Mehdi Oualha

Jean-François Boitiaux

Jean-Marc Tadié

Aurélie Cazes

Marc Riquet

Emmanuel Naline

Dominique Israël-Biet

Dr Christophe Delclaux
email: christophe.delclaux@egp.aphp.fr

Association of ex vivo vascular and bronchial dysfunctions in smokers

Keywords: zaprinast, acetylcholine, bronchial ring, arterial ring, endothelial dysfunction

Introduction

Recent researches on the pathogenesis of chronic obstructive pulmonary disease (COPD) suggest that perturbations in the vasculature may occur early in COPD [START_REF] Barr | Impaired flow-mediated dilation is associated with low pulmonary function and emphysema in exsmokers: the Emphysema and Cancer Action Project (EMCAP) Study[END_REF][START_REF] Mcallister | Arterial stiffness is independently associated with emphysema severity in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Mills | Increased arterial stiffness in patients with chronic obstructive pulmonary disease; a mechanism for increased cardiovascular risk[END_REF]. Nitric oxide (NO)-mediated, endothelium-dependent relaxation provoked by adenosine diphosphate and acetylcholine is attenuated in the excised pulmonary arteries of patients with COPD compared with those of smoking and non-smoking control subjects [START_REF] Peinado | Endothelial dysfunction in pulmonary arteries of patients with mild COPD[END_REF]. Barr and colleagues demonstrated that impaired flow-mediated dilation, assessed by brachial artery flow-mediated dilation, is associated with low pulmonary function and emphysema in ex-smokers [START_REF] Barr | Impaired flow-mediated dilation is associated with low pulmonary function and emphysema in exsmokers: the Emphysema and Cancer Action Project (EMCAP) Study[END_REF]. In this latter study, a linear relationship was demonstrated between flow-mediated dilation and postbronchodilator FEV 1 , and the magnitude of this association was the same in former smokers who had not developed COPD (FEV 1 /FVC ≥ 70%) as in the overall sample (mild to severe COPD). The authors postulated that the systemic nature of endothelial function made likely the alteration of endothelial reactivity in the pulmonary circulation. Based on this assumption, one may hypothesize that endothelial dysfunction of pulmonary arteries would be associated with impaired airway function. Accordingly, Peinado and colleagues showed that maximal relaxation of pulmonary arterial rings induced by acetylcholine is correlated with the FEV 1 /FVC ratio of COPD patients [START_REF] Peinado | Endothelial dysfunction in pulmonary arteries of patients with mild COPD[END_REF]. Inasmuch as endothelial dysfunction is associated with dysregulated nitric oxide (NO) metabolism, we speculated that NO-mediated bronchodilation would also be impaired. We therefore conducted this study to assess whether endothelial dysfunction of pulmonary arteries was associated with modifications of the level of resting tone of isolated airways and their cGMP-mediated dilation. Both parameters are NOdependent, and the former has previously been suggested to be associated with the level of airflow limitation [START_REF] Tadie | Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease[END_REF].

Material and Methods

Patients

Consecutive patients referred for lung surgical procedure (lobectomy or pneumonectomy for lung tumour) were eligible. Since impaired flow-mediated systemic dilation has been associated with low pulmonary function in former smokers, only ex-smokers were included (>15 Pack-Year). Based on the post-bronchodilator value of pre-operative pulmonary function test, patients were classified according to GOLD criteria [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF]. All patients were informed of these biological experiments (bioethics' law of august 2004). This study was approved by our Institutional Review Board.

Pharmacological studies

Just after resection, segments of pulmonary arteries and bronchi were taken from an area as far as possible from the tumour and were dissected free of parenchyma. Arterial rings were evaluated immediately after dissection, whereas bronchial rings were placed in oxygenated Krebs's solution and stored overnight at 4°C, as previously described [START_REF] Tadie | Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Henno | Pulmonary vascular dysfunction in end-stage cystic fibrosis: role of NF-kappaB and endothelin-1[END_REF][START_REF] Henno | Tobacco smoking and pulmonary endothelial dysfunction[END_REF].

Intralobar arterial rings (inside diameter of 1.5-2 mm) or bronchial rings (inside diameter 1-2 mm) were suspended on tissue hooks in 5 ml organ baths containing Krebs-Henseleit solution (NaCl, 119 mM; KCl, 5.4 mM; CaCl2, 2.5 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; NaHCO3, 25 mM; glucose, 11.7 mM), gassed with 95% O 2 : 5% CO 2 and maintained at 37°C (pH 7.4). Each preparation was connected to a force displacement transducer (Statham UF-1) and isometric tension changes recorded in a computer (acquisition system: EMKA France, Bourre, France). All conditions were run in duplicate (when the duplicate experiments show discordant values, no result is given).

Arterial experiments:

Endothelium-dependent relaxation was assessed by measuring the responses to acetylcholine (ACh, Pharmacie Centrale des Hôpitaux, Paris, France) in vessels preconstricted with Lphenylephrine dichloride (10 -5 M). Relaxation to ACh was expressed as a percentage of relaxation of phenylephrine induced tone. The absence of ACh-induced dilation or even a contractile response defined endothelial dysfunction, which constitutes a stringent definition that is based on the upper normal limit of the response in non smokers with normal lung function in our laboratory. accordingly to previous experiments done in an historical group of non smokers. We previously demonstrated that the decrease in vascular reactivity was not due to endothelial damage during manipulations [START_REF] Henno | Pulmonary vascular dysfunction in end-stage cystic fibrosis: role of NF-kappaB and endothelin-1[END_REF].

Bronchial experiments:

After the initial loading of 2.5 g and subsequent equilibrium period, an initial resting tension (RT1) was obtained, bronchial viability was assessed using 10 -3 M ACh. Then a new resting tension (RT2) was obtained, and a dose response curve to zaprinast (10 -8 to 10 -5 M) was conducted. At the end of the experiment, after an equilibrium period to resting tension, maximal dilation (10 -3 M theophylline) was assessed, as previously described [START_REF] Naline | Effects of RP 73401, a novel, potent and selective phosphodiesterase type 4 inhibitor, on contractility of human, isolated bronchial muscle[END_REF]. The absence of epithelial damage has also been checked in preliminary experiments [START_REF] Tadie | Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease[END_REF]. Results were expressed as raw values (g) or percentage of theophylline-induced relaxation, as specified.

The investigators performing bronchial experiments were blinded for the results of vascular experiments.

Statistical analyses

Results are expressed as median [interquartile range]. Data were analyzed using Statview 5.0 (SAS Institute, Inc, North Carolina). For intergroup comparisons, categorical variables were compared using the chi-squared test or Fisher's exact test, and continuous variables were compared using the Mann-Whitney U test or Kruskal Wallis test as appropriate. For intragroup comparisons, continuous variables were compared using the Wilcoxon paired test. The Spearman's rank correlation coefficient was used to examine possible correlation between two variables. A P value less than 0.05 was considered as significant.

Results

The clinical and functional characteristics of the 20 patients who were included are described in table 1. The characteristics of patients with and without dysfunction were similar. The figure 1 shows acetylcholine response of preconstricted arterial rings in patients with (n=5) and without endothelial dysfunction (n=15).

The degree of airflow limitation was significantly correlated with the level of initial resting tension (RT1) (Rho= -0.49; p=0.032), and tended to be correlated with theophylline-induced maximal dilation (Rho= -0.38, p=0.096).

The response to acetylcholine (constriction to dilation) of arterial rings was correlated with the degree of zaprinast-induced bronchodilation (figure 2), and tended to be correlated with the FEV 1 /FVC ratio (with ACh 10 -5 M, Rho=0.37, p=0.11).

The figure 3 shows that patients with endothelial dysfunction (n=5), as compared to those without dysfunction (n=15), were characterized by a higher level of resting tone after acetylcholine-induced contraction (RT2) and higher levels of tension under each single concentration of zaprinast (figure 3, upper panel). Since the level of RT2 was significantly different between the two groups of patients, their response to zaprinast was further evaluated as percentage of theophylline-induced maximal dilation (similar for the two groups). The impaired response to zaprinast is further confirmed (figure 3, lower panel), suggesting an impairment of cGMP-mediated dilation in COPD patients with endothelial dysfunction.

Discussion

Background

It has been shown that an impairment of flow-mediated dilation in systemic circulation occurs early in COPD, and is associated with airflow limitation, independently of smoking history, further suggesting a link between vascular and bronchial functions [START_REF] Barr | Impaired flow-mediated dilation is associated with low pulmonary function and emphysema in exsmokers: the Emphysema and Cancer Action Project (EMCAP) Study[END_REF]. We suggest that in former smokers, endothelial dysfunction of pulmonary arteries is associated with impairment of both spontaneous resting tone and cGMP-mediated dilation of airways, establishing a biological link between pulmonary arterial and bronchial functions. To our best knowledge, comparison of arterial and bronchial pharmacological functions obtained from the same lung sample has never been made, and the description of dysfunction of pulmonary arteries has been assessed in a very limited number of studies [START_REF] Peinado | Endothelial dysfunction in pulmonary arteries of patients with mild COPD[END_REF][START_REF] Barbera | Pulmonary hypertension in chronic obstructive pulmonary disease[END_REF][START_REF] Dinh-Xuan | Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease[END_REF]. A recent pulmonary perspective supported by the National Heart, Lung, and Blood Institute stated that there is a need for methods linking in vitro observations to in vivo disease state with regard to airway smooth muscle function [START_REF] Panettieri | Airway smooth muscle in bronchial tone, inflammation, and remodeling: basic knowledge to clinical relevance[END_REF]. Along this line we show an inverse relationship between the level of resting tone and FEV 1 , which is consistent with the fact that increased smooth muscle tone may contribute to airflow limitation in COPD, as previously suggested [START_REF] Tadie | Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Lambert | Functional significance of increased airway smooth muscle in asthma and COPD[END_REF][START_REF] Saez | Peripheral airway smooth muscle mechanics in obstructive airways disease[END_REF]. In our limited series of patients, airflow limitation was similar whether or not endothelial dysfunction was present. Several factors have been shown to contribute to systemic endothelial dysfunction, and our study was not powered to describe clinical and functional factors associated with pulmonary endothelial dysfunction.

Endothelial dysfunction

There is no consensual definition of pulmonary endothelial dysfunction, but we do think that the absence of acetylcholine-induced dilation or even contractile response constitutes a drastic definition. We observed that one quarter of our COPD patients displayed pulmonary endothelial dysfunction, which is in agreement with our subsequent results observed in a larger series [START_REF] Henno | Tobacco smoking and pulmonary endothelial dysfunction[END_REF]. No data are available in the literature for further comparison, but a ~30% incidence of pulmonary hypertension in COPD patients has been reported [START_REF] Barbera | Pulmonary hypertension in chronic obstructive pulmonary disease[END_REF]. In the study of Peinado and colleagues in mild COPD patients (mean ± SD: 24.9 ± 22.9 %), the dilator response to acetylcholine was diminished as compared to non smokers (40.1 ± 22.0 %), without reaching statistical significance. The standard deviations of their results suggest that some COPD patients depicted a constrictor response to acetylcholine while almost all non smokers have a vasodilator response. Obviously, our results need to be confirmed in a larger sample of patients due to the restricted size of our sample with endothelial dysfunction.

Bronchial dysfunction

The nil to moderate degree of cGMP-mediated bronchial dilation that is evidenced is consistent with the results of other investigators [START_REF] Lagente | A nitric oxidereleasing salbutamol elicits potent relaxant and anti-inflammatory activities[END_REF][START_REF] Rabe | Phosphodiesterase isozymes modulating inherent tone in human airways: identification and characterization[END_REF][START_REF] Torphy | Identification, characterization and functional role of phosphodiesterase isozymes in human airway smooth muscle[END_REF], emphasising the confounding effect of underlying pathology (such as endothelial dysfunction) and the modest contribution of cGMP-mediated dilation in airways. The presence of a systemic endothelial dysfunction suggests an impairment of NO-mediated relaxation in COPD. Our results further argue for a generalized impairment of NO-dependent functions, affecting both vascular and bronchial lung compartments. Airway blood flow reactivity has also been shown to be impaired in exsmokers [START_REF] Mendes | Airway blood flow reactivity in healthy smokers and in ex-smokers with or without COPD[END_REF]. Mullershausen and colleagues have demonstrated a similar mechanism of desensitization of NO/cGMP signaling in smooth muscle of both aorta and bronchi of rats, involving phosphodiesterase (PDE) 5 activation [START_REF] Mullershausen | Desensitization of NO/cGMP signaling in smooth muscle: blood vessels versus airways[END_REF], giving insight into the underlying mechanisms of generalized endothelial dysfunction. An increased expression of PDE 5 in models of pulmonary hypertension has been demonstrated [START_REF] Murray | Increased expression of the cGMP-inhibited cAMPspecific (PDE3) and cGMP binding cGMP-specific (PDE5) phosphodiesterases in models of pulmonary hypertension[END_REF].

Figure 3 shows that resting tension after ACh was higher in the group with endothelial dysfunction that could already be related to an impaired relaxation after contraction. The bronchial relaxation induced by the nonselective phosphodiesterase (PDE) inhibitor (theophylline) was similar whatever the presence or absence of impaired cGMP-dependent relaxation (see Figure 3). It first suggests that cAMP-dependent relaxation is preserved whatever the degree of impairment of cGMP-mediated relaxation. In human bronchial smooth muscle, the presence of PDE I, II, III, IV (cAMP specific) and V (cGMP specific) have been identified [START_REF] Rabe | Phosphodiesterase isozymes modulating inherent tone in human airways: identification and characterization[END_REF][START_REF] Barnes | Cyclic nucleotides and phosphodiesterases and airway function[END_REF][START_REF] Rabe | Theophylline and selective PDE inhibitors as bronchodilators and smooth muscle relaxants[END_REF]. One should have expected that theophylline-induced relaxation would be lower in presence of an impairment of cGMP relaxation. Since theophylline, in addition to its PDE inhibitory activity, is an antagonist at adenosine receptors, blockade of adenosine activity has been proposed as a possible mechanism for theophylline's effects. Accordingly, Ethier and Madison have shown that, in addition to its well-established indirect effects on smooth muscle cells, adenosine also has direct effects on contractile signalling pathways since it mobilizes intracellular calcium stores [START_REF] Ethier | Adenosine A1 receptors mediate mobilization of calcium in human bronchial smooth muscle cells[END_REF]. This may explain why theophylline has a very potent bronchodilating effects ex vivo using a high concentration.

Clinical perspectives

Establishing a formal relationship between pulmonary vascular and bronchial functions is an important issue. We further suggest that NO-related dysfunction may participate in this link.

Whether pulmonary endothelial dysfunction is associated with COPD morbidity warrants further studies. But we recently evidenced a frequent endothelial dysfunction of ex vivo pulmonary arteries of patients suffering from cystic fibrosis (that was associated with pulmonary hypertension), and we further demonstrated that criteria of mild pulmonary vascular disease were evidenced in some patients with cystic fibrosis that participated in exercise limitation [START_REF] Hubert | Exhaled nitric oxide in cystic fibrosis: relationships with airway and lung vascular impairments[END_REF].

Finally, it will deserve assessing the effects of statins on bronchial functions because reductions in COPD morbidity and mortality, and improvements in lung function, have been attributed to statin use. Insights into cellular mechanisms indicate that statins promote vasorelaxation partly by upregulating the expression of endothelial NO synthase [START_REF] Tesfamariam | The effects of HMG-CoA reductase inhibitors on endothelial function[END_REF].

Conclusion

This is the first study suggesting that endothelial dysfunction of pulmonary arteries is associated with increased resting tone and impaired cGMP-mediated dilation of airways in former smokers, suggesting common underlying mechanisms of pulmonary arterial and bronchial dysfunctions. Acetylcholine-induced dilation of arterial rings is expressed as percentage of the constrictor response to phenylephrine (PE), whereas zaprinast-induced dilation of bronchial rings is expressed as percentage of maximal response to theophylline (theo). An equivalent molar concentration (10 -5 M) for both drugs is selected for the figure, but the relationship between zaprimast (10 -5 M) response and ACh was of borderline significance for ACh 10 -7 M (p= 0.057) and significant for ACh 10 -6 M to 10 -4 M (p= 0.022, figure and p= 0.024, respectively).

Grey circles are patients with endothelial dysfunction (negative acetylcholine-induced dilation = constriction, n=5), whereas white circles are patients without endothelial dysfunction (n=15). Upper panel: Levels of tension (g) according to the condition, initial resting tone (RT1), constrictor response to acetylcholine (10 -3 M Ach), resting tone after contraction (RT2), dose response to zaprinast (10 -8 to 10 -5 M), and maximal dilation induced by theophylline (10 -3 M theo). # denotes a P value < 0.05, * denotes a P value < 0.01.

Patients without endothelial dysfunction depicted a weak but highly significant dilator response to zaprinast as compared RT-2 level: 10 -8 M zaprinast, p=0.009; 10 -7 M, p=0.020; 10 -6 M, p=0.011; 10 -5 M, p=0.002 (Wilcoxon paired test), whereas patients with dysfunction had no significant dilator response to zaprinast (p>0.05 for all concentrations, Wilcoxon test).

Lower panel: Response to zaprinast expressed as percentage of maximal theo-induced dilation (10 -8 to 10 -5 M). A significant dilator response is evidenced in patients without endothelial dysfunction (trend for 10 -7 M, significant for 10 -5 M).

Figure 1 :

 1 Figure 1: Acetylcholine-response of preconstricted arterial rings.

Figure 2 :

 2 Figure 2: Relationship between acetylcholine-induced arterial and zaprinast-induced

Figure 3 :

 3 Figure 3: Zaprinast-induced bronchodilation.

Table 1 .

 1 Characteristics of the patients. as median [interquartile range] ED denotes endothelial dysfunction; NS non significant FEV 1 denotes forced expiratory volume in one second (post-bronchodilator value)

Acknowledgments

Mehdi Oualha was supported by a grant from the Fondation pour la Recherche Médicale, Jean-François Boitiaux was supported by a grant from the Société de Pneumologie de Langue Française and Jean-Marc Tadié was supported by a grant shared by the Société de Pneumologie de Langue Française and the Société de Réanimation de Langue Française.

Conflict of Interest

The authors declare no conflict of interest.


Maria G Matera

Luigino Calzetta

Paola Rogliani

Floriana Bardaro

Clive P Page

Mario Cazzola
email: mario.cazzola@uniroma2.it

Evaluation of the effects of the R-and S-enantiomers of salbutamol on equine isolated bronchi

Keywords: salbutamol, enantiomers, equine isolated bronchi, RAO

Background: Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with some similarities to human asthma and COPD, which represents a major cause of morbidity and loss of lung performance. Salbutamol has been widely used for the treatment of human airway diseases and has usually been prepared as the racemic form of the drug. However, recently the R-enantiomer of salbutamol has been introduced into clinical practice in the treatment of asthma in humans and this has been suggested to be an improvement on the racemic form of the drug; therefore thus the S-enantiomer has been demonstrated to have adverse effects in the lung and thus using the R-enantiomer may improve the therapeutic ratio. However, little is known about the properties of the R-and S-enantiomers of salbutamol in equine airways and the present study has evaluated the relaxant effects of racemic β 2 agonists in comparison with the R-and S-enantiomers in isolated equine isolated bronchi, as well as the bronchoprotective effects of these drugs on cholinergic and histaminergic pathway.

Methods:

We have studied the effects of the R-and S-enantiomers of salbutamol on bronchi isolated from RAO-affected or unaffected horses. The first study assayed the relaxant effects of R-and S-salbutamol on isolated bronchial rings contracted with carbachol or histamine at a submaximal concentration (EC70). A second study evaluated the effects of R-and S-salbutamol on semi-logarithmic cumulative concentration response curves induced by carbachol or histamine. Specific software was used to calculate statistical significance and the appropriate sigmoidal curve-fitting model.

Results:

Neither enantiomers of salbutamol caused a relaxant effect on the sub-maximal plateau contractile effects of carbochol; in fact, both R-and S-salbutamol induced a slight, but significant contraction (P≤0.05) compared to the controls. In contrast, R-salbutamol induced a significant relaxation of bronchi precontracted with histamine (RAO-unaffected: 92.06%±2.00; RAO-affected 100.20±3.99; P≤0.01). S-salbutamol induced a weak relaxation (RAO-unaffected: 15.81%±5.65; RAO-affected1 2.36±5.15) when compared to that induced by papaverine. The incubation with either R-or S-salbutamol shifted rightward (P≤0.001) the carbachol contraction curve in RAO-unaffected bronchi, but not in RAOaffected bronchi, compared to control tissues. R-salbutamol induced a reduction in Emax values (C: 9.07gr±0.68; R-salb.: 6.36gr±0.21; P≤0.01) in normal bronchi. On the contrary it reduced the histamine potency in RAO-affected bronchi (EC50 7.10 µM ±0.35, P<0.001).

The incubation with S-salbutamol shifted leftward the histamine concentration curve in both normal bronchi (C: 7.00µM±0.29; S-salb.: 2.25µM±0.19; P≤0.001) and bronchi from RAOaffected horses (C: 2.80µM±0.26; S-salb.: 1.50µM±0.80; P≤0.05).

Conclusion:

Our studies have demonstrated that S-salbutamol elicited a modest increase in contraction of equine airway smooth muscle induced by carbachol and induced a significant hyperresponsiveness to histamine. These results confirm the ability of the Senantiomer of salbutamol to potentiate the contractile effect of certain spasmogens on airway smooth muscle. Such an adverse effect would be determined in the airways of horses with RAO and suggest that if salbutamol is to be used in the treatment of symptoms of RAO in horses, the R-enantiomer, rather than the racemic mixture should be considered.

Introduction

There is an increasing amount of evidence demonstrating an association of inhaled β 2 agonists with a rise in asthma-related deaths and life-threatening experiences, as well as an ability of β 2 -agonists to induce bronchial hyperresponsiveness when used clinically, particularly when administered as monotherapy [START_REF] Cazzola | Safety of long-acting β 2 -agonists in the treatment of asthma[END_REF]. The mechanisms contributing to the adverse effects in the airways are not known, but it has been suggested that some of these undesirable effects maybe due to the S-enantiomer as most commonly available β 2 agonists are produced as racemic mixtures of R-and S-enantiomers in equal ratios [START_REF] Page | Contrasting properties of albuterol stereoisomers[END_REF].

The S-enantiomer of salbutamol has been considered to be largely inert or at best to have weak β 2 -agonist activities. However, recent studies have documented that S-salbutamol can potentiate the effects of spasmogens in airway smooth muscle from both guinea-pigs and humans , with a number of clinical studies also reporting worsening of airways hyperresponsiveness in animals [START_REF] Gupta | Evidence based review on levosalbutamol[END_REF] and in subjects with asthma [START_REF] Perrin | Differential responses of asthmatic airways to enantiomers of albuterol. Implications for clinical treatment of asthma[END_REF]. It has also been documented that the metabolic clearance of S-salbutamol occurs much less rapidly than Rsalbutamol [START_REF] Spitzer | The use of β-agonists and the risk of death and near death from asthma[END_REF], and thus the paradoxical responses may persist after the beneficial effects of (R)-salbutamol have waned [START_REF] Page | Contrasting properties of albuterol stereoisomers[END_REF], particularly after chronic use of the drug. S-salbutamol has also been reported to have proinflammatory effects that could negatively affect the airways [START_REF] Agrawal | S)-Albuterol activates pro-constrictory and pro-inflammatory pathways in human bronchial smooth muscle cells[END_REF].

Chemically, all β-agonists are racemates, or drugs composed of two non-superimposable mirror image molecules in a 50/50 ratio [START_REF] Handley | The pursuit of precision pharmaceuticals: divergent effects of β 2 agonist isomers[END_REF]. These molecules are classified as R-or Saccording to their molecular configuration, and as 'lev' or 'dex' based on the rotation of polarised light as it passes through the molecule.

Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with certain similarities to human asthma and COPD and represents a major cause of morbidity and loss of lung performance in this species [START_REF] Robinson | The pathogenesis of chronic obstructive pulmonary disease of horses[END_REF][START_REF] Matera | Innervation of equine airways[END_REF]. Whilst β 2 -agonists are used in the treatment of RAO, little is known about the properties of the enantiomers of salbutamol in the horse. The present study has therefore evaluated effects of racemic β 2 -agonists in isolated equine bronchi in comparison with the pure Rand S-enantiomers.

Materials and Methods

Equine lungs, derived from healthy horses following humane killing in an abattoir, were obtained from six RAO-unaffected horses (3 male and 3 female; aged 2.3 ± 0.1 years; weighted kg 375 ± 54.3) and four RAO-affected horses (2 male and 1 female; aged 2.5 ± 0.2 years; weighted kg 388 ± 46.5). Lungs were rapidly transported to our laboratory in a Krebs-Henseleit solution (KH) (composition, mM: NaCl 117.5. KCl 5.60. MgSO 4 1.18. CaCl 2 2.50. NaH 2 P0 4 1.28. NaHCO 3 25.00 and glucose 5.5, pregassed with =O 2 /CO 2 95:5% v/v. pH7.4). Segmental bronchi, typically 3-5 mm in diameter, were derived from each set of lungs and dissected free from connective tissue and cut into rings. Epithelium intact bronchial rings were transferred into organ baths containing KH-buffer (37 °C), treated with indomethacin (10 µM) in order to inhibit cyclo-oxygenase enzymes and so prevent prostaglandin formation and any intrinsic tissue activity. Tissues were continuously gassed with O 2 /CO 2 95:5 v/v. Bronchial rings were connected to an isometric force displacement transducer and were allowed to equilibrate for 90 min containing modified with the KH buffer being changed every 10 min. During equilibration (2 h) optimal passive tension were determined by gentle stretching of tissue (≅ 2 g).The isometric changes in tension was measured with a transducer Fort 10 WPI (Basile Instruments.

Italy).

The tissue responsiveness was assessed using acetylcholine (ACh) 100 µM; when the response reached a plateau, rings were washed three times and allowed to equilibrate for a further 60 min.

Study design

Protocol 1: Relaxant effect of salbutamol enantiomers on submaximal contractions induced by carbachol or histamine.

Following equilibration of the tissues, semi-logarithmic concentration-response curves induced by carbachol (dose from 10 nM to 100 µM) or histamine (10 nM to 100 µM) were constructed in order to establish the baseline contractility of tissues and the concentration inducing a sub-maximal contraction (70%). The bronchial rings were washed a further three times and allowed to stabilize for about 30 min following the last washing.

In order to evaluate the relaxant effects of salbutamol enantiomers, in all rings contracted with carbachol and histamine at the sub maximal concentration (EC70), a concentration semi-logarithmic curve to salbutamol enantiomers or the racemic mixture was constructed at concentrations ranging from 0.1nM to 100µM for each compound. Each concentrationresponse curve was obtained by the cumulative addition of R-, S-or racemic salbutamol at intervals of 5-15 min to reach a stable level of relaxation before the next addition was made. In the control groups a cumulative concentration of vehicle was added in the organbaths and used as a time control. At the end of the experiments papaverine (500 µM) was added to the bronchial rings to determine the maximal relaxant response achievable for each isolated bronchus. Similar experiments of protocol 1 were repeated in bronchi from RAO-affected horses.

Protocol 2: Effects of salbutamol enantiomers on smooth muscle contraction induced by carbachol or histamine.

In order to evaluate the effects of salbutamol enantiomers on carbachol and histamineinduced contraction , concentration-contraction semi-logarithmic curves to carbachol or histamine (10 nM to 100 µM) were constructed from bronchi pre-incubated with R-, S-or racemic salbutamol (100 µM) for 90 min. The control bronchi were pre-incubated with vehicle which also served as a time control.

Analysis

All experiments were carried out on bronchi isolated from the lungs of six different horses (n=6). Appropriate curve-fitting to a sigmoidal model was used to calculate the effect, the maximal response , the dose inducing 50% maximal effect and the dose inducing 70% maximal effect (E, Emax, EC50 and EC70 respectively). The equation used was log(agonist) vs. response, Variable slope, expressed as Y=Bottom + (Top-Bottom)/{1+10^[(LogEC50-X)*HillSlope]}. The relaxant responses were expressed as percentage of papaverine (500 µM) induced relaxation. All values are presented as mean ± SEM of n subject in each treatment group. Statistical significance was assessed by Student's t test or one-or two-way analysis of variance (ANOVA), with Dunnett's or Bonferroni post-tests respectively. The level of statistical significance was defined as P ≤ 0.05. All data analyses were performed using computer software (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA).

Drugs

Acetylcholine, carbachol and histamine were dissolved in KH solution; papaverine was dissolved in distilled water; R-, S-and racemic salbutamol were dissolved in distilled water; indomethacin was dissolved in ethanol and the maximal amount of ethanol (0.02%) did not influence the response of isolated bronchi. Stock solutions (1 mM) of each of the tested substances were prepared in distilled water, diluted with KH solution and prepared fresh on each day of study. The substances were obtained from Sigma-Aldrich (St. Louis, USA), R-S-and racemic salbutamol were obtained from Stirling Products Ltd (Sydney, Australia) .

Results

Protocol 1: Relaxant effect of salbutamol enantiomers on submaximal contractions induced by carbachol or histamine

The baseline tension of bronchial tissues did not change throughout the experiments carried out with S-Salbutamol or the racemic mixture.

In the control bronchi, papaverine completely relaxed equine bronchial rings contracted with a sumaximal concentration of carbachol (CCh: +4.37gr±0.42; P:-4.64gr±0.45) or histamine (His: +6.59gr±0.98; P: -7.93gr±0.85). In contrast, salbutamol enantiomers did not show direct relaxant effects on the sub-maximal plateau contractile response induced by carbachol in either RAO-affected or RAO-unaffected bronchi. On the contrary, both enantiomers of salbutamol induced a slight but significant increase (P≤0.05) of the carbachol baseline. Furthermore, racemic salbutamol showed a similar trend to the induced enantiomers (Figure 1; Table 1). R-salbutamol induced a significant (P<0.05) relaxation of the histamine sub-maximal plateau contractile response (RAO-unaffected: Emax 92.06%±2.00, EC50 0.55µM±0.06; RAO-affected: 100.20%±3.99, EC50 0.40µM±0.04) . S-salbutamol induced a weak relaxation (RAO-unaffected: 15.81%±5.65; RAO-affected: 12.36±5.15) when compared to that induced by papaverine on the histamine response that was significant (P≤0.01) only at the higheest concentrations used. Moreover, the racemic compound was analogous to the R-salbutamol effect, but eliciting a smaller Emax, particularly in bronchi from RAO-affected horses (Figure 1; Table2). In all bronchial rings the vehicle did not induce any relaxation of the bronchi.

Protocol 2: Effects salbutamol enantiomers on smooth muscle contraction induced by carbachol or histamine

Carbachol and histamine induced a concentration-dependent contraction of equine isolated bronchi; the incubation with KH did not elicit any effect on equine isolated bronchial rings (figure 2). The EC70 of carbachol and histamine are presented in Table 3. The incubation with R-or S-salbutamol shifted to the right the carbachol contraction curve compared to the controls (P≤0.001) at concentrations ranging from 0.3 µM to 100 µM; both R-and Ssalbutamol showed a significant reduction in Emax (R-salb.: P≤0.001; S-salb.: P≤0.05) with a significant increase in the EC50 (R-salb.: P≤0.001; S-salb.: P≤0.05); the trend of the carbachol induced contraction curves after the incubation with racemic mixture of salbutamol was analogous to effects elicited by the individual enantiomers (figure 2, table 3). 3).

The contractile response to carbachol did not change in bronchi from RAO-affected horses, whereas the contractile potency of histamine was significantly enhanced in bronchi from RAO-affected horses (EC50 2.8µM±0.26), compared to tissue obtained from RAOunaffected horses. In RAO-affected bronchi, R-and S-enantiomers of salbutamol and the racemic mixture did not alter the concentration response curve to carbachol compared to control tissues (P>0.05). On the contrary, the contractile response to histamine was shifted leftwards by S-salbutamol (EC50 1.50µM±0.80, P<0.05) whereas it was shifted rightward by R-salbutamol (EC50 7.10 µM ±0.35, P<0.001). In bronchi from RAO-affected horses the racemate did not change the contraction induct by histamine (P>0.05) (figure 2, table 3).

Discussion

Inhaled β 2 -agonists remain the first line treatment of acute symptoms of airway obstructive diseases in horses. Most commonly available β 2 -agonist preparations are racemic, i.e. they are composed of an equimolar mixture of R-and S-stereoisomers. Traditionally the Senantiomers were considered inert or at best weak β 2 -agonists [START_REF] Penn | Comparison of R-, S-, and RS-albuterol interaction with human β 1 -and β 2 -adrenergic receptors[END_REF], but it is now recognized that the S-enantiomer can have adverse effect in the airways [START_REF] Page | Contrasting properties of albuterol stereoisomers[END_REF]. Thus, it has been documented in in vitro and in vivo experimental studies that S-salbutamol can be proinflammatory and have pro-constrictor effects in airways. These results are in agreement with clinical studies that have demonstrated that S-salbutamol can induce airway hyperresponsiveness in patients with asthma. It has been suggested that these paradoxical responses may be reduced by removing the dose of S-enantiomers [START_REF] Spitzer | The use of β-agonists and the risk of death and near death from asthma[END_REF][START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF]. Besides, Rsalbutamol is now commonly available for the treatment of obstructive lung disease and further R-isomers preparations of β 2 -agonist are under development in man [START_REF] Gupta | Evidence based review on levosalbutamol[END_REF] Like human obstructive bronchial diseases, equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO), is a common equine chronic pulmonary disease, in which the prominent symptom is airway obstruction which is treated with β 2 agonists [START_REF] Robinson | The pathogenesis of chronic obstructive pulmonary disease of horses[END_REF][START_REF] Matera | Innervation of equine airways[END_REF]. Our present study shows that S-salbutamol has adverse proconstrictory effects in equine bronchi, particularly in bronchi from RAO-affected horses.

Previous studies carried out with isolated equine trachea have demonstrated that β 2agonists facilitate acetylcholine release from prejunctional cholinergic nerves by the stimulation of prejunctional β 2 -receptors localized on parasympathetic nerve endings [START_REF] Cazzola | Inhaled β 2 -adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease[END_REF][START_REF] Zhang | β 2 -adrenoceptor activation augments acetylcholine release from tracheal parasympathetic nerves[END_REF]. Moreover, the S-enantiomers of two β 2 -agonists (salbutamol and formoterol) elicited a significant increase in acetylcholine release in the presence of M 2 receptor blockade, and suggested a potential deleterious effect of (S)-β 2 agonists in the lungs of horses with RAO [START_REF] Zhang | Excitatory prejunctional β 2 -adrenoceptor distribution within equine airway cholinergic nerves[END_REF]. To our knowledge, this is the first paper to focus on the effects of the R-and S-enantiomers of salbutamol on isolated bronchi obtained from Either normal or RAO affected horses.

Our results are consistent with an effect of acetylcholine release, as demonstrated by Zhang and co-workers [START_REF] Zhang | β 2 -adrenoceptor activation augments acetylcholine release from tracheal parasympathetic nerves[END_REF][START_REF] Zhang | Excitatory prejunctional β 2 -adrenoceptor distribution within equine airway cholinergic nerves[END_REF][START_REF] Zhang | Effects of enantiomers of β 2agonists on ACh release and smooth muscle contraction in the trachea[END_REF]. Furthermore, it has also been postulated that S-salbutamol has a possible agonistic effect on M 3 muscarinic receptors postjunctionally, via a mechanism that involves an increase of intracellular calcium in airway smooth muscle [START_REF] Zhang | Excitatory prejunctional β 2 -adrenoceptor distribution within equine airway cholinergic nerves[END_REF][START_REF] Zhang | Effects of enantiomers of β 2agonists on ACh release and smooth muscle contraction in the trachea[END_REF]. According with this hypothesis, and considering that the affinity of S-isomer for βadrenoceptors is >100 fold lower than R-isomer, Mitra and coworkers suggested that Ssalbutamol could work as an agonist for muscarinic receptors cross-reacting with M 3 receptors, acting also as a calcium agonist [START_REF] Zhang | Effects of enantiomers of β 2agonists on ACh release and smooth muscle contraction in the trachea[END_REF]. Moreover, several studies have shown that an exaggerated cholinergic stimulation of airway smooth muscle causes a reduction in the ability of β 2 -agonists to relax airway smooth muscle [START_REF] Van Amsterdam | Role of phosphoinositide metabolism in functional antagonism of airway smooth muscle contraction by β-adrenoceptor agonists[END_REF][START_REF] Boterman | Protein kinase C potentiates homologous desensitization of the β 2 -adrenoceptor in bovine tracheal smooth muscle[END_REF]. These results could suggest that both enantiomers of salbutamol could have deleterious effects in equine airways, particularly in RAO-affected horses. However, our results have demonstrated that Rsalbutamol functionally antagonized the carbachol concentration contraction curve in normal bronchi, but not in bronchi obtained from horses with RAO. Furthermore, Rsalbutamol postjunctionally inhibited the smooth muscle tone induced by both histamine and carbachol in a significant manner in both normal and RAO bronchi. In addition, on equine carbachol precontracted tracheal strips, we have provided evidence that Ssalbutamol had a modest, but significant, bronchoconstrictor effect [START_REF] Zhang | Excitatory prejunctional β 2 -adrenoceptor distribution within equine airway cholinergic nerves[END_REF][START_REF] Johansson | Effects of albuterol enantiomers on in vitro bronchial reactivity[END_REF]. These data may support the deleterious role of S-salbutamol, particularly in RAO-affected horses, in which the β 2 -adrenergic receptor-G s -protein-adenylate cyclase system is significantly impaired in membranes of peripheral lung and bronchi, when compared with the segments from control animals [START_REF] Abraham | Agonist-independent alteration in βadrenoceptor-G-protein-adenylate cyclase system in an equine model of recurrent airway obstruction[END_REF].

In contrast with the observations on carbachol precontracted equine bronchi, the results of our study demonstrated that R-salbutamol relaxed equine isolated airways contracted with histamine. Moreover, the cumulative concentration-contraction curve to histamine was significantly shifted rightward with a significant increase of its EC50 when both normal and RAO equine bronchi were incubated with R-salbutamol, compared to controls tissues.

Our findinge are in line with results obtained in bovine tracheal smooth muscle, in which the contraction induced by muscarinic agonists was relatively resistant to relaxation by β 2agonists, compared to the contraction induced by histamine [START_REF] Boterman | Protein kinase C potentiates homologous desensitization of the β 2 -adrenoceptor in bovine tracheal smooth muscle[END_REF]. In fact, this spasmogen induced a less marked calcium mobilization and influx when compared to that induced by methacholine [START_REF] Van Amsterdam | Role of phosphoinositide metabolism in functional antagonism of airway smooth muscle contraction by β-adrenoceptor agonists[END_REF], which is less susceptible to elevations of c-AMP [START_REF] Hoiting | Modulation of agonist-induced phosphoinositide metabolism, Ca 2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms[END_REF].

As expected, we observed a less marked relaxant effect of S-salbutamol, that was significant only at the highest concentration and certainly less effective than papaverine. In addition, we found a significant leftward shift of the histamine cumulative concentration contraction curve, with an increase of histamine potency, when normal airways or bronchi from RAO-affected horses were incubated with S-salbutamol, compared to control tissues.

In line with these results, it has been documented that detrimental effects of the racemic mixture of β 2 -agonists are strictly related to the S-isomers [START_REF] Page | Contrasting properties of albuterol stereoisomers[END_REF][START_REF] Agrawal | S)-Albuterol activates pro-constrictory and pro-inflammatory pathways in human bronchial smooth muscle cells[END_REF][START_REF] Hoshiko | Exacerbation of airway hyperreactivity by (+/-)salbutamol in sensitized guinea pig[END_REF][START_REF] Mazzoni | Hyperresponsiveness of the airways following exposure of guinea-pigs to racemic mixtures and distomers of β 2 -selective sympathomimetics[END_REF]. Although, Ssalbutamol induced a weak relaxation of equine isolated bronchi pre-contracted by histamine, it induced an increase in airway smooth muscle responsiveness to the cumulative dose response to this spasmogen in both normal and RAO bronchi. In agreement with these findings, racemic salbutamol induced an exaggerated conctractile response to histamine in airway tissues obtained from sensitized from guinea pigs [START_REF] Hoshiko | Exacerbation of airway hyperreactivity by (+/-)salbutamol in sensitized guinea pig[END_REF]. It has been speculated that the pro-inflammatory and pro-constrictory activity of S-isomer, are mediated by the activation of transcriptional nuclear factor κB (NF-κB) as has been demonstrated in human isolated airways [START_REF] Abraha | S,S)-formoterol increases the production of IL-4 in mast cells and the airways of a murine asthma model[END_REF],. These findings were also confirmed in other animal models of bronchial hyperresponsiveness. Thus it has been reported that Ssalbutamol potentiates the pro-constrictory activity of histamine and IL-4 in sensitized guinea pigs [START_REF] Cho | S)-albuterol increases the production of histamine and IL-4 in mast cells[END_REF], by stimulating the production of IL-2 and IL-13 in T-cells [START_REF] Baramki | Modulation of T-cell function by (R)and (S)-isomers of albuterol: anti-inflammatory influences of (R)-isomers are negated in the presence of the (S)-isomer[END_REF]. Over all, these cytokines shift the coupling of the β 2 -adrenergic receptors to pro-constrictory G proteins such as G iα-1 protein [START_REF] Abraham | Agonist-independent alteration in βadrenoceptor-G-protein-adenylate cyclase system in an equine model of recurrent airway obstruction[END_REF]. Considering all these data, it is possible to hypothesize an analogous pro-inflammatory and pro-constrictory activity of S-salbutamol also in equine airways after only 90 min. of incubation,enhancing the potency of histamine, as has been demosntrated in human bronchi after passive sensitization [START_REF] Watson | Role of IgE in hyperresponsiveness induced by passive sensitization of human airways[END_REF].

In conclusion, our studies demonstrated that S-salbutamol elicited a weak contraction of equine airway smooth muscle, but induced a significant hyperresponsiveness to histamine that could be detrimental particularly in RAO-affected horses [START_REF] Scott | β-adrenergic blockade in ponies with recurrent obstructive pulmonary disease[END_REF][START_REF] Hare | Pulmonary eosinophilia associated with increased airway responsiveness in young racing horses[END_REF][START_REF] Wong | Comparison of results for intradermal testing between clinically normal horses and horses affected with recurrent airway obstruction[END_REF][START_REF] Deaton | Early onset airway obstruction in response to organic dust in the horse[END_REF]. Our results suggest that it may be prudent to administrate the R-isomer alone in RAO horses to avoid detrimental effect of S-isomer.

R

  -salbutamol caused a significant shift to the right of the cumulative concentrationcontraction curve of histamine, with a significant reduction in Emax values (C: 9.07gr±0.68; R-salb: 6.36gr±0.21; P≤0.01) but without any significantly change on EC50 values (C: 7.00µM±0.29; R-salb: 7.25µM ±1.06; P<0.05). In contrast the incubation with S-salbutamol shifted leftward the histamine concentration curve when compared to the left when compared with controls, with a significant reduction of EC50 values (C: 7.00µM±0.29; Ssalb: 2.25µM±0.19; P≤0.001) but without any significant change on Emax values (C: 9.07gr±0.68; S-salb: 8.12gr±0.51; P<0.05). The trend of the histamine induced contraction curves after incubation with the racemic mixture of salbutamol was similar to the effects induced by the S-isomer, but with Emax values significantly different when compared to control tissues (figure 2, table

Figure 1 -

 1 Figure 1 -Relaxant effects of R, S and racemic mixture of salbutamol on isolated bronchi from RAO-unaffected (A) and RAO-affected (B) horses, pre-contracted with submaximal dose of carbachol (CCH) or histamine (His). All values are mean ± SEM of six (A) and four (B) samples. * P ≤0.05, # P ≤0.01, § P ≤0.001, vs CCh or His baseline plateau; @ P ≤0.05 vs bronchi from normal horses.

Figure 2 -

 2 Figure 2 -Effects of R, S and racemic mixture of salbutamol on the dose response curve to carbachol (CCh, A and B) and histamine (His, C and D) in bronchi from RAO-unaffected (A and C) and RAO-affected (B and D) horses. All values are mean ± SEM of at the six (A and C) and four (B and D) samples. * P ≤0.05, # P ≤0.01, § P ≤0.001 vs control.

Table 1 . Relaxant effects of R, S and racemic mixture of salbutamol on equine isolated bronchi (normal and RAO-affected horses) contracted with carbachol (negative values mean a contraction).

 1 All values are mean ± SEM of six (normal bronchi) and four (bronchi from RAO horses) samples. * P≤0.05 vs baseline; ° P ≤0.05 vs normal bronchi; nd: not detectable.

				Treatment		Papaverine	SubMax
			Emax (%)	Emax (g)	EC50 (µM)	relaxation Emax (g)	contraction CCh (g)
		R salbutamol	*-8.98±0.71	*-0.35±0.03	nd	3.97±0.54	3.94±0.55
	Normal bronchi	S salbutamol	*-12.34±0.50 *-0.63±0.02	nd	5.08±0.52	5.12±0.54
		Racemic	*-9.98±0.84	*-0.45±0.03	nd	4.53±0.39	4.69±0.41
		R salbutamol	-9.90±0.81	-0.43±0.02	nd	4.33±0.42	4.02±0.46
	Bronchi
	from RAO	S salbutamol	°-17.71±0.89	°-0.46±0.03	nd	4.86±0.71	4.68±0.45
	horses
		Racemic	-12.25±0.74	-0.51±0.03	nd	4.51±0.58	4.24±0.48

Table 2 -Relaxant effects of R-and S-salbutamol on equine isolated bronchi (normal and RAO-affected horses) contracted with histamine.

 2 All values are mean ± SEM of six (normal bronchi) and four (bronchi from RAO horses) samples. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001 vs baseline; ° P ≤0.05 and °° P ≤0.01 vs normal bronchi; nd: not detectable.

				Treatment		Papaverine	SubMax
			Emax (%)	Emax (g)	EC50 (µM)	relaxation Emax (g)	contraction His (g)
		R salbutamol ***92.06±2.00	***8.55±0.18 ***0.55±0.06	9.29±0.31	7.56±1.00
	Normal bronchi	S salbutamol	**15.81±5.65	**1.16±0.41	nd	7.40±0.97	5.07±1.06
		Racemic ***81.12±3.67	***7.13±0.74 ***0.30±0.04	8.79±0.92	6.68±0.83
		R salbutamol °100.20±3.99	°9.43±0.29	°°0.40±0.04	9.42±0.29	8.98±0.86
	Bronchi
	from RAO	S salbutamol	12.36±5.15	1.03±0.33	nd	8.30±0.85	8.02±1.12
	horses
		Racemic	°64.08±3.55	°5.54±0.56	°°0.20±0.03	8.65±0.98	8.50±0.98

Table 3 -Effect of R, S and racemic mixture of salbutamol on equine isolated bronchi concentration-dependent curve to carbachol (CCh) and histamine (His).

 3 All values are mean±SEM of six samples. * P ≤0.05, ** P ≤0.01, *** P ≤0.001 vs controls; # P ≤0.001 vs normal bronchi.

				CCDR CCh			CCDR His
			Emax (g)	EC50 (µM)	EC70 (µM)	Emax (g)	EC50 (µM)	EC70 (µM)
		Control	6.97±0.15	0.80±0.03	1.63±0.05	9.07±0.68	7.00±0.29	13.8±0.97
	Normal	R salb. incub. §5.32±0.31	§2.50±0.24		**6.36±0.21	7.25±1.06
	bronchi
		S salb. incub.	*5.67±0.44	*2.35±0.69		8.12±0.51	***2.25±0.19
		Racemic incubation	**5.42±0.39 **2.44±0.52		*7.31±0.40	***2.00±0.25
		Control	6.96±0.18	1.00±0.16	1.55±0.09	8.34±0.28	#2.80±0.26	#5.50±0.45
	Bronchi	R salb. incub.	7.07±0.26	1.30±0.12		8.37±0.27	***7.10±0.35
	from RAO
	horses	S salb. incub.	6.84±0.21	0.90±0.08		7.91±0.34	*1.50±0.80
		Racemic incubation	6.89±0.31	1.20±0.11		7.78±0.30	2.50±0.34


Olivier Herbinet
email: olivier.herbinet@ensic.inpl-nancy.fr

Joffrey Biet

Mohammed Hichem Hakka

Valérie Warth

Pierre- Alexandre Glaude

André Nicolle

Frédérique Battin-Leclerc

Modeling study of the low-temperature oxidation of large methyl esters from C 11 to C 19

Keywords: Methyl esters, Methyl palmitate, Biodiesel fuels, Modeling, Oxidation

The modeling of the low-temperature oxidation of large saturated methyl esters really representative of those found in biodiesel fuels has been investigated. Models have been developed for these species and then detailed kinetic mechanisms have been automatically generated using a new extended version of software EXGAS, which includes reactions specific to the chemistry of esters. A model generated for a binary mixture of n-decane and methyl palmitate was used to simulate experimental results obtained in a jet-stirred reactor for this fuel. This model predicts very well the reactivity of the fuel and the mole fraction profiles of most reaction products. This work also shows that a model for a middle size methyl ester such as methyl decanoate predicts fairly well the reactivity and the mole fractions of most species with a substantial decrease in computational time. Large n-alkanes such as n-hexadecane are also good surrogates for reproducing the reactivity of methyl esters, with an important gain in computational time, but they cannot account for the formation of specific products such as unsaturated esters or cyclic ethers with an ester function.

Introduction

Due to the depletion of fossil fuels, the development of renewable energy is more vital than ever [1] and [2]. The production of biofuels such as methyl esters is encouraged by the European Union in order to incorporate them in existing diesel fuels. Molecules produced from vegetable oils are methyl esters with a long carbon chain such as methyl palmitate (C 17 H 34 O 2 ) and methyl stearate (C 19 H 38 O 2 ). Due to the large size of the related kinetic mechanisms, it is necessary to determine the optimal model in terms of size and performance which could be used to simulate the oxidation of these large methyl esters and to investigate the differences with the combustion of alkanes induced by the ester function. While biodiesel fuels deriving from soybean and rapeseed oils contain mainly methyl esters from C 17 to C 19 [3], there is almost no study of the oxidation of compounds really representative of these species.

Herbinet et al. [4] developed the first model for the oxidation of a middle size saturated methyl ester, methyl decanoate, including all pertinent reactions to low and high temperature regions. The model has been validated against limited available data and suggests that large methyl esters and n-alkanes of similar size have very similar reactivity. This model was reduced and used to model extinction and ignition of laminar non-premixed flames containing methyl decanoate [5]. Very recently, Glaude et al. [6] performed an experimental study of the oxidation of methyl decanoate in a jet-stirred reactor. They showed that their results were well reproduced by the model of Herbinet et al. [4], and they used also these data to validate a new version of EXGAS for the automatic generation of detailed kinetic model of methyl ester oxidation (EXGAS-ALKANES-ESTER 1 called EXGAS in this paper).

The purpose of this paper is to describe and test detailed kinetic mechanisms for the oxidation of large methyl esters from C 11 to C 19 automatically generated with software EXGAS [6]. A model thus generated is compared with experimental results of the oxidation of methyl palmitate (blended with n-decane) obtained in a jet-stirred reactor [7]. The models for methyl esters from C 11 up to C 19 are then used to compare the reactivity and the mole fractions of reaction products and to investigate if methyl esters such as methyl decanoate can be good surrogates for larger methyl esters.

Models generated with EXGAS

Models for the oxidation of methyl esters from methyl decanoate (C 11 H 22 O 2 ) to methyl stearate (C 19 H 38 O 2 ) were generated with software EXGAS. The rules used for the generation have already been comprehensively described in [6] and only the main features are given here. Models generated with EXGAS are made of three parts: a comprehensive primary mechanism, where the only molecular reactants considered are the initial organic compounds and oxygen; a C 0 -C 2 reaction base, including all the reactions involving radicals or molecules containing less than three carbon atoms [8]; and a lumped secondary mechanism, containing the reactions consuming the molecular products of the primary mechanism, which do not react in the C 0 -C 2 reaction base. The molecules formed in the primary mechanism and consumed in the secondary mechanism, with the same molecular formula and the same functional groups, are lumped into one unique species, without distinguishing between the different isomers [9]. This avoids an explosion of the number of molecules in the models. Globalized reactions in the secondary mechanism were considered in order to promote the formation of radicals having more than two carbon atoms, the reactions of which are already included in the primary mechanism [10].

The rules used for the generation of the primary mechanism are the same as the ones which are used in the case of the oxidation of alkanes [11] and no new specific reaction was considered. However some changes were made in the estimation of the kinetic data in order to take into account the presence of the ester group. These changes affect:

1. The activation energies of the unimolecular initiations which involve the breaking of the C-C bonds located in the α-and β-positions from the ester function (bond dissociation energies were taken equal to 93.4 and 85.0 kcal/mol, respectively, according to the values recently calculated by El-Nahas et al. [12]).

2. The activation energy of oxidations of radicals (yielding an unsaturated molecule and HO 2 ) and metatheses involving an H-atom carried by the atom of carbon just neighboring the ester function. Since the energy of the corresponding C-H bond (94.3 kcal/mol [START_REF] Luo | Handbook of Bond Dissociation Energies in Organic Compounds[END_REF]) is close to that of a tertiary H-atom (e.g., 95.7 kcal/mol in isobutene [START_REF] Luo | Handbook of Bond Dissociation Energies in Organic Compounds[END_REF]), the activation energies used for oxidations were the same as the ones for tertiary H-atoms. For H-atom abstractions, the correlations were taken the same as those used for the abstraction of a tertiary H-atom from alkanes, except in the case of the abstractions by •O• and •H atoms and by •OH, •HO 2 and •CH 3 radicals. In this case, the rate parameters have been evaluated using an Evans-Polanyi correlation proposed by Dean and Bozzelli [START_REF] Dean | Combustion Chemistry of Nitrogen[END_REF]. Evans-Polanyi correlations have been proposed to give more accurate kinetic parameters as they take into account the real bond energies of the C-H bonds involved in the reaction. As the C-H bond energy in the α position of the ester function is slightly lower than the C-H bond energy in the case of a tertiary H-atom, the kinetic constants evaluated using the Evans-Polanyi correlation are larger and reactions faster (of about a factor 1.7 at 650 K and 2 at 1000 K).

3. The activation energy of radical intramolecular isomerizations. The activation energy for abstracting an H-atom from the carbon atom located in the α-position from the ester function has been taken equal to that for the abstraction of a tertiary H-atom in an alkyl radical. Quantum calculations were carried out to estimate the strain energy in cyclic transition states including the atoms of the ester group [6].

4. The activation energy of radical decompositions by β-scission. Quantum calculations were performed to estimate the activation energy of the reactions of decomposition involving the different types of bonds of the ester group [6].

New rules were implemented in the secondary mechanism for the consumption of the species formed in the primary mechanism and which did not exist in the case of alkanes: hydroperoxides, cyclic ethers, ketones and aldehydes bearing an ester function, as well as unsaturated methyl esters. As in the case of alkanes [10], the general idea is to promote the formation of radicals, the reactions of which are already included in the primary mechanism, i.e., C 2 + alkyl radicals and C 2 + methyl ester alkyl radicals. The rules of decomposition of the species formed in the primary mechanism as well as the associated kinetic parameters are presented in detail in [6]. Models generated with EXGAS were already validated from available shock tube, rapid compression machine, and jet-stirred reactor experimental data for methyl esters from C 5 to C 11 [6] and [START_REF] Hakka | [END_REF]. Models used in this study were generated with the same version of EXGAS and using the same set of kinetic parameters.

Because of the loss of the symmetry due to the presence of the ester group, models for the oxidation of methyl esters are much larger than models for the oxidation of n-alkanes. As an example the model generated for methyl decanoate (C 11 H 22 O 2 ) is composed of 1247 species and 7775 reactions against 655 species and 4435 reactions for n-undecane (C 11 H 24 ). The methyl decanoate model of Herbinet et al. [4], which was build in a different way than models generated with EXGAS, is much larger than the one generated in this study: it includes 3012 species and 8820 reactions. The numbers of species and reactions (Table 1) increase drastically with the size of the molecule resulting in a large increase in CPU time for the largest methyl esters. An increase of two in the number of carbon atoms in the ester molecule results in an increase by a factor of 2.4 for the computational time in a PSR, e.g., a factor of 33 from methyl decanoate to methyl stearate.

Validation against methyl palmitate experimental data

Very little experimental data are available for the validation of kinetic models in the case of large esters. However, experimental data of the oxidation of methyl palmitate (blended with n-decane) in a jet-stirred reactor were recently published [7]. We present here a simulation of these data using a model generated by EXGAS for a binary mixture composed of methyl palmitate and n-decane ([Fig. 1] and [Fig. 2]). Experiments were carried out at a pressure of 106 kPa, a residence time of 1.5 s and at stoichiometric conditions. The initial mole fraction of the fuel was 0.002 (dilution in helium) and the mole composition of the blend was 0.74 n-decane and 0.26 methyl palmitate. Simulations were performed with the code PSR of the software package Chemkin [START_REF] Kee | Chemkin II: A Fortran Chemical Kinetics Package for the Analysis of a Gas-phase Chemical Kinetics[END_REF]. As far as the reactivity is concerned, the model captures well the mole fraction profiles of methyl palmitate, n-decane and oxygen (Fig. 1). The conversion of both reactants is over-predicted at low temperature (around 650 K) and in the region following the negative temperature coefficient (NTC) zone. The model is too reactive at high temperature (above 1000 K) as shown by the too high consumption of intermediate species (e.g., carbon monoxide, alkenes, and unsaturated esters).

The model also reproduces well the mole fraction profiles of most reaction products such as 1-alkenes (e.g., 1-heptene, and 1-nonene in Fig. 2) and unsaturated esters with the double bond at the end of the chain (e.g., 5-hexene methyl ester in Fig. 2). It can be seen that the formation of some intermediate species is strongly over-predicted in the low temperature region. This is the case for species such as acetaldehyde (Fig. 1), 1-octene, and 13-tetradecene methyl ester (Fig. 2). These discrepancies are due to the rules of decomposition of lumped species in the secondary mechanism.

As This reaction channel to n-octyl radical contributes then to the over-estimation of the formation of an important amount of 1-octene. The discrepancies in the mole fractions of some reaction products are also due to the change in the initial mole fraction of the fuels made to keep the carbon content constant when going from methyl decanoate to methyl stearate. Note that the lumping technique has little effect on the reactivity as the rate of consumption of a lumped species is the sum of the rates of consumption of all possible isomers. The lumping strategy is not affected by the fact that methyl esters are not symmetric: there are twice more isomers than in the case of linear alkanes of similar size but their individual amounts are twice lower. Thus the concentrations of the lumped species and their consumption rates are about the same for symmetric and non-symmetric species.

The model predicts well the global amount of C 17 H 32 O 3 five-membered ring cyclic ethers (Fig. 2). All possible isomers are formed in similar amounts except for the cyclic ether with the ring including the four atoms of carbon located at the end of the alkyl chain (which is less formed than the other ones) and for the cyclic ether including the four atoms of carbon just neighboring the ester function (the formation of which is favored by the lower energy of the C-H bonds in the α-position of the ester group). The larger amount of this ether is not in agreement with the experimental data as only very small quantities were observed [7].

According to the kinetic analysis performed at 650 K under the conditions of the experimental study, methyl palmitate is quasi-integrally consumed by H-abstractions by OH radicals. Note that the abstraction of the H-atoms in the α-position of the carbonyl group is easier than the ones of other Hatoms and that 18.5% of the fuel is consumed through this reaction. Chain branching is obtained through the sequence of reactions: addition of radicals from the reactant to O ketohydroperoxide molecule due to the large length of the chain, but the main ones are sequences which involves first and second isomerizations through a six-membered ring transition state.

The experimental conversion of methyl oleate, an ester with one double bond in the middle of the chain, which has been studied in a jet-stirred reactor [START_REF] Bax | [END_REF], is also reported in Fig. 1. It can be seen that reactivities of both esters are very similar except above the NTC where methyl oleate is slightly more reactive than methyl palmitate. Consequently the model generated for methyl palmitate also reproduces well the conversion of methyl oleate. The comparison with the reaction products is not presented in this paper, but it can be deduced from the comparison between methyl palmitate and methyl oleate experimental data [START_REF] Bax | [END_REF] that the generated model can also well reproduce the mole fraction profiles of the main species such as CO, CO 2 , and ethylene. Note that the present model cannot account for the formation of products specific to unsaturated esters, such as dienes and esters containing two double bonds.

Comparison of the reactivity of large esters in a jet-stirred reactor

Models for neat methyl esters from C 11 to C 19 (with an odd number of atoms of carbon), generated with EXGAS, were used to compare their reactivity and the mole fractions of some reaction products in a jet-stirred reactor. Simulations were performed at temperatures ranging from 500 to 1100 K, at a pressure of 106 kPa and under stoichiometric conditions with dilution in helium. In the case of methyl decanoate, the inlet mole fraction of the fuel was 0.0023. Inlet mole fractions of other methyl esters were calculated in order to keep constant the carbon content of each reacting mixture. Simulations were also run with the model for n-hexadecane [10] in order to compare a large n-alkane with large methyl esters.

All these species exhibit a very similar conversion curves with the S shape due to the NTC (Fig. 3). Their reactivities are very close over the range of studied temperatures with the main differences observed around 780 K, where the conversion is minimum due to the NTC behavior. The reactivity increases from methyl decanoate to methyl stearate with that of other intermediate methyl esters lying in between. The reactivity of n-hexadecane is also very similar to the reactivity of large methyl esters. The conversion of this n-alkane is slightly larger than the ones of the studied methyl esters at low temperature (below 750 K), but lower at the minimum of conversion in the NTC zone. There is almost no difference at high temperature (above 800 K). Dagaut et al. also showed that the oxidation of a rapeseed biodiesel fuel can be successfully modeled by a detailed kinetic of the oxidation of nhexadecane [18]. Figure 3 also displays the mole fraction profiles of some reaction products, CO, ethylene, propene, 5-hexene methyl ester and the sum of five-membered ring cyclic ethers with the same skeleton as the reactant. Mole fraction of CO, ethylene, and propene are very similar whatever the methyl esters. The main differences can be observed for 5-hexene methyl ester (due to some decomposition routes globalized in the secondary mechanism and favoring the formation of species with n/2 carbon atoms, n being the number of carbon atoms in the reacting species) and for cyclic ethers. For these last species, the mole fractions are different but the carbon contents are very similar due to the difference in the numbers of carbon atoms in the molecules. As an example, the amount of cyclic ethers computed with the methyl decanoate model is about twice larger than the one computed with the methyl stearate model but methyl decanoate has about twice less carbon atoms than methyl stearate. Methyl decanoate seems thus to be a good surrogate for the reactivity of larger unsaturated methyl esters with a substantial decrease in computational time.

Mole fraction profiles obtained with the n-hexadecane model are also very similar to those obtained with the models for methyl esters. The main differences are observed for ethylene and propene. Their mole fractions are smaller in the simulations performed with the ester models due to the loss of symmetry in the initial reactants (esters lead to alkenes and unsaturated esters, whereas n-hexadecane only leads to alkenes). n-Hexadecane seems also to be a good surrogate for the reactivity of large unsaturated methyl esters but it cannot reproduce the mole fraction profiles of products specific to esters.

Conclusions

Detailed kinetic mechanisms for the oxidation of large saturated methyl esters from C 11 to C 19 were automatically generated with the software EXGAS including the reactions specific to the chemistry of esters and using a single set of kinetic parameters. These models contain many species and reactions because of the size of the reactants and because of the loss of the symmetry due to the ester function.

The data computed with the model generated for methyl palmitate were compared with experimental mole fraction profiles obtained in a jet-stirred reactor. The agreement between computed and experimental results was satisfactory for the conversion of the reactants and the mole fraction of most reaction products. This study showed that methyl esters larger than methyl decanoate are very good surrogates for the large saturated, and to a lesser extent unsaturated, esters found in biodiesel fuels as they predict well the reactivity and the mole fraction profiles of most species. This study also confirmed that large n-alkanes can be used to model the reactivity of large saturated esters, but that they cannot account for the formation of some specific reaction products such as unsaturated esters.

Biodiesel fuels also contain unsaturated methyl esters such as methyl oleate (C 19 H 36 O 2 ) and methyl linoleate (C 19 H 34 O 2 ). The present version of EXGAS is not able to account for the specific chemistry due to the presence of double bonds in the chains and further works about esters could consist in implementing specific rules in the software. Nevertheless previous work [START_REF] Bax | [END_REF] shows that the presence of one double bond in the middle of the alkyl chain has very little effect on the reactivity of this type of large molecules.

Fig. 1 .

 1 Fig. 1. Comparison of computed and experimental conversions of reactants and mole fractions of products for the oxidation of a blend of methyl palmitate and n-decane in a jet-stirred reactor (• and ×: experimental data; -: methyl palmitate model).

Fig. 2 .

 2 Fig. 2. Comparison of computed and experimental mole fractions of products (Part II) for the oxidation of a blend of methyl palmitate and n-decane in a jet-stirred reactor (•: experimental data;-: methyl palmitate model).

Fig. 3 .

 3 Fig. 3. Comparison of the conversion and of the mole fractions of some reaction products for methyl esters from C 11 to C 19 (with an odd number of atoms of carbon) and n-hexadecane in a jet-stirred reactor. Mole fractions are only presented for methyl decanoate, methyl tetradecanoate, methyl stearate, and n-hexadecane.

Table 1 .

 1 Numbers of species and reactions in the models generated with EXGAS.

		C 11 H 22 O 2	C 13 H 26 O 2	C 15 H 30 O 2	C 17 H 34 O 2	C 19 H 38 O 2
	species	1247	2012	3061	4442	6203
	reactions	7775	13004	20412	30425	43444

  an example, all the C 17 H 32 O 5 ketohydroperoxides formed in the low temperature chain branching reactions are lumped into one species called C 17 H 32 O 5 KPS in the model. This species decomposes into an OH radical, a CO molecule, a large C 8 H 14 O 3 ester with an aldehyde function and an n-octyl radical.

  2 forming ROO radicals; isomerizations of ROO radicals to QOOH radicals through H-shift; second additions of QOOH radicals to O 2 forming OOQOOH radicals; and isomerizations to a lumped C 17 H 32 O 5 ketohydroperoxide molecule plus OH. Note that many pathways lead to the formation of the lumped C 17 H 32 O 5

Software EXGAS-ALKANES-ESTERS automatically generates detailed kinetic mechanisms for the oxidation of linear and branched alkanes, and linear methyl esters and is freely available for academic researchers (valerie.warth@ensic.inpl-nancy.fr).

Acknowledgments

This study was supported by the ADEME in collaboration with IFP Energies nouvelles, PSA Peugeot Citroën and TOTAL and by the European Commission through the "Clean ICE" Advanced Research Grant of the European Research Council.