are increased in capo patients; this could account for the elevated CALV levels because these cells are able to express iNOS in response to proinflammatory cytokines.?? However, because aminoguanidine has only lO-fold in vitro selectivity? for iN as vs nNOS together with a 45% inhibition following its inhalation, our data suggest that aminoguanidine may also inhibit other NOS isoforms (eg, nNOS) in the lung. nNOS expression is significantly increased in peripheral lungs from capo patients compared with nonsmokers.P' suggesting that nNOS may also contribute to the increase in CALV in capo, but the reduced inhibitory effect of aminoguanidine against this isoform may explain why CALV was not reduced more effectively in capo patients. Increased expression of nNOS has also been reported in skeletal muscles of capo patients by Barreiro et al;32 who showed that the severity of airway obstruction correlates with vastus lateralis nNOS protein levels, suggesting that nNOS might contribute to the development of nitrosative stress in capo. The lack of a complete inhibitory effect of nebulized aminoguanidine on capo could be explained by an insufficient dmg delivery in peripheral airways, the most critical in terms of gas exchange. However, this is unlikely because the nebulizer used delivers particles in the range of 2 to 5 urn in diameter, which should reach in a significant proportion the peripheral airways. Nevertheless, it cannot be excluded that the dose used was insufficient to influence CALV values. In fact, although the dose administered was the same as in previous studies.s" this dosage was established empirically. As the decrease in CALV lasted 24 h in capo patients, the duration of effects in lowering CALV following a single dose suggests that the dosing regimen could be adjusted to permit daily dosing. In addition, the inhibitor was well tolerated and had no effect on BP or heart rate, suggesting either that aminoguanidine does not inhibit endothelial NOS due to reasonable selectivity, or that inhaled aminoguanidine has no systemic effect. Several biomarkers of oxidative and nitrosative stress have been detected in EBC in patients with COPD.:33 For the evaluation of oxidative/nitrosative stress, BAL, induced sputum, and EBC have been employed:34.:3.5 However, there is no "gold standard" for noninvasive assessment of nitration and oxidation. Thus, it is unclear which technique is "better" or "more accurate." This study presented the opportunity to evaluate the concentrations of some biomarkers in different biological fluids obtained by CHEST /135/2/ FEBRUARY. 2009

363

noninvasive techniques of sampling lung secretions. Higher levels of all markers were observed in sputum supernatant as compared to EBC, and several possible explanations might account for such results. Induced sputum is likely to be less diluted with water than EBC; in fact, it is known that EBC consists of aerosolized particles diluted with a large amounts of condensed water vapor.?" In this regard, it is acknowledged that a proper analytical comparison between compounds in different biological fluids would require the knowledge of dilution factors of each fluid in order to compare the absolute number of moles rather than concentrations. However, for hoth induced sputum and EBC, at this stage information regarding a "gold standard" factor of dilution is not yet available.F Some attempts have been made to assess the dilution of alveolar lining fluid in EBC samples and to standardize by using exhaled volume,:1S exhaled ions,:lfUfJ,40 urea,:16,41 protein concentration.t- or conductance of lyophilized samples as "internal standards,">' or by using external dilution markers. Furthermore, it would be of interest having a comparison marker (unaltered hy oxidation), to compare the oxidation products with. This would not be a dilution marker but rather a compound that could serve as an internal monitor for oxidative imbalance. In addition, the sputum can be considered as a "biogel,' with a high concentration of different types of cells and enzymes.v' while EBC can be considered an aqueous solution, with a low concentration of salts, lipids, and proteins. Therefore, the degradation and/or biotransformation of markers in the medium could be different. At last, it can be speculated that the technique of induced sputum samples predominantly the proximal airways, where most of the airway secretions are likely to be more concentrated, whereas EBC is thought to more closely reflect the composition of alveolar lining fluid because of the much greater surface area from which generation of aerosols may OCCUr. 44 A practical conclusion coming from these data is that EBC and sputum must be considered as independent measurements. 8-Isoprostane, a marker of oxidative stress, is formed in vivo hy free-radical peroxidation of arachidonic acid but may also be produced by cyclooxygenase-l and cyclooxygenase-2 activation in some cells and tissue in vitro; however, in vivo 8-isoprostane enzymatic synthesis in humans seems to be negligible. 4 .5 Oxidative stress is a major component of inflammation in COPD and cigarette smoking is the major risk factor for COPD. COPD patients and healthy smokers were matched for smoking habit, but the former had twofold-higher 8-isoprostane levels, indicating a higher level of oxidant stress in COPD. 8-Isoprostane levels in sputum were significantly elevated in both 364

healthy smokers and COPD patients and significantly correlated with lung function, as previously reported;" Since 8-isoprostane is a chemically stable end product of a metabolic pathway, it was not surprising not to find changes after aminoguanidine inhalation. A simultaneous higher production of O 2 - and NO may occur in COPD, potentially leading to the increased production of ONOO-, a powerful oxidant responsible of many cytotoxic effects.F However the cellular source of ONOO- in COPD is unclear. A previous study4s found that the production of ONOO- was increased in airway macrophages and neutrophils in COPD patients compared with healthy control subjects. Kanazawa and Yoshikawa-? found that ONOO- stress Significantly correlated with airflow limitation in COPD, suggesting that the severity of COPD might progress as a result of decreases in ONOO- inhibitory activity. In this regard, because epithelial cells are an important source of antioxidant capacities, the reduction of ONOO- inhibitory activity may reflect the injury or death of epithelial cells. In tum, an inadequate supply of ONOO- inhibitory capacities would render epithelial cells vulnerable to ONOO- -mediated cellular injury. Moreover, the half-life of ONOO- has heen reported>" to be only 1 s at pH 7.4 and 37°C. We have detected for the fist time ONOO- levels in EBC and sputum, and the values correlated with CALV and with airflow limitation. As ONOO- is formed by reaction of O 2 - and NO, increased NO from peripheral lung might cause the elevation of ONOO-. Aminoguanidine decreased 0 N00 - levels in COPD patients and healthy smokers, confirming an important role for enhanced ONOO- stress in COPD pathogenesis. Nevertheless, the levels of N0 2 - INO:1 - were elevated in EBC of COPD patients and healthy smokers compared with nonsmokers. This is in agreement with previous studies·5 1,52 showing an increase in NO metabolites in plasma of smokers. It is tempting to speculate that the observed high levels are related to an increase of scavenged NO gas into N0 2 - and NO:l - due to the high concentration of reactive oxygen species generated in the particulate-free gas phase of the main stream in cigarette smoke. In healthy smokers, we observed high N0 2 - INO:1 levels in EBC and sputum, suggesting an increased nitrosative stress induced by smoke, which may force the transformation into stahle bioactive oxidation end products of NO pathway. In patients with COPD, NO z - INO: 3 - values in EBC and sputum were higher, possibly related to an enhanced oxidative stress. In fact, in airways of patients with COPD, there is an increased number of neutrophils, which Original Research

produce numerous free radicals, causing the oxidation of part of NO gas into NO 2 -IN 0:3 - • After placebo inhalation, we observed an increase in N0 2 - IN0 3 - in healthy smokers and COPD patients, which might be explained by the fact that NO is trapped at the epithelial surface of human lower respiratory tract in formation of bioequivalent oxides of nitrogen and the trapping mechanism is redox sensitive.P Superoxide released from activated neutrophils can decrease NO by conversion to NO.3 -, suggesting a potential mechanism for modulation of NO in vivo. 54 Smoking-induced acute oxidative stress may therefore lead to transiently increased oxidation in the airways, facilitating oxidation of NO and resulting in an increase in NO oxidative end products. Conversely, after aminoguanidine, NO metabolites are likely to be reduced and slowly return to baseline levels; obviously, this recovery might be more difficult in COPD. Nitrotyrosine is considered a useful biomarker of reactive nitrogen species (RNS) generated during airway inflammation and two pathways are thought to be involved in RNS-dependent tyrosine nitration: one is ONOO--mediated tyrosine nitration.P and the other is nitration mediated by peroxidase-dependent N0 2 - oxidation.P" Aminoguanidine did not have an effect on nitrotyrosine levels; this could be due to the fact that the formation during inflammatory processes of NOderived RNS that are capable of inducing nitration involves multiple distinct mechanisms so that the nitrotyrosine production is the end product of this complex metabolic pathway. Alternatively, nitrotyrosine release may be also a step of the degradation of nitrated protein. If more complex pathways are involved, multiple doses of aminoguanidine will be required to inhibit nitrotyrosine release. Increased nitrotyrosine formation has been detected in biopsy samples of different respiratory disorders including COPD,57 suggesting the involvement of nitrosative stress in the pathogenesis of this disease, particularly in the progressive impairment of lung function. Tyrosine nitration could cause airflow obstruction via amplification of nitrativeloxidative stress-mediated inflammatory process-" and degradation of extracellular matrix, resulting in alveolar wall destruction and small airways collapse.s? Ichinose et al48 reported that the number of macrophages and neutrophils nitrotyrosine positive are increased in the sputum of COPD patients compared with healthy control subjects. Furthermore, we found increased amounts of nitrotyrosine in EBC and sputum in COPD, suggesting that both central and peripheral lung compartment are exposed to nitrosative stress. It remains to be established www.chestjournal.org

whether nitrotyrosine is merely a biomarker of RNS or whether it contributes to cellular dysfunction and development of airway inflammation in COPD. In this respect, the discovery of enzymatic "nitrotyrosine denitrase" activitf°·61 may further hint at the potential significance of nitration (and denitration) as a signaling of the inflammatory mechanisrn.V The increased production of NO from peripheral airways, its incomplete inhibition following aminoguanidine inhalation, and the increased ONOOlevels not completely suppressed by aminoguanidine in COPD patients suggest that there might be a different "NOS-induced NO production" in asthma and in COPD. Studies combining novel agents with direct measurements of inflammatory markers should in time provide further information regarding the significance of iNOS/nNOS dysregulation in COPD and the potential of NOS isoforms as a target for therapeutic intervention.

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19

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21 22 23

24

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