Nicola ; Tasinato
email: tasinato@unive.it

Andrea Pietropolli Charmet

Paolo Stoppa

Santi Giorgianni

Determination of the Vinyl Fluoride line intensities by TDL spectroscopy: the Object Oriented approach of Visual Line Shape Fitting Program to the line profile analysis

Keywords: Vinyl fluoride, Line parameter determination, Line shape fitting software, Weak and strong collision models, Voigt and Dicke narrowed profiles Vinyl fluoride, Line parameter determination, Line shape fitting software, Weak and strong collision models, Voigt and Dicke narrowed profiles Vinyl fluoride, Line parameter determination, Line shape fitting software, Weak and strong collision models, Voigt and Dicke narrowed profiles

In this work the self-broadening coefficients and the integrated line intensities for a number of ro-vibrational transitions of vinyl fluoride have been determined for the first time by means of TDL spectroscopy. The spectra recorded in the atmospheric window around 8.7 μm appear very crowded with a density of about 90 lines per cm -1 .

In order to fit these spectral features a new fitting software has been implemented. The program which is designed for laser spectroscopy, can fit many lines simultaneously on the basis of different theoretical profiles (Doppler, Lorentz, Voigt, Galatry and Nelkin -Ghatak). Details of the Object Oriented implementation of the application are given.

The reliability of the program is demonstrated by determining the line parameters of some ro-vibrational lines of sulphur dioxide in the ν 1 band region around 9 μm. Then the software is used for the line profile analysis of vinyl fluoride. The experimental line shapes show deviations from the Voigt profile which can be well modelled by using a Dicke narrowed line shape function. This leads to the determination of the selfnarrowing coefficient within the framework of the strong collision model.

Introduction

The study of the Earth and planetary atmospheres and of the interstellar medium by means of spectroscopic techniques has rapidly grown up since the earlier experiments in the second half of the 1900. Among the various techniques, infrared spectroscopy plays a significant role, in particular for remote sensing of the terrestrial atmosphere. In fact, IR remote sensing permits to monitor and accurately retrieve the concentration of the atmospheric constituents and trace pollutants. Though, in order to obtain reliable measurements, an accurate knowledge of the line parameters, such as line centres, intensities and pressure broadening coefficients, is required.

Thanks to the availability of always more powerful computers, the time consuming task of inverting the experimental spectra to extract spectroscopic parameters has become increasingly assisted by computer programs. Compared to the huge number of software available for extracting the Hamiltonian's parameters from the assigned line positions (a list of freely available devices is given in [1]), some programs specifically designed to perform the line shape analysis by fitting the experimental spectrum have been presented (see, for example, [2 -6]). It is worthwhile to note that these programs appear specifically designed to deal mostly with FTIR spectra. As a consequence, during the fitting procedure, they explicitly take into account the step of the parameterization of the FTIR instrumental line shape. These software, based on the multi-spectrum fitting technique, are the best choice when the large number of lines recorded by the interferometer in a single scan needs to be analyzed.

On the other hand, the spectrometers based on laser sources have a simpler instrumental contribution, generally modelled as a Gaussian or Lorentzian function. Furthermore, the spectral region which can be recorded in a scan is of the order of some wavenumbers, thus greatly reducing the amount of lines to be accounted for in a single spectrum. In many cases, the line shape analysis is carried out by using home made programs which are sometimes designed to fit the experimental features to a single theoretical function. Further, some programs are able to fit only one absorption line at time and still run from a command line window, thus avoiding an immediate comparison between the experimental data and the theoretical model. Commercial packages, such as IGOR Pro [7] and Origin [8], can be employed as well, although they are not completely well suited for this purpose.

Vinyl Fluoride, CH 2 CHF, is widely used by industry mainly as monomer for the production of synthetic materials such as poly-vinyl fluoride. The potential role of halogenated alkenes as air pollutants has motivated a number of spectroscopic investigations and studies about their reactivity toward hydroxyl radical and ozone (see Refs. [9,10] and references therein).

The infrared spectra of vinyl fluoride have been deeply investigated by our research group (see Refs. [11 -13] and references therein). The studies have mainly dealt with the analysis of the high resolution spectra and the obtained results have led to the determination of accurate spectroscopic parameters for a number of fundamental and overtone vibrations and a thorough understanding of the observed perturbations.

Recently, the medium resolution spectrum has been studied in the 400 -8000 cm -1 region and the integrated absorption cross sections have been determined for the first time [13]. In addition, ab-initio calculations with large basis sets have been performed at the CCSD(T) level of theory leading to the determination of harmonic and anharmonic force fields. Despite the industrial importance of vinyl fluoride, there exist no studies on the determination of the line parameters of the ro-vibrational absorption lines. The lack of literature data is partly motivated by the complex structure of the high resolution spectrum. In the present work we report the very first results obtained from the line shape analysis in the ν 7 band region around 1148 cm -1 by means of tunable diode laser (TDL) spectroscopy. In this region, the spectrum of vinyl fluoride appears very crowded even at low pressures. Hence, the analysis strongly requires a fitting software capable to simultaneously fit all the absorptions present in the spectral micro-windows acquired by the TDL system. For this reason we implemented a new software for the line shape fitting and the retrieval of the line parameters. The program, called Visual Line Shape Fitting Program (VLSFP), can fit several lines at once using different theoretical line shape functions. These include the Doppler, Lorentz and Voigt profiles, as well as the Galatry and Nelkin -Ghatak functions. The reliability of VLSFP has been tested by retrieving the sulphur dioxide line parameters and comparing them with the literature values. In this paper we also describe the program implementation and validation. In Section 2 the adopted experimental procedure is outlined while Sections 3 and 4 are concerned with the description of the fitting software and its test. Then in Section 5 we illustrate the results of the line shape analysis of vinyl fluoride. Conclusions and future work are addressed in Section 6.

Experimental procedure and data inversion

The high resolution spectra of either sulphur dioxide or vinyl fluoride were acquired on our tunable diode laser spectrometer employing a 92.3 (± 0.2) cm path respectively, between the filling of the cell and the spectrum acquisition was adopted.

The spectra were calibrated employing the SO 2 line frequencies obtained by high resolution FTIR measurements. The SO 2 and CH 2 CHF gas samples were provided by Sigma -Aldrich (99.9% purity) and Peninsular Chemical Research Inc. (99% purity), respectively, and were used without any further purification; no buffer gasses were employed. The SO 2 spectra were acquired at 297 (± 1) K and the pressures were varied in the range between 15 and 800 Pa. The measurements on CH 2 CHF were carried out at 299 (± 1) K with pressures ranging from 7 to 68 Pa.

Since the instrumental response of our TDL spectrometer is well described by a Gaussian function, its contribution to the line shapes was explicitly taken into account during the fitting procedure by fixing the Doppler half width to an effective value, ,

given by procedure using the Levenberg -Marquardt algorithm [14,15]. The algorithm has been implemented following the pseudo-code described in [16] and then modified in order to set bounds to the variability of the parameters or to keep the parameter values constrained, as described by Kanzow et al. [17]. In the current implementation, the algorithm terminates when the maximum number of iterations is completed or when one of the convergence criteria is met. These criteria are related to the square of the Euclidean norm of the residuals, its gradient and to the variation of the parameters between two subsequent iterations. The fitting routine runs as a background process avoiding the program interface to be frozen during the execution of the fitting task.

eff D γ ( ) ( ) 2 2 TDL D sample D eff D γ γ γ + = (1
The computational kernel, schematically depicted in Figure 1, has been designed following an Object Oriented Programming approach [18]. This programming technique, born around 1960s, uses objects and their interactions to design computer

programs. An application is then composed of a collection of cooperating objects, rather than an ensemble of subroutines that compute specific tasks. Each box of Figure 1 is referred to as class and it represents an "object". The class defines the properties (the class variables) of the corresponding object and its behaviour (the class methods). The object is able to process data and mostly to interact with other objects by receiving and sending messages. In a pictorial representation, every single object can be seen as a little Referring to Figure 1, the classes Vector, Matrix and Complex implement vector, matrix and complex algebra, respectively. They are used by the other objects of the project to store and process the data. The matrices have been implemented as onedimensional arrays thus speeding up the access to their elements. The fitting routines have been encapsulated inside the class LevMar and they have been designed to deal with a generic object of the type Line Shape. Line Shape represents an abstract class, that is an abstract object which defines the common characteristics possessed by all the line shapes (for example, the computed line profile, the line parameters array and the Jacobian matrix). The single line shape functions have been derived from the main abstract class and within each class a specific implementation of the methods has been made. As a consequence, new line shape functions, such as the speed dependent profiles, can be implemented without changing neither the LineShape nor the LevMar classes.

The line shape functions have been implemented in the normalized form.

Denoting the generic line function by f(p), it is normalized to unitary area:

(2)

( ) ∫ +∞ ∞ - = 1 dx f p
where p = [x, y, z,…] is the vector of dimensionless parameters which are described in

D D γ α = (3)
where γ D is the dimensional Doppler half width given by [20]

2 / 1 A B 0 2 ln 2 ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ = m T N k c D ν γ (4)
where ν 0 is the resonant frequency; c is the speed of light; k B is the Boltzman's constant; N A is the Avogadro's number; T (K) is the absolute temperature, m (a. m. u.) is the mass of the absorbing gas.

According to Beer -Lambert's law, the transmission of monochromatic radiation travelling a path of length through a uniform gas at pressure p is given by l where S is the integrated absorption coefficient (also referred to as line intensity) and f(ν -ν 0 ) is the line shape function as defined in equation ( 2). The dimensionless line shape functions implemented in VLSFP are listed in Table 2. During the fitting procedure, VLSFP refines the line intensity S, the line frequency centre v 0 and the remaining line parameters, which clearly depend on the specific line shape function.

The Voigt profile is widely used for modelling the spectral line profiles and in other scientific areas ( [21] and references therein), and hence the issue of its computation has been dealt over long time and intensive efforts have led to the development of many algorithms [22 -27]. A comprehensive survey of the different computational approaches has been reported by Schreier [28]. In VLSFP, the Voigt function is computed employing the optimized version of Humliček's code proposed by Wells [29]. Since this algorithm computes also the imaginary part of the complex probability function, it can be conveniently used for the computation of the Nelkin -Ghatak profile [30,31], as well.

Concerning the Galatry function [32], the profile is computed by the Fourier transform of the corresponding correlation function, Φ(y, z, t), following the algorithm proposed by Ouyang and Varghese [33]:

( ) ( ) [ { t z y z y s x f , , Re π 1 , , Φ ℑ = - ]} (7)
where ℑ indicates the Fourier transform operator. The derivatives are in turn computed via the Fourier transform of the appropriate correlation function. As a consequence, a discrete fast Fourier transform routine (DFFT) has been implemented. VLSFP has been tested by retrieving the parameters of some ro-vibrational lines of sulphur dioxide in the region around 9 μm. This region is characterized by the absorptions of the ν 1 fundamental corresponding to the symmetric stretching normal mode. In this section the results for a number of lines are presented in order to demonstrate the reliability of the application.

An example of the fits obtained for an isolated absorption line is given in Figure 2. When fitted to a Voigt profile, this line seems to show the effect of Dicke narrowing as suggested by the shape of the residuals. As a consequence, the line has also been fitted employing the weak and strong collision models. As can been seen from the plots of the residuals, the models accounting for the velocity changing collision effects reproduce properly the observed line shape.

A wider spectral region is shown in Figure 3. In this case, the lines have been fitted to a Voigt profile, employing the multi -line fitting procedure. In general, all the lines have been well reproduced although the residuals show some regular trends. This behaviour can be partly explained by the presence of very weak lines underneath the stronger ones. These lines, being not resolved at all, can not be taken into account during the fits but at the same time cause asymmetries in the observed absorptions. For the same reason, the Dicke narrowed profiles are not able to account for all the deviations. Anyway, even in such situation, the physical meaning of the parameters is preserved. This has been confirmed by plotting the collisional half widths and the line intensities as a function of the gas pressure: the line parameters lie on straight lines passing through the origin. The obtained line parameters are listed in Table 3 together with the more recent literature values [34,35] whose comparison is shown in Figure 4. As it can be seen, the present data and the literature values agree in a very satisfactory manner, indicating the reliability of the retrieved line parameters and hence validating the computational kernel of VLSFP.

Determination vinyl fluoride line shape parameters

The spectrum of vinyl fluoride in the 8.7 μm atmospheric region is characterized by the ro-vibrational transitions belonging to the ν 7 band which corresponds to the C-F stretching. As pointed out in the introduction, this spectral region is very crowded: the high density of lines, about 90 lines per cm -1 , can be appreciated in Figure 5 (a). Besides the stronger absorption of the ν 7 normal mode, the majority of the weakest lines is due to the ro-vibrational transitions of the ν 7 + ν 9 -ν 9 hot band. Due to their intensity these lines strongly affect the main features and therefore they must be accounted during the fitting procedure.

When the spectral lines are fitted to the Voigt profile, the shape of the residuals shows significant deviations from this model, as illustrated in Figure 5 (b). For this reason the lines have also been fitted employing the Nelkin -Ghatak profile. By using this strong collision model there is a dramatic improvement of the residuals as it is shown in Figure 5 (c): the standard deviation of the fit decreases by about a factor of two. Therefore, in the pressure range investigated the self-broadened vinyl fluoride sample appears as system strongly affected by the motional narrowing effect in which The retrieved line parameters for the ν 7 lines are listed in Table 4. The line parameters of the hot band have been determined as well, but they are not given here because of the lack of a reliable assignment of the ro-vibrational transitions at present. Again, we preferred to not report the self-broadening coefficient of three lines because their accuracy is too low. This is due to the small range of vinyl fluoride pressures adopted in the experiments which limits the amount of pressure broadening for some of the lines. On the other hand, the use of low pressures is strongly recommended in order to avoid the saturation of the absorptions, and hence working in the experimental conditions of applicability of the Beer -Lambert law. As can be seen from Table 4, the obtained self-broadening coefficients ranges from 0.4 to 0.9 cm -1 atm -1 . Such a wide interval is shown, for instance, by water vapour, whose self-broadening parameters are between 0.1 and 0.5 cm -1 atm -1 [36]. Besides, to a first investigation no dependence on the quantum numbers have been observed. This is understandable by considering the reduced set of data in which the majority of lines belong to the Q P subband. Therefore, in order to draw a more reliable conclusion about the quantum number dependence of the self-broadening coefficients, the analysis need to extended to a spectral region as wide as possible including the B-type transitions, as well.

The fits employing the Nelkin -Ghatak profile led to the determination of the narrowing parameter which result to be 0.40 (± 0.08) cm -1 atm -1 . As can be seen, the magnitude of the self-broadening and self-narrowing have a similar extent. The present paper gives an overview of the work that we are carrying out at present. Indeed, on the basis of the good results obtained from the analysis of the vinyl fluoride crowded spectra, we are going to extend the investigation in a spectral region as wide as possible with the aim to derive the vinyl fluoride line parameters in the 8 -9 μm atmospheric window. Future work will also be devoted toward a definitive assignment of the transitions belonging to the ν 7 + ν 9 -ν 9 hot band and to an improvement of the accuracy for some of the broadening coefficients.

) ( ) ( ) ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ + - - + - = - ζ ζ ζ ζ y s x w y s x w y s x f , π 1 , Re π 1 , ,
0 self γ [cm -1 atm -1 ] S 0 [10 -22 cm⋅molecule -1 ] J' K a ' K c ' J" K a " K c " 0 ν ~ [cm -1 ]
This b From Ref. [34].

c From Ref. [35]; the quoted uncertainties have been derived from the error bars of Figures 4, 6 and8.

d From Ref. [35].

e Belonging to ν 1 + ν 2 -ν 2 of 32 SO 2 ; an overlapping with 16 13,3 17 14,4 of 34 The objects interact with each other to make the application: the arrow means "derives from", the diamond means "makes use of" (see text for discussion).

0 self γ [cm -1 atm -1 ] J' K a ' K c ' J" K a " K c " 0 ν ~ [cm -1 ] Voigt Nelkin -Ghatak S 0 [10

Introduction

The study of the Earth and planetary atmospheres and of the interstellar medium by means of spectroscopic techniques has rapidly grown up since the earlier experiments in the second half of the 1900. Among the various techniques, infrared spectroscopy plays a significant role, in particular for remote sensing of the terrestrial atmosphere. In fact, IR remote sensing permits to monitor and accurately retrieve the concentration of the atmospheric constituents and trace pollutants. Though, in order to obtain reliable measurements, an accurate knowledge of the line parameters, such as line centres, intensities and pressure broadening coefficients, is required.

Thanks to the availability of always more powerful computers, the time consuming task of inverting the experimental spectra to extract spectroscopic parameters has become increasingly assisted by computer programs. Compared to the huge number of software available for extracting the Hamiltonian's parameters from the assigned line positions (a list of freely available devices is given in [1]), some programs specifically designed to perform the line shape analysis by fitting the experimental spectrum have been presented (see, for example, [2 -6]). It is worthwhile to note that these programs appear specifically designed to deal mostly with FTIR spectra. As a consequence, during the fitting procedure, they explicitly take into account the step of the parameterization of the FTIR instrumental line shape. These software, based on the multi-spectrum fitting technique, are the best choice when the large number of lines recorded by the interferometer in a single scan needs to be analyzed.

On the other hand, the spectrometers based on laser sources have a simpler instrumental contribution, generally modelled as a Gaussian or Lorentzian function. 3 Furthermore, the spectral region which can be recorded in a scan is of the order of some wavenumbers, thus greatly reducing the amount of lines to be accounted for in a single spectrum. In many cases, the line shape analysis is carried out by using home made programs which are sometimes designed to fit the experimental features to a single theoretical function. Further, some programs are able to fit only one absorption line at time and still run from a command line window, thus avoiding an immediate comparison between the experimental data and the theoretical model. Commercial packages, such as IGOR Pro [7] and Origin [8], can be employed as well, although they are not completely well suited for this purpose.

Vinyl Fluoride, CH 2 CHF, is widely used by industry mainly as monomer for the production of synthetic materials such as poly-vinyl fluoride. The potential role of halogenated alkenes as air pollutants has motivated a number of spectroscopic investigations and studies about their reactivity toward hydroxyl radical and ozone (see Refs. [9,10] and references therein).

The infrared spectra of vinyl fluoride have been deeply investigated by our research group (see Refs. [11 -13] and references therein). The studies have mainly dealt with the analysis of the high resolution spectra and the obtained results have led to the determination of accurate spectroscopic parameters for a number of fundamental and overtone vibrations and a thorough understanding of the observed perturbations.

Recently, the medium resolution spectrum has been studied in the 400 -8000 cm -1 region and the integrated absorption cross sections have been determined for the first time [13]. In addition, ab-initio calculations with large basis sets have been performed at the CCSD(T) level of theory leading to the determination of harmonic and anharmonic force fields.

Experimental procedure and data inversion

The high resolution spectra of either sulphur dioxide or vinyl fluoride were acquired on our tunable diode laser spectrometer employing a 92.3 (± 0.2) cm path procedure using the Levenberg -Marquardt algorithm [14,15]. The algorithm has been implemented following the pseudo-code described in [16] and then modified in order to set bounds to the variability of the parameters or to keep the parameter values constrained, as described by Kanzow et al. [17]. In the current implementation, the algorithm terminates when the maximum number of iterations is completed or when one of the convergence criteria is met. These criteria are related to the square of the Euclidean norm of the residuals, its gradient and to the variation of the parameters between two subsequent iterations. The fitting routine runs as a background process avoiding the program interface to be frozen during the execution of the fitting task.

The computational kernel, schematically depicted in Figure 1, has been designed following an Object Oriented Programming approach [18]. This programming technique, born around 1960s, uses objects and their interactions to design computer

programs. An application is then composed of a collection of cooperating objects, rather than an ensemble of subroutines that compute specific tasks. Each box of Figure 1 is referred to as class and it represents an "object". The class defines the properties (the class variables) of the corresponding object and its behaviour (the class methods). The object is able to process data and mostly to interact with other objects by receiving and sending messages. In a pictorial representation, every single object can be seen as a little Referring to Figure 1, the classes Vector, Matrix and Complex implement vector, matrix and complex algebra, respectively. They are used by the other objects of the project to store and process the data. The matrices have been implemented as onedimensional arrays thus speeding up the access to their elements. The fitting routines have been encapsulated inside the class LevMar and they have been designed to deal with a generic object of the type Line Shape. Line Shape represents an abstract class, that is an abstract object which defines the common characteristics possessed by all the line shapes (for example, the computed line profile, the line parameters array and the Jacobian matrix). The single line shape functions have been derived from the main abstract class and within each class a specific implementation of the methods has been made. As a consequence, new line shape functions, such as the speed dependent profiles, can be implemented without changing neither the LineShape nor the LevMar classes.

The line shape functions have been implemented in the normalized form.

Denoting the generic line function by f(p), it is normalized to unitary area:

( ) ∫ +∞ ∞ - = 1 dx f p (2)
where p = [x, y, z,…] is the vector of dimensionless parameters which are described in Table 1 following the notation of Varghese and Hanson [19]. As it can be seen, the line parameters are normalized by the effective Doppler half width defined as

D D γ α = (3)
where γ D is the dimensional Doppler half width given by [20]

2 / 1 A B 0 2 ln 2       = m T N k c D ν γ (4)
where ν 0 is the resonant frequency; c is the speed of light; k B is the Boltzman's constant; N A is the Avogadro's number; T (K) is the absolute temperature, m (a. m. u.) is the mass of the absorbing gas.

According to Beer -Lambert's law, the transmission of monochromatic radiation travelling a path of length l through a uniform gas at pressure p is given by ( )

( ) l p k I I ν ν - = e 0 (5)
where I 0 is the unattenuated intensity of the radiation having frequency ν. The quantity k(ν) is called the absorption coefficient and, for a single absorption line, it can be expressed in the form: where S is the integrated absorption coefficient (also referred to as line intensity) and f(ν -ν 0 ) is the line shape function as defined in equation ( 2). The dimensionless line shape functions implemented in VLSFP are listed in Table 2. During the fitting procedure, VLSFP refines the line intensity S, the line frequency centre v 0 and the remaining line parameters, which clearly depend on the specific line shape function.

( ) ( ) 0 ν ν ν - = f S k (6)
The Voigt profile is widely used for modelling the spectral line profiles and in other scientific areas ( [21] and references therein), and hence the issue of its computation has been dealt over long time and intensive efforts have led to the development of many algorithms [22 -27]. A comprehensive survey of the different computational approaches has been reported by Schreier [28]. In VLSFP, the Voigt function is computed employing the optimized version of Humliček's code proposed by Wells [29]. Since this algorithm computes also the imaginary part of the complex probability function, it can be conveniently used for the computation of the Nelkin -Ghatak profile [30,31], as well.

Concerning the Galatry function [32], the profile is computed by the Fourier transform of the corresponding correlation function, Φ(y, z, t), following the algorithm proposed by Ouyang and Varghese [33]:

( ) ( ) [ ] { } t z y z y s x f , , Re π 1 , , Φ ℑ = - (7)
where ℑ indicates the Fourier transform operator. The derivatives are in turn computed via the Fourier transform of the appropriate correlation function. As a consequence, a discrete fast Fourier transform routine (DFFT) has been implemented. VLSFP has been tested by retrieving the parameters of some ro-vibrational lines of sulphur dioxide in the region around 9 µm. This region is characterized by the absorptions of the ν 1 fundamental corresponding to the symmetric stretching normal mode. In this section the results for a number of lines are presented in order to demonstrate the reliability of the application.

An example of the fits obtained for an isolated absorption line is given in Figure 2. When fitted to a Voigt profile, this line seems to show the effect of Dicke narrowing as suggested by the shape of the residuals. As a consequence, the line has also been fitted employing the weak and strong collision models. As can been seen from the plots of the residuals, the models accounting for the velocity changing collision effects reproduce properly the observed line shape.

A wider spectral region is shown in Figure 3. In this case, the lines have been fitted to a Voigt profile, employing the multi -line fitting procedure. In general, all the lines have been well reproduced although the residuals show some regular trends. This behaviour can be partly explained by the presence of very weak lines underneath the stronger ones. These lines, being not resolved at all, can not be taken into account during the fits but at the same time cause asymmetries in the observed absorptions. For the same reason, the Dicke narrowed profiles are not able to account for all the deviations. Anyway, even in such situation, the physical meaning of the parameters is preserved. This has been confirmed by plotting the collisional half widths and the line intensities as a function of the gas pressure: the line parameters lie on straight lines passing through the origin. The obtained line parameters are listed in Table 3 together with the more recent literature values [34,35] whose comparison is shown in Figure 4. As it can be seen, the present data and the literature values agree in a very satisfactory manner, indicating the reliability of the retrieved line parameters and hence validating the computational kernel of VLSFP.

Determination vinyl fluoride line shape parameters

The spectrum of vinyl fluoride in the 8.7 µm atmospheric region is characterized by the ro-vibrational transitions belonging to the ν 7 band which corresponds to the C-F stretching. As pointed out in the introduction, this spectral region is very crowded: the high density of lines, about 90 lines per cm -1 , can be appreciated in Figure 5 (a). Besides the stronger absorption of the ν 7 normal mode, the majority of the weakest lines is due to the ro-vibrational transitions of the ν 7 + ν 9 -ν 9 hot band. Due to their intensity these lines strongly affect the main features and therefore they must be accounted during the fitting procedure.

When the spectral lines are fitted to the Voigt profile, the shape of the residuals shows significant deviations from this model, as illustrated in Figure 5 The retrieved line parameters for the ν 7 lines are listed in Table 4. The line parameters of the hot band have been determined as well, but they are not given here because of the lack of a reliable assignment of the ro-vibrational transitions at present.

Again, we preferred to not report the self-broadening coefficient of three lines because their accuracy is too low. This is due to the small range of vinyl fluoride pressures adopted in the experiments which limits the amount of pressure broadening for some of the lines. On the other hand, the use of low pressures is strongly recommended in order to avoid the saturation of the absorptions, and hence working in the experimental conditions of applicability of the Beer -Lambert law. As can be seen from Table 4, the obtained self-broadening coefficients ranges from 0.4 to 0.9 cm -1 atm -1 . Such a wide interval is shown, for instance, by water vapour, whose self-broadening parameters are between 0.1 and 0.5 cm -1 atm -1 [36]. Besides, to a first investigation no dependence on the quantum numbers have been observed. This is understandable by considering the reduced set of data in which the majority of lines belong to the Q P subband. Therefore, in order to draw a more reliable conclusion about the quantum number dependence of the self-broadening coefficients, the analysis need to extended to a spectral region as wide as possible including the B-type transitions, as well.

The fits employing the Nelkin -Ghatak profile led to the determination of the narrowing parameter which result to be 0.40 (± 0.08) cm -1 atm -1 . As can be seen, the magnitude of the self-broadening and self-narrowing have a similar extent. The present paper gives an overview of the work that we are carrying out at present. Indeed, on the basis of the good results obtained from the analysis of the vinyl fluoride crowded spectra, we are going to extend the investigation in a spectral region as wide as possible with the aim to derive the vinyl fluoride line parameters in the 8 -9

µm atmospheric window. Future work will also be devoted toward a definitive assignment of the transitions belonging to the ν 7 + ν 9 -ν 9 hot band and to an improvement of the accuracy for some of the broadening coefficients. The objects interact with each other to make the application: the arrow means "derives from", the diamond means "makes use of" (see text for discussion).

F

  KBr windows. The gas sample pressures were measured employing an Alcatel ARD 1003 capacitance gauge with a full scale range of 1000 Pa and a quoted manufacturer's accuracy of 1.5 Pa. Before each measurements the cell was evacuated down to about 10 -4 Pa; an elapse time of 10 and 20 minutes, for SO 2 and CH 2 CHF

3 .

 3 and instrumental Doppler half widths, respectively. The value of was retrieved by fitting the spectral lines recorded at low pressure (less than 30 Pa and 10 Pa for SO Visual Line Shape Fitting Program implementation: an Object Oriented approach VLSFP has been written combining Microsoft Visual Basic and C# for the graphical interface and the computational kernel, respectively. The determination of the line parameters within a given theoretical model is performed through an iterative

  own task. The set of distinct machines then cooperate to accomplish an objective: in our case the fit of the experimental line shape by means of different theoretical profiles.

  the unattenuated intensity of the radiation having frequency ν. The quantity k(ν) is called the absorption coefficient and, for a single absorption line, it can be expressed in the form:

4 .

 4 Test of VLSFP with SO 2 lines

  changing collisions and the internal state -dephasing collisions play an almost equal role in determining the shape of the spectral lines. Consequently, the line profiles accounting for the Dicke narrowing effect must be employed to properly model the experimental features.

6 .

 6 Conclusions and OutlookThe very first results of the line shape analysis of the vinyl fluoride infrared absorptions have been presented and discussed. In addition, the details of the implementation and validation of the fitting software (VLSFP) used for the analysis have been given.The program has been specifically designed to deal with the spectra recorded by means of laser based spectrometers in which the instrumental function can be modelled as a Gaussian or Lorentzian functions. Besides these two functions, the spectral absorptions can be fitted employing the Voigt profile as well as the weak and strong collision models which account for the Dicke narrowing effect. The application has been implemented following a strongly Object Oriented Programming approach which leads to an easier maintenance and future developments. The program runs under Windows environment and it has a standard graphical interface according to the version of the operation system currently in use. In order to test the program, the line parameters of some sulphur dioxide lines have been determined and compared with the literature data. Once validated, VLSFP has been used to obtain the line parameters of CH 2 CHF.The vinyl fluoride spectra in the 1148 cm -1 atmospheric region have been recorded with our TDL spectrometer at 299 K. The measurements have been carried out on a self-broadened CH 2 CHF sample with pressures in the range between 7 and 68 Pa: even at these low gas pressures the spectrum presents a high density of absorption lines. The self-broadening and integrated absorption coefficients for 13 lines belonging to the R branch of the ν 7 normal mode have been determined for the first time. The experimental line shapes showing deviations from the Voigt profile have also been 13 Page 13 of 51 URL: http://mc.manuscriptcentral.com/tandf/using the Nelkin -Ghatak profile. This has led to the determination of the selfnarrowing coefficient.

Table 1 .

 1 List of the dimensionless parameters of the normalized line shape functions. -1 ]. a α D denotes the 1/e Doppler half width. b The upper script 0 indicates quantities normalized to a pressure of 1 atm. 18 Page 18 of 51 URL: http://mc.manuscriptcentral.com/tandf/

  Broadening by thermal motion and internal state perturbing collisions; strong collision model for velocity changing collisions.

(

  a w(x, y) is the complex probability function. 19 Page 19 of 51 URL: http://mc.manuscriptcentral.com/tandf/

SO 2 ν 1 band also occurs. 20 Page

 20

Figure 1 .

 1 Figure 1. Block diagram representing the implementation of VLSFP. Each box

Figure 2 .

 2 Figure 2. A single line fit obtained employing VLSFP. Row (a) shows the

Figure 3 .Figure 4 .

 34 Figure 3.Multi -line fit of the SO 2 spectrum between 1089.69 and 1089.87 cm -1 :

Figure 5 .

 5 Figure 5.Vinyl fluoride spectrum between 1148.14 and 1148.6 cm -1 : (a)

  importance of vinyl fluoride, there exist no studies on the determination of the line parameters of the ro-vibrational absorption lines. The lack of literature data is partly motivated by the complex structure of the high resolution spectrum. In the present work we report the very first results obtained from the line shape analysis in the ν 7 band region around 1148 cm -1 by means of tunable diode laser (TDL) spectroscopy. In this region, the spectrum of vinyl fluoride appears very crowded even at low pressures. Hence, the analysis strongly requires a fitting software capable to simultaneously fit all the absorptions present in the spectral micro-windows acquired by the TDL system. For this reason we implemented a new software for the line shape fitting and the retrieval of the line parameters. The program, called Visual Line Shape Fitting Program (VLSFP), can fit several lines at once using different theoretical line shape functions. These include the Doppler, Lorentz and Voigt profiles, as well as the Galatry and Nelkin -Ghatak functions. The reliability of VLSFP has been tested by retrieving the sulphur dioxide line parameters and comparing them with the literature values. In this paper we also describe the program implementation and validation. In Section 2 the adopted experimental procedure is outlined while Sections 3 and 4 are concerned with the description of the fitting software and its test. Then in Section 5 we illustrate the results of the line shape analysis of vinyl fluoride. Conclusions and future work are addressed in Section 6.

3 .

 3 KBr windows. The gas sample pressures were measured employing an Alcatel ARD 1003 capacitance gauge with a full scale range of 1000 Pa and a quoted manufacturer's accuracy of 1.5 Pa. Before each measurements the cell was evacuated down to about 10 -4 Pa; an elapse time of 10 and 20 minutes, for SO 2 and CH 2 CHF respectively, between the filling of the cell and the spectrum acquisition was adopted.The spectra were calibrated employing the SO 2 line frequencies obtained by high resolution FTIR measurements. The SO 2 and CH 2 CHF gas samples were provided by Sigma -Aldrich (99.9% purity) and Peninsular Chemical Research Inc. (99% purity), respectively, and were used without any further purification; no buffer gasses were employed. The SO 2 spectra were acquired at 297 (± 1) K and the pressures were varied in the range between 15 and 800 Pa. The measurements on CH 2 CHF were carried out at 299 (± 1) K with pressures ranging from 7 to 68 Pa.Since the instrumental response of our TDL spectrometer is well described by a Gaussian function, its contribution to the line shapes was explicitly taken into account during the fitting procedure by fixing the Doppler half width to an effective value, and instrumental Doppler half widths, respectively. The value of eff D γ was retrieved by fitting the spectral lines recorded at low pressure (less than 30 Pa and 10 Pa for SO 2 and CH 2 CHF, respectively) to a Gaussian profile. Visual Line Shape Fitting Program implementation: an Object Oriented approach VLSFP has been written combining Microsoft Visual Basic and C# for the graphical interface and the computational kernel, respectively. The determination of the line parameters within a given theoretical model is performed through an iterative

  own task. The set of distinct machines then cooperate to accomplish an objective: in our case the fit of the experimental line shape by means of different theoretical profiles.

4 .

 4 Test of VLSFP with SO 2 lines

  (b). For this reason the lines have also been fitted employing the Nelkin -Ghatak profile. By using this strong collision model there is a dramatic improvement of the residuals as it is shown in Figure5(c): the standard deviation of the fit decreases by about a factor of two. Therefore, in the pressure range investigated the self-broadened vinyl fluoride sample appears as system strongly affected by the motional narrowing effect in which changing collisions and the internal state -dephasing collisions play an almost equal role in determining the shape of the spectral lines. Consequently, the line profiles accounting for the Dicke narrowing effect must be employed to properly model the experimental features.

6 .

 6 Conclusions and OutlookThe very first results of the line shape analysis of the vinyl fluoride infrared absorptions have been presented and discussed. In addition, the details of the implementation and validation of the fitting software (VLSFP) used for the analysis have been given.The program has been specifically designed to deal with the spectra recorded by means of laser based spectrometers in which the instrumental function can be modelled as a Gaussian or Lorentzian functions. Besides these two functions, the spectral absorptions can be fitted employing the Voigt profile as well as the weak and strong collision models which account for the Dicke narrowing effect. The application has been implemented following a strongly Object Oriented Programming approach which leads to an easier maintenance and future developments. The program runs under Windows environment and it has a standard graphical interface according to the version of the operation system currently in use. In order to test the program, the line parameters of some sulphur dioxide lines have been determined and compared with the literature data. Once validated, VLSFP has been used to obtain the line parameters of CH 2 CHF.The vinyl fluoride spectra in the 1148 cm -1 atmospheric region have been recorded with our TDL spectrometer at 299 K. The measurements have been carried out on a self-broadened CH 2 CHF sample with pressures in the range between 7 and 68 Pa: even at these low gas pressures the spectrum presents a high density of absorption lines.The self-broadening and integrated absorption coefficients for 13 lines belonging to the R branch of the ν 7 normal mode have been determined for the first time. The experimental line shapes showing deviations from the Voigt profile have also been Nelkin -Ghatak profile. This has led to the determination of the selfnarrowing coefficient.

  model) Collisional narrowing, Ω; Collisional narrowing parameter, 0 Ω [cm -1 atm -1 ]. shift, δ; Line shift coefficient, 0 δ [cm -1 atm -1 ].a α D denotes the 1/e Doppler half width.b The upper script 0 indicates quantities normalized to a pressure of 1 atm.

  x, y) is the complex probability function.

Figure 1 .

 1 Figure 1. Block diagram representing the implementation of VLSFP. Each box

Figure 2 .

 2 Figure 2. A single line fit obtained employing VLSFP. Row (a) shows the

Figure 3 . 23 Figure 4 .

 3234 Figure 3.Multi -line fit of the SO 2 spectrum between 1089.69 and 1089.87 cm -1 :

Figure 5 .

 5 Figure 5.Vinyl fluoride spectrum between 1148.14 and 1148.6 cm -1 : (a)

Table 1

 1 following the notation of Varghese and Hanson[19]. As it can be seen, the line parameters are normalized by the effective Doppler half width defined

as 7 Page 7 of 51 URL: http://mc.manuscriptcentral.com/tandf/tmph Molecular Physics

	ln	2
	F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y

Table 2 .

 2 Normalized line shape function implemented in VLSFP.

	Profile and Physical features	Equation a
	Doppler
	Inhomogeneous broadening by
	random thermal motion.

Table 3 .

 3 Obtained line parameters of the ro-vibrational transitions of the ν 1 band of SO 2 a employed to test VLSFP.

Table 4 .

 4 Obtained line parameters of the ro-vibrational transitions of the ν 7 band of CH 2 CHF a .

-21 cm⋅molecule -1 ]

								Molecular Physics
	10	1	10	11	1	11	1148.1499(8)	0.80(10)	0.78(2)	2.8(2)
	10	7	3	11	7	4	1148.1963(4)	-	0.69(6)	3.05(4)
	10	8	2	11	8	3	1148.30268(13)	0.48(4)	0.88(4)	2.88(2)
	9 8 10 9	1 0 9 2	8 8 1 7 F o 10 9 11 10 r	1 1 9 2	9 9 2 8	1148.3454(2) 1148.3689(2) 1148.4210(2) 1148.4327(2)	0.74(2) -0.31(2) 0.75(2)	0.80(2) -0.54(6) 0.39(3)	4.253(13) 0.72(2) 2.59(2) 4.29(2)
	9 20 10 9	1 1 10 3	8 19 0 6	9 20 P 2 2 11 10 e 10 3 e 7 18 1 7 r	1148.4574(4) 1148.5396(3) 1148.5513(3) 1148.5644(3)	0.51(5) --0.47(6)	0.87(10) --0.45(5)	0.63(3) 0.70(4) 0.85(2) 3.30(5)
	9 9	3 2	7 8	10 10	3 2	8 9 R	1148.5742(3) 1148.5792(3)	--	0.65(10) 0.84(6)	4.6(2) 5.3(3)
						e
						v i e
							w
							O n l
								y

a Figures in parentheses are one standard deviations. 21 Page 21 of 51 URL: http://mc.manuscriptcentral.com/tandf/tmph

Table 1 .

 1 List of the dimensionless parameters of the normalized line shape functions.

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 .

 2 Normalized line shape function implemented in VLSFP.

	Profile and Physical features	Equation a
	Doppler
	Inhomogeneous broadening by
	random thermal motion.

Table 3 .

 3 Obtained line parameters of the ro-vibrational transitions of the ν 1 band of SO 2 a employed to test VLSFP. From Ref.[35]; the quoted uncertainties have been derived from the error bars of Figures4, 6 and 8. Belonging to ν 1 + ν 2 -ν 2 of 32 SO 2 ; an overlapping with 16 13,3 17 14,4 of 34 SO 2 ν 1 band also occurs.

	0 self γ [cm -1 atm -1 ]	S 0 [10 -22 cm⋅ ⋅ ⋅ ⋅molecule -1 ]

a Figures in parentheses are one standard deviations. b From Ref. [34]. c d From Ref. [35].

e URL: http://mc.manuscriptcentral.com/tandf/tmph

Table 4 .

 4 Obtained line parameters of the ro-vibrational transitions of the ν 7 band of CH 2 CHF a . Figures in parentheses are one standard deviations.

	0 self γ [cm -1 atm -1 ]

a URL: http://mc.manuscriptcentral.com/tandf/tmph

URL: http://mc.manuscriptcentral.com/tandf/tmph Molecular Physics

Acknowledgments

The authors thank Prof. P. L. Varghese for the code for the computation of the Galatry function. The authors also gratefully acknowledge Prof. J. M. Flaud for the SO 2 data about ν 1 and ν 1 + ν 2 -ν 2 upon which the assignments have been made. This work was financially supported by PRIN 2007 (project: "Trasferimenti di energia, carica e molecole in sistemi complessi").

Acknowledgments

The authors thank Prof. P. L. Varghese for the code for the computation of the Galatry function. The authors also gratefully acknowledge Prof. J. M. Flaud for the SO 2 data about ν 1 and ν 1 + ν 2 -ν 2 upon which the assignments have been made. This work was financially supported by PRIN 2007 (project: "Trasferimenti di energia, carica e molecole in sistemi complessi").


MD Nicola Pietro Consilvio

MD Sabrina Di Pillo

MD Marcello Verini

MD Tommaso De Giorgis

MD Anna Cingolani

MD Valentina Chiavaroli

PhD Francesco Chiarelli

MD. Angelika Mohn
email: amohn@unich.it

G D'annunzio

The reciprocal influences of asthma and obesity on lung

Keywords: Childhood Asthma, Obesity, Fractional exhaled Nitric Oxide, Lung Function Testing, Airways Hyperresponsiveness Obesity, Asthma, Fractional exhaled Nitric Oxide, Lung Function Testing, Airways Hyperresponsiveness Body Mass Index-Standard Deviation Score, obesity, asthma, Fractional exhaled Nitric Oxide, Lung Function Testing, Airways Hyperresponsiveness

INTRODUCTION

Over several decades the prevalence of obesity has steadily increased reaching epidemic proportions. Current estimates of childhood obesity coming from the National Health and Nutrition Examination Survey (NHANES) revealed an increase from 13.8% in 1999-2000 to 16.0% in 2003-2004 for American females and from 14.0% to 18.2% in American males subjects. 1 In Europe, Italy together with other Mediterranean countries explicates the highest rate of obesity in children aged between 7 and 11 years, reaching almost 17%. 2 This has been accompanied by a similar rise in the rates of asthma. In fact, the CDC National Surveillance for Asthma revealed that its prevalence in children has risen from 3.5% to 7.5% over a period ranging from 2001-2003. 3 Similarly in developing countries undergoing their epidemiological transition phase comparable significant rises have been observed. 4 Accordingly, the World Health Organization (WHO) includes both obesity and asthma among the major chronic disorders representing world-wide public health priorities. 5 Given this dramatic rise in the prevalence of obesity and asthma, it is not surprising that there has been an increasing body of literature on the association between Body Mass Index (BMI) and asthma. 6 Obesity may have effects on both the upper and lower airway, 7 and this hypothesis is supported to a certain extent by the observation that BMI, the best single measure of body adiposity, 8 seems to be a reliable predictor of allergic respiratory symptoms and airways hyperresponsiveness (AHR). 9 In fact, recent literature emphasises the role of obesity in inducing a pro-inflammatory state due to increased levels of different pro-inflammatory cytokines. This might be implicated in the genesis of chronic inflammation of the airway system 10 similar to those observed in asthma which is characterized by persistent infiltration of eosinophils, T-lymphocytes and mast cells combined with different cytokines. In this respect exhaled Nitric Oxide (eNO) has been shown to tightly reflect this airway inflammation 11 and its fractional concentration in exhaled air (FeNO) has been recently accepted by the PRACTALL Consensus Report for the Diagnosis, Management and Treatment of Childhood Asthma as a logical complementary item in the follow-up of bronchial inflammation. The reported association between increased BMI and onset of AHR has been elegantly demonstrated by Litonjua et al. 13 in a longitudinal study of aging and has been further confirmed by two large cross-sectional studies performed in adults. 14,15 This is in contrast with other epidemiological studies performed in childhood where this association has not been universally observed. 16,17,18 This represents an important discordance of the recent literature as it might indicate that different factors such as duration of obesity and subsequent physiological changes of airway structures might play an important role in the development of obesity induced AHR.

Pre-pubertal children are a special study population as the shorter duration combined with better control of both diseases eliminates confounding factors commonly present in older subjects. In fact, this kind of population offers the exclusive opportunity to tangle whether obesity and asthma explicate an independent effect on lung function testing or influence themselves in a reciprocal manner.

Therefore, with the primary aim to establish the possible dependent or independent role of asthma and obesity on airway system function we evaluated AHR and bronchial inflammation expressed by FeNO in a group of obese and non obese asthmatic children in comparison to non-asthmatic obese and normal-weight pre-pubertal children. Asthma was classified according to Global Initiative for Asthma (GINA) guidelines 19 as intermittent asthma, mild persistent, moderate persistent and severe persistent asthma. The diagnosis was established by a single paediatric respiratory physician on the basis of a clinical history of repeated episodes of coughing, dyspnoea and wheezing, according to American Thoracic Society -European Respiratory Society (ATS-ERS) criteria. 20 Only subjects who fulfilled the definition of intermittent/mild persistent asthma were recruited. Low dose inhaled corticosteroids (ICSs) therapy was withdrawn for not more than weeks before the study evaluation. Over this period all children were closely monitored by the medical staff of the Paediatric Allergy and Respiratory Clinic, none experienced any adverse event and at the time of the study evaluation all subjects were symptom-free.

Obesity was defined as Body Mass Index [BMI] above 2 Standard Deviations (SD) for the mean of age and sex. 21 In all subjects endocrine diseases as cause for obesity were excluded prior to study entry. None of the non-asthmatic obese children had a personal history for asthma and allergic diseases.

We recruited as controls a group of healthy children, comparable for age, gender, and pubertal stage, who were admitted to the Department of Paediatrics for minor diseases. None of the children had a personal history for asthma and allergic diseases, and all were otherwise in a good health.

Anthropometric measurement and respiratory function were assessed only after complete recovery of the disease. Exclusion criteria for all children were: presence of upper or lower respiratory infections including asthma during the previous 4 weeks, congenital abnormalities of cardio-respiratory system, chest or skeletal deformities, or neuromuscular system disease, chronic and autoimmune disease, gastrooesophageal reflux, presence of medical condition in which exercise was forbidden. None of the children was taking any medication.

Based on the presence or absence of asthma and according to BMI, the study population was divided into four groups: Written informed consent was obtained from all parents and oral consent from all children.

The study was approved by Ethical Committee of University of Chieti.

Methods

Anthropometric measurements

Body weight was determined to the nearest 0.1 Kg, and height was measured in triplicate with Harpenden stadiometer to the nearest 0.1 cm. As fatness indices, we used BMI (the weight in kilograms divided by the square of the height in meters). SDS-height and BMI-SDS were calculated

Spirometric and Flowmetric Measurements: Baseline Flow/Volume curves

Lung function evaluation was performed by Flow/Volume curves (KitMicro Spirometer 7.3a -COSMED) in the standing position with nose-clip until two consecutive technically acceptable curves were achieved, according to ATS/ERS Guidelines. 23 The main spirometric parameters included: Forced Expiratory Volume in 1 sec (FEV 1 ), Forced Vital Capacity (FVC), FEV 1 /FVC, and Forced Expiratory Flows between 25% and 75% of the FVC (FEF 25-75 ) and the results are expressed as Z-scores according to Stanojevic S et al. 24 Peak Expiratory Flow (PEF) and Maximal Expiratory Flows at 75% of the FVC (MEF 75 ) were expressed as a percentage of theoretical values for age, gender, and height according to Quanjer PH et al. 25

Six-Minute Walk Test (6MWT)

In order to estimate the bronchial hyperresponsiveness, after baseline lung function testing, a 6MWT was performed according to standard protocol. 26 The target of the test was to achieve 6 minutes of free walk until the hearth-rate was 80-90% of the predicted maximum (calculated as 220 -age in years), according to ATS/ERS recommendations for exercise challenge test in older children. 27 Spirometry was performed in duplicate sets at 1, 3, 5, 10 and 20 minutes post-exercise.

The better of the two curves was selected as the representative value at each interval, but differences between the two values of FEV 1 had to be <5%. Exercise response was defined as the greatest decrease in FEV 1 expressed as a percentage of the baseline values. A bronchial obstruction response to exercise was considered positive when the FEV 1 reduction from baseline was greater than 12%. 28 29,30

Allergic sensitization

Skin

Respiratory inflammation marker

FeNO was determined with an on-line method using a single breath exhalation and a sensitive chemiluminescence assay (Ecomedics CLD 88), according to ATS-ERS. 31 Patients made an inspiration of eNO-free air via a mouthpiece immediately followed by full exhalation at a constant rate (50 ml/sec) for at least 5 seconds. The mean of three readings at the end of the expiration (plateau phase) was taken as the representative value for each measurements. 12 ppb or more were considered positive, according to ATS-ERS criteria. 32

Statistical analysis

All values were expressed as Median and Interquartile Range. Kruskal Wallis Test was performed for evaluating differences between the four groups (ObA, NwA, Ob, Nw). Statistical significance level was p<0.05. Subsequently, differences within the single groups were tested by Mann-Whitney Test, adjusting the significance level by p<0.008. Differences in functional respiratory baseline parameters and after 6MWT within each group were analyzed using Wilcoxon Signed Ranks Test. Categorical variables were tested by Pearson's chi-square (X 2 ) test.

A multiple analysis of covariance (MANCOVA) was performed to investigate the independent effect of obesity and asthma on respiratory indices (PEF, MEF 75 and FEV 1 /FVC) and bronchial inflammation (FeNO), after adjusting for gender, age and height.

RESULTS

Anthropometric measurements and respiratory functional data of the whole study population are reported in Table 1. The four groups were similar for age, gender, and SDS-height.

As expected BMI-SDS was similar between ObA and Ob children, but was significantly different between obese children and normal-weight children (ObA vs NwA p= 0.0004; ObA vs Nw: p=0.0008; Ob vs NwA p=0.0001; Ob vs Nw: p=0.0002).

All the asthmatic children were sensitized to Dermatophagoides Pteronyssinus.

Baseline Respiratory Functional Parameters

No significant differences were found for FVC, FEV 1 , and FEF 25-75 Z-scores between the four groups, whereas FEV 1 /FVC Z-score resulted significantly different (Tab 1). In fact, this parameter was significantly lower in the ObA group compared to the Nw group (p=0.005). Moreover a significant difference was detected between the four groups in terms of PEF (p=0.03) and MEF 75 (p=0.001) ( Fig 1). By comparing the four groups directly no further significances could be detected for PEF, whereas MEF 75 was significantly lower in the ObA group and the Ob group compared to the Nw group (p=0.004 and p=0.0001, respectively)

Respiratory Functional Parameters after 6MWT

After 6MWT no significant differences were found in post-FVC and post-FEF 25-75 Z-scores among the four groups (Tab 1). Although post-FEV 1 /FVC Z-scores resulted significant different between the four groups, no further differences were detected by comparing the four groups directly. In

Respiratory inflammation marker

FeNO values were significantly different across all four groups (p=0.0002) (Tab. 1).

In fact FeNO values were significantly higher in the ObA group and the NwA group compared to the Ob group (p=0.008 and p=0.0002, respectively) and to the Nw group (p=0.0001 and p=0.0003, respectively).

Furthermore FeNO was significantly higher in the Ob group when compared to the Nw group (p=0.0004). In contrast no differences were detected between the two asthmatic groups.

Effect of asthma and obesity on respiratory functional parameters and bronchial inflammation

In order to investigate the role of asthma and obesity on resulted significant different baseline respiratory variables and FeNO, a multiple analysis of covariance was performed. Asthma was significantly and independently related to FeNO values, whereas no association was found for obesity. In contrast obesity was significantly and independently related to PEF and MEF 75 . In the same model no relationship was found for asthma and obesity on FEV 1 /FVC Z-score (Table 3).

DISCUSSION

Although the effect of obesity on pulmonary function tests has been extensively investigated in adults, the number of similar studies in children is limited and confirm the controversial results observed in adults between obesity and AHR. In this study we compare for the first time obese asthmatic with obese non-asthmatic pre-pubertal children and healthy controls with the aim to analyse the reciprocal influences of obesity and asthma on lung function and bronchial inflammation. Our findings clearly demonstrate that while an asthmatic status is tightly and directly involved in AHR and bronchial inflammation, obesity mainly affects the upper airways flows.

As obese children often experience dyspnoea and breathlessness during physical activities it is a crucial clinical question as to whether these symptoms are effectively related to AHR such as in asthmatic children. In order to explore this possibility a 6MWT was performed. While basal values of spirometric indices were normal in all examined groups, in ObA and NwA children exercise challenge induced AHR documented by the reduction of functional respiratory parameters. In contrast in Ob and Nw children no changes of any of the obtained variables were detected. This is confirmed by the number of children exhibiting a positive response to exercise defined as fall in FEV1 greater than 12%. In fact within the asthmatic children 12 subjects met this definition compared to only 2 obese children. In agreement with our data, epidemiological studies do not uniformly support the conclusions that obesity leads to airflow limitation, AHR or asthma. In this respect Schachter and co-workers showed in a group of 1971 adults, that although BMI was associated with a clinical diagnosis of asthma and subjective symptoms of dyspnoea and wheeze, airflow obstruction or AHR subsequent to histamine challenge could not be detected in the sole obese subjects. 16 Similar findings were obtained by Bibi et al. 17 who demonstrated in a large population of pre-pubertal children that bronchodilation could not be induced by salbutamol inhalation in non-asthmatic obese subjects. 33 These data demonstrate that obesity should not be considered as being a risk factor for AHR in pre-pubertal children. Furthermore asthma like symptoms especially during physical activity should not be overestimated by parents and medical Interestingly in the ObA and Ob groups we documented lower basal values of MEF 75 when compared to healthy controls. Furthermore PEF values were significantly different across the four groups with a tendency of lower values in the two obese groups. In fact, by performing a multiple analysis of covariance obesity was significantly and independently related to MEF 75 and to PEF, while this association was not found for asthma. Similar data were documented by two previous studies 34,35 where obesity was associated with a significant reduction in FEF 25-75. These data suggests that obesity might play a primary role on upper airway flows, and despite being possibly explained by fatty infiltration of muscles narrowing directly the upper airway, recent literature suggest that this functional parameter might be used as an early predictor of sub-clinical asthma. 36 In order to obtain a complete evaluation of the bronchial status we performed also FeNO analysis.

In effect, recent studies revealed that FeNO is a potentially useful measure to evaluate the role of airways inflammation in asthma, as it represents the forerunner of the most important event namely remodelling of bronchial airways, 37 offering therefore a logical complementary item to the only use of clinical and functional data. In our study, as expected, both groups of asthmatic children presented highly elevated FeNO values when compared to Ob children and normal controls reflecting the underlying bronchial inflammation process. However, data from animal and human studies clearly demonstrate that the enhancement of normal adipose tissue in obesity leads to a systemic pro-inflammatory state. 38 Adipose tissue from obese individuals expresses a number of pro-inflammatory molecules, such as leptin, tumor necrosis factor-α, IL-6, transforming growth factor-β1, and C-reactive protein, and there appears to be significant overlap between the immune function of adipocytes and the function of T-lymphocytes and macrophagues, particularly with respect to the elaboration of inflammatory cytokines. 39,40 Although a conclusive relationship between obesity and airway inflammation has not yet to been described, it could be argued that the 41 who found a positive correlation between FeNO and BMI in healthy children.

However it needs to be acknowledged that in the multiple analysis of covariance obesity was not a predictor for increased FeNO values as only asthmatic status remained as such. This apparent contrasting findings might be partially explained by the age group of our study population as obesity related inflammation might not have reached its full implication and a possible involvement of the respiratory system might require a long standing history of obesity.

In this respect an important limitation of this study is the cross sectional design which does not allow to speculate on the full clinical implications of our findings. In fact, as puberty is a well known risk factor for developing obesity related complications it might be of crucial interest to obtain longitudinal data starting in pre-puberty and lasting through all pubertal stages. This would allow to fully explore this intriguing relationship and to not underestimate the possible role of obesity on the development of bronchial inflammation.

In conclusion, while AHR and airways inflammation are clearly associated to an asthmatic status, obesity seems to induce reduction of upper airway flows combined with a certain degree of early pro-inflammatory airways changes. Longitudinal data are required in order to clarify whether these changes might represent risk factor for asthma later on in life. MEF75% was significantly lower in obese asthmatic and non-asthmatic children compared to healthy controls and the influence of obesity on MEF75% was confirmed by multiple linear regression. After the "free-run" Exercise Challenge Test all analysed respiratory parameters decreased significantly in asthmatic obese and non-obese children, in contrast in non-asthmatic obese children no differences were found. FeNO analysis demonstrate significantly higher values in asthmatic children compared to obese non-asthmatic and healthy controls. Furthermore FeNO was significantly higher in obese non-asthmatic children when compared to healthy controls.

In conclusion, while AHR and airways inflammation are clearly associated to an asthmatic status, obesity seems to induce early proinflammatory airways changes combined with reduction of higher intrathoric airway flows. Examination Survey (NHANES III) revealed a prevalence of obesity -defined as a body mass index (BMI) in excess of 95% of the age specific distribution -of 14% in children aged 6 -11 surveyed between 1988 and 1994. 1 In comparison NHANES II, conducted between 1976 and 1980, found a prevalence of obesity of 7.6% for the same population. This has been accompanied by a similar rise in the association rates of asthma. The CDC self-reported prevalence of asthma in children aged 5 -14 rose from 42.8 per 1000 in 1980 to 74.4 per 1000 in 1994. 2 Epidemiologists have reported that both obesity and asthma are "endemic" in developed countries 3,4 and "epidemic" in developing countries undergoing their epidemiological transition phase. 5 Accordingly, the World Health Organization (WHO) includes both obesity and asthma among the major chronic disorders representing world-wide public health priorities. 6 Given the dramatic rise in the prevalence of obesity and asthma, it is not surprising that there has been an increasing body of literature on the association between BMI and asthma. 7 Obesity may have effects on both the upper and lower airway; 8 and this hypothesis is supported to a certain extent by the observation that BMI, the best single measure of body adiposity, 9 seems to be a reliable predictor of allergic respiratory symptoms and airways hyperresponsiveness (AHR). 10,11,12 Infiltration by eosinophils, mast cells and T lymphocytes is a key feature of airway inflammation in asthma 13 and an increased eosinophil number in induced sputum of asthmatic children is tightly linked to current asthma symptoms and AHR. 14 Recently, exhaled Nitric Oxide (eNO) has been shown to have a good correlation with airway eosinophilic inflammation 15 and its fractional concentration in exhaled air (FeNO) has been accepted to be a useful indicator of airway inflammation. On the other hand recent literature emphasises the role of obesity in inducing a proinflammatory state as obesity is associated with increased levels of leptin and proinflammatory 4 cytokines such as interleukin (IL)-6 and IL-1 which might be implicated in a possible up-regulate inflammation of the airway system. 16 The reported association between increased BMI and onset of AHR has been elegantly demonstrated by Litonjua et al. 17 in a longitudinal study of aging; and has been further confirmed by two large cross-sectional studies. 18,19 This is in contrast with epidemiologic studies where this Children with values between -2 and 2 SD were defined normal-weight, while children with BMI above 2 SD were defined obese. 20,21 -Asthma was classified according to GINA guidelines [START_REF] Lindgren | Swedish population standards for height, weight and body mass index attained at 6 to 16 years (girls) or 19 years (boy)[END_REF] as intermittent asthma, mild persistent, moderate persistent and severe persistent asthma. All subjects had been admitted for intermittent/mild persistent asthma and the diagnosis was made by a pediatric respiratory physician on the basis of clinical history of repeated episodes of coughing, dyspnea and wheezing, according to ATS-ERS criteria. 23 To evaluate the bronchial reactivity was made an Exercise Challenge Test.

Moreover, to evaluate the bronchial inflammation all children underwent FeNO analysis. To evaluate the allergic sensitization we made Skin Prick Test (SPT) and serum specific IgE measurements for the most common respiratory allergens. Asthmatic children had received an inhaled corticosteroids (ICS) therapy, but were free from therapy for at least 2 weeks before the study evaluation; and all obese children were therapy-free.

All the subjects were symptom-free and had no clinical evidence of cardiopulmonary disease in a preceding evaluation. Exclusion criteria from the study for all groups were: presence of upper or lower respiratory infection or exacerbation of asthma during the previous 4 weeks, not well controlled asthma according to current Global Initiative for Asthma (GINA) classification of asthma, 24 congenital abnormalities of cardiorespiratory system, chest or skeletal deformities, or neuromuscular system disease, chronic and autoimmune disease, gastroesophageal reflux, presence of medical condition in which exercise was forbidden.

Written informed consent was obtained from all parents and oral consent from all children.

The study was approved by Ethical Committee of University of Chieti.

Methods

Anthropometric measurements

Body weight was determined to the nearest 0.1 Kg, and height was measured in triplicate with Harpenden stadiometer to the nearest 0.1 cm.

We used BMI for fatness indexes (the weight in kilograms divided by the square of the height in meters); and also BMI expressed as a continuous variable in standard deviation score (BMI-SDS) units (z-score), determined as difference between the individual observed value and the reference mean for age and sex, divided by the corresponding standard deviation. 25

Functional respiratory data

Evaluations were made by Flow/Volume curves (KitMicro Spirometer 7.3a -COSMED), according ATS/ERS Guidelines, 26 and the main spirometric tests were considered: Forced Expiratory Volume in 1 sec (FEV1), Forced Vital Capacity (FVC), Peack Expiratory Flow (PEF) and Maximal To evaluate the bronchial hyperresponsiveness measurements are performed in the standing position, with nose-clip until two consecutive technically acceptable curves are achieved. After the free-run, spirometry was measured in duplicate sets at 1, 3, 5, 10 and 20 minutes post-exercise. The better of the two curves was selected as the representative value at each interval, but differences between the two values of FEV1 had to be <5%. The target was to achieve 6 minutes of "free run" to increase hart-rate to 80% in the maximum for at least 4 minutes according to ATS/ERS recommendations for exercise challenge test in older children. 27,28 Exercise response was defined as the greatest decrease in FEV1 expressed as a percentage of the baseline values. A bronchoconstriction response to exercise was considered as positive when the FEV1 decrease from baseline was greater than 13%. 29

Allergic sensitization

Was evaluated by Skin Prick Test (SPT) and serum specific IgE measurements for the most common respiratory allergens: Dust Mite (Dermatophagoides Pteronyssinus and Farinae), Grass, Parietaria, Artemisia Vulgaris, Olive, Cypress, Lime, Stone, Elm, Plane, Cat and Dog dander, Alternaria Alternata and Aspergillus Fumigatus (moulds). Determination of Allergen-Specific IgE was made by an Immunoenzymatic Allergo-sorbent Test (Cap test Pharmacia). 30,31

Respiratory inflammation marker

FeNO was determined with an on-line method using a single breath exhalation and a sensitive chemiluminescence assay (Ecomedics CLD 88), according to ATS-ERS. 32 Patients made an inspiration of eNO-free air via a mouthpiece immediately followed by full exhalation at a constant rate (50 ml/sec) for at least 5 seconds. The mean of three readings at the end of the expiration F o r P e e r R e v i e w 8 (plateau phase) was taken as the representative value for each measurements. 12 ppb or more were considered positive, according to ATS-ERS criteria. 33

Statistical analysis

All values were expressed as Means and SD. Kruskal Wallis Test was performed for evaluating differences between the four groups (ObA, NwA, Ob, Nw). Statistical significance level was p<0.05. Subsequently, differences within the single groups were tested by Mann-Whitney Test, adjusting the significance level by p<0.0125. Differerences in functional respiratory parameters preand post-"free-run" exercise Challenge Test were analysed using Wilcoxon Signed Ranks Test within each group.

A multiple linear regression analysis was performed to asses the independent contribution of obesity (BMI-SDS) and asthma on respiratory inflammation and respiratory functional parameters.

FeNO, PEF% and MEF75% were the dependent variables and BMI-SDS and asthma were the

RESULTS

Anthropometric measurements and respiratory functional data are reported in table 1.

The four groups were similar for age, gender, and height.

All the asthmatic children were allergic to Dermatophagoides Pteronyssinus (Df. pt).

As expected BMI and BMI-SDS was similar between obese asthmatic and obese non-asthmatic children, but was significantly different between obese and normal-weight children (ObA vs Ob: p>0.05; ObA vs NwA: p= 0.0004; ObA vs Nw: p=0.0008; Ob vs NwA: p=0.0001; Ob vs Nw: p=0.0002).

Baseline Respiratory Functional Parameters

No significant differences were found for FVC%, FEV1%, MEF50% and MEF25% between the four groups.

Despite detecting a trend of difference in PEF% between the four groups, no differences were found when performing sub-analysis between ObA vs NwA, ObA vs Ob, ObA vs Nw, NwA vs Ob, NwA vs Nw, and Ob vs Nw.

We found that MEF75% was significantly lower in OA group and OnA group compared to NnA group (p=0.004 and p=0.0002, respectively) (Table 1).

Respiratory Functional Parameters after "free run" Exercise Challenge Test

No significant differences were found in FEV1%, FVC%, MEF50% and MEF25% after the exercise test among the four groups.

After the "free-run" exercise Challenge Test we found statistically significant differences in PEF% and MEF75%. In fact NnA had significantly higher values of these two parameters compared to OA (p=0.001 for PEF% and p=0.002 for MEF75%); furthermore obese non-asthmatic children had significantly lower value of MEF75% compared to the NnA group (p=0.007) (Table 1). 2, analysing functional respiratory data within each group after the "free-run" exercise Challenge Test, we found that all analysed parameters, with the exception of FVC% for the OA group and of MEF25% for the NA group, decreased significantly in asthmatic obese and nonobese children (without clinical and functional evidence of obstruction -all indices were in the normal range for age and height). In contrast in non-asthmatic obese children we did not detect any statistical significant differences in all functional respiratory parameters after "free run" exercise Challenge Test.

Respiratory inflammation marker

FeNO values were significantly different across all four groups (p=0.0002).

In fact FeNO values were significantly higher in the ObA group compared to Ob group and to the Nw group (p=0.011 and p=0.0001, respectively); and were significantly higher in the NwA group compared to Ob group and to the Nw group (p=0.0002 and p=0.0003, respectively). We did not detect any statistical significant difference in FeNO values between the asthmatic children (p=0.201). Furthermore FeNO was significantly higher in the Ob group when compared to the Nw group (p=0.0004) (Figure 1).

Independent contribution of BMI-SDS and asthma on respiratory inflammation and respiratory functional parameters

In a multiple linear regression analysis with FeNO as the dependent variable and BMI-SDS and asthma (asthmatic vs non-asthmatic children) as the independent variables, asthma was significantly and independently related to FeNO values, whereas no association was found for obesity (BMI-SDS). In a second multiple linear regression model with PEF% as the dependent variable and BMI-SDS and asthma as the independent variables, BMI-SDS was significantly and independently related to PEF% values. A further multiple linear regression analysis with MEF75% as the 11 dependent variable and BMI-SDS and asthma as the independent variables, showed again that BMI-SDS was significantly and independently related to MEF75% values (Table 3). In order to evaluate the relationship between obesity and AHR and to detect whether breathlessness experienced by obese children during physical activities is effectively due to the airway hyperresponsiveness we performed a "free-run" Exercise Challenge test. While basal values of spirometric indexes were normal in all examined groups, in asthmatic obese and non-obese children free run induced AHR documented by reduction of all functional respiratory parameters. In contrast in obese children no changes in these functional respiratory parameters were detected. In addition, in a multiple linear regression we found that asthma remained the only predictor for AHR. In agreement to our data, epidemiological studies do not uniformly support the conclusions that obesity leads to airflow limitation, AHR or asthma. In this respect Schachter and coworkers showed in a group of 1971 adults, that although BMI was associated with a clinical diagnosis of asthma and subjective symptoms of dyspnea and wheeze, airflow obstruction or AHR subsequent to histamine challenge could not be detected in the sole obese subjects. 34 Similar findings were obtained by Bibi et al. 35 who demonstrated in a large population of prepubertal children that broncodilation could not be induced by salbutamol inhalation in non-asthmatic obese subjects. 36 These data demonstrate that obesity should not be considered as being a risk factor for AHR in prepubertal children.

Furthermore asthma like symptoms especially during physical activity should not be overestimated by parents and medical staff. This is of paramount relevance as it might induce inappropriate treatment and refrain children from performing sports leading to a vicious circle impeding improvement of both obesity and respiratory symptoms. Interestingly in obese asthmatic children and obese non-asthmatic children we documented lower basal values of MEF75% when compared to healthy controls. Furthermore by performing a multiple linear logistic regression analysis obesity, defined as BMI-SDS, was significantly and independently related to MEF75%; while no association was found for asthma. Similar data were documented by two previous studies 37,38 where obesity was associated with a significant reduction in FEF 25-75. These data suggests that obesity might play a primary role on higher intrathoracic airway flows and despite being possibly explained by fatty infiltration of muscles narrowing directly the upper airway, recent literature suggest that this functional parameter might be used as an early predictor of subclinical asthma. In order to obtain a complete evaluation of bronchial status in this study we performed also FeNO analysis. In fact recent studies revealed that FeNO is a potentially useful measure to evaluate the role of airways inflammation in asthma, as it represents the forerunner of the most important event namely "remodelling" of bronchial airways. 39 In fact FeNO, as recommended in the Consensus Report Practall for the Diagnosis, Management and Treatment of Childhood Asthma 40 offers a logical complementary item to the only use of clinical and functional data, evaluating the bronchial eosinophilic inflammation. In our study, as expected, both groups of asthmatic children presented highly elevated FeNO values when compared to normal controls reflecting the underlying bronchial inflammation process. Data from animal and human studies clearly demonstrate that the enhancement of normal adipose tissue functions in obesity leads to a systemic proinflammatory state. 41 Adipose tissue from obese individuals expresses a number of proinflammatory molecules, such as leptin, tumor necrosis factor-α (TNF-α), IL-6, transforming growth factor-β1 (TGF-β1), and C-reactive protein (CRP), and there appears to be significant overlap between the immune function of adipocytes and the function of T-lymphocytes and macrophagues, particularly with respect to the elaboration of inflammatory cytokines. [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF][START_REF] Wellen | Obesity-induced inflammatory changes in adipose tissue[END_REF] Although a conclusive relationship between obesity and airway inflammation has not yet to been described, it could be argued that the role of the proinflammatory state underlying the main metabolic and cardiovascular complications in obesity might play a pivotal role in determining airways study where FeNO values were significantly higher in obese non-asthmatic children compared to healthy controls and the findings of Santamaria et al. [START_REF] Santamaria | Relationship between exhaled nitric oxide and body mass index in children and adolescent[END_REF] who found a positive correlation between FeNO and BMI in healthy children. However it needs to be acknowledged that in the multiple regression analysis obesity was not a predictor for increased FeNO values. This apparent contrasting findings might be partially explained by the age group of our study population as obesity related inflammation might not have reached its full implication and a possible involvement of the respiratory system might require a long standing history of obesity. As puberty is a well known risk factor for developing obesity related complications it might be of crucial interest to obtain longitudinal data starting in prepuberty and lasting through all pubertal stages in order to not underestimate the possible role of obesity on the development of bronchial inflammation and therefore to fully explore this intriguing relationship.

In conclusion, while AHR and airways inflammation are clearly associated to an asthmatic status, obesity seems to induce early proinflammatory airways changes combined with reduction of higher intrathoric airway flows. Longitudinal data are required in order to clarify whether these changes might represent risk factor for asthma later on in life. We thank you and the referees for the detailed review and for the precious comments, which we have addressed thoroughly thereby improving our manuscript substantially.

We have tried to answer the major comments of referee 1 and 2 and please find enclosed a point-bypoint answer sheet for each of them.

In agreement with your suggestion we have now calculated and expressed the results of the lung function tests as Z-score, according to the equations proposed by Stanojevic et al. (AJRCCM 2008).

However, we continue to present the values of MEF 75 and PEF as %-expected because for these parameters the equations for Z-score were not available and the main differences detected in our study regarded these indices of lung function.

Furthermore, as requested we added all necessary information concerning our inclusion and exclusion criteria in the method section. We have modified the introduction in order to define in a clear way the main hypothesis of our study. Finally the most important limitation consisting of the cross sectional nature of this study has been discussed.

Please find enclosed the revised version of our manuscript where the changes have been marked-up.

We hope that our findings on this intriguing relationship between obesity and asthma are of interest to your readership and hope that the manuscript is now acceptable for publication in your prestigious

12

 12

  measurements and respiratory parameters of a group of 118 Caucasian prepubertal children referred to the Children Obesity Clinic, to the Paediatric Allergy and Respiratory Clinic and to the Department of Paediatrics, University of Chieti, Italy, were sequentially evaluated between May 2008 and August 2008.

1 .

 1 Obese Asthmatic Children [ObA] (sixteen boys and 14 girls); 2. Normal-weight Asthmatic Children [NwA] (sixteen boys and 14 girls); 3. Non-Asthmatic Obese Children [Ob] (fourteen boys and 16 girls); 4. Non-Asthmatic Normal-weight Children [Nw] (fourteen boys and 14 girls). All children underwent a complete physical examination including anthropometric parameters [height, weight]. Pubertal stage was defined on the basis of breast development in girls and genital development in boys according to the criteria of Tanner (all subjects had pre-adolescent characteristics corresponding to stage 1). Respiratory function was assessed by standard Flow/Volume curves at baseline and after Six-Minute Walk Test (6MWT), and respiratory inflammatory status was assessed by FeNO. Only asthmatic children performed allergy evaluation by Skin Prick Test (SPT), and blood sample was taken for specific IgE measurement.

  the individual observed value and the reference mean for age and sex, divided by the corresponding standard deviation.[START_REF] Lindgren | Swedish population standards for height, weight and body mass index attained at 6 to 16 years (girls) or 19 years (boy)[END_REF]

  Statistical significance level was p<0.05. All calculations were made with the computer program SPSS (Statistical Package for the Social Science), version 16.0 software for Windows.

  contrast, post-FEV 1 Z-score resulted significantly lower in NwA children when compared to Ob children and Nw children (p=0.006 and p=0.006, respectively). Post-PEF and post-MEF 75 resulted significantly different between the four groups (p=0.01, both). In fact, post-PEF was significantly lower in ObA children when compared to Nw children (p=0.002). This was associated with significantly lower values of post-MEF 75 in the ObA group and the Ob group when compared to the Nw group (p=0.003 and p=0.007, respectively).

  parameters after 6MWT within each group are shown in Table2. Post-FEV 1 /FVC Z-score was significantly lower in the ObA group, while in NwA children a significant reduction was detected for post-FEV 1 , post-FVC and post-FEF 25-75 Z-scores. In contrast no changes were detected for the Ob and Nw groups.A positive response to exercise challenge was found in 5 ObA children and 7 NwA children compared to only 2 Ob children. As expected none of the healthy control children experienced a fall in FEV 1 greater than 12%.

  is of paramount relevance as it might induce inappropriate treatment and refrain children from performing sports leading to a vicious circle impeding improvement of both obesity and respiratory symptoms.

  pro-inflammatory state underlying the main metabolic and cardiovascular complications in obesity might play a pivotal role in determining airways inflammation independently of any allergic mechanisms. This might explain the findings of our study where FeNO values were significantly higher in Ob children when compared to healthy controls, and the findings of Santamaria et al.

1 2 ABSTRACT

 12 The reciprocal influences of asthma and obesity on lung function in prepubertal children.Consilvio Nicola Pietro, MD; Di Pillo Sabrina, MD; Verini Marcello, MD; Tommaso de Giorgis, MD; Anna Cingolani, MD; Fabio Masuccio, MD; Francesco Chiarelli, PhD; Angelika Mohn, MD.As the incidence of obesity and asthma increase consistently, the reciprocal influences of both on lung function, airways hyperresponsiveness (AHR) and bronchial inflammation were analysied in a group of 118 caucasian prepubertal children.Anthropometric measurements and respiratory parameters (Flow/Volume curves, "free run" Exercise Challenge Test, FeNO) were evaluated and the study population was divided into four groups according to Body Mass Index and the presence or absence of asthma: Obese Asthmatic, Normal-weight Asthmatic, Obese non-Asthmatic, Normal-weight non-Asthmatic children.

INTRODUCTION

  Over the past 20 years the incidence and prevalence of obesity have steadily increased. The most recent data from the Centers for Disease Control's (CDC) Third National Health and Nutrition

  association has not been universally observed, indicating that different factors such as duration of obesity, physiological changes of airway structures might play an important role on the development of this relationship. Prepubertal children might represent a special study population in order to investigate the reciprocal and independent influences of obesity and asthma on lung function testing, and bronchial indices of flogosis as both diseases should not necessarily having caused physiological changes commonly observed in older subjects. We therefore evaluated AHR and bronchial inflammation expressed by FeNO in obese and non obese asthmatic children in comparison with non asthmatic obese and normalweight prepubertal children. measurements and respiratory parameters of a group of 118 Caucasian prepubertal children referred to the Department of Pediatrics, Children Obesity Clinic and to the Allergological and Pneumological Unity of the Pediatric Department, University of Chieti, Italy, between May 2008 and August 2008, were evaluated. According to Body Mass Index [BMI], and based on the presence or absence of asthma, the study population was divided into four groups: 1. Obese Asthmatic Prepubertal Children [ObA] (16 M and 14 F); 2. Normal-weight Asthmatic Prepubertal Children [NwA] (16 M and 14 F); 3. Non-Asthmatic Obese Prepubertal Children [Ob] (14 M and 16 F); 4. Non-Asthmatic Normal-weight Prepubertal Children [Nw] (14 M and 14 F) -Pubertal stage was defined on the basis of breast development in girls and genital development in boys according to the criteria of Tanner (all subjects had pre-adolescent characteristics corresponding to stage 1). -A complete physical examination including [height, height Standard Deviation Score (SDS), weight, weight-SDS, BMI, BMI-SDS].

7 Expiratory

 7 Flows at 75%, 50% and 25% of the Forced Vital Capacity (MEF75%, MEF50%, MEF25%). All the tests were expressed as a percentage of theoretical values for eight.

  independent variables. Statistical significance level was p<0.05. All calculations were made with the computer program SPSS (Statistical Package for the Social Science), version 16.0 software for Windows.

DISCUSSION

  This is the first study comparing obese asthmatic with obese non-asthmatic children and healthy controls with the aim to analyse the reciprocal influences of obesity and asthma on lung function in prepubertal children. Although the effect of obesity on pulmonary function tests has been extensively investigated in adults, the number of similar studies in children is limited and confirm the controversial results between obesity and AHR observed in adults.

  any allergic mechanisms. This might explain the findings of our

Page 40 of 42 John

 42 i a t r i c D e p a r t m e n t (Chief: Prof. Francesco Chiarelli) Chieti, Italy, March 6 th 2010 Dear Editor, Thank you for giving us the possibility of revising and resubmitting our manuscript PPUL-09-0317 entitled "The reciprocal influences of asthma and obesity on lung function testing, AHR and airway inflammation in prepubertal children."

  University of Chieti Ospedale PoliclinicoVia dei Vestini 5 66100 Chieti, Italy Fax: +39 0871-574831 E-mail: amohn@unich.it

  Prick Test (SPT) were performed and serum specific IgE were measured for the most common

	Artemisia Vulgaris, Olive, Cypress, Lime, Stone, Elm, Plane, Cat and Dog dander, Alternaria
	Alternata and Aspergillus Fumigatus (moulds). Determination of Allergen-Specific IgE was made
	by an Immunoenzymatic Allergo-sorbent Test (Cap test Pharmacia).
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	respiratory allergens: Dust Mite (Dermatophagoides Pteronyssinus and Farinae), Grass, Parietaria,
	8

Table 1 .

 1 Anthropometric measurements and respiratory parameters of the study population.

		ObA	NwA	Ob	Nw	p †
	Clinical and Anthropometric characteristics
	Gender (M/F)	16/14	16/14	14/16	14/14	NS*

Table 2 .

 2 Comparison of the respiratory parameters after the 6MWT within each group.

	ObA	NwA	Ob	Nw
	FVC (Z-score)

Table 3 .

 3 Independent effect of asthma and obesity on lung function and bronchial inflammation. FeNO= Fractional exhaled Nitric Oxide; MEF 75 = Maximal Expiratory Flow at 75% of the Forced Vital Capacity; PEF= Peak Expiratory Flow; FEV 1 /FVC= Forced Expiratory Volume in 1 sec/ Forced Vital Capacity FEV 1 /FVC ratio are expressed as Z-score. MEF 75 and PEF are expressed as percentage of theoretical values for age, gender, and height.

	Source	Dependent Variable Type III Sum of Squares	F	p #
	Asthma	FeNO	655,174	3,957	0.048
		MEF 75 (%)	505,481	1,038	NS
		PEF (%)	33,466	0,184	NS
		FEV 1 /FVC (Z-score)	18,871	0,623	NS
	Obesity	FeNO	119,116	0,719	NS
		MEF 75 (%)	4156,102	8,534	0.005
		PEF (%)	1131,609	6,232	0.015
		FEV 1 /FVC (Z-score)	5,290	0,175

NS

NS, no significant difference (P > 0.05). # Multiple analysis of covariance (MANCOVA)

John Wiley & Sons, Inc.Pediatric Pulmonology

John Wiley & Sons, Inc.

Acknowledgements

We thank all nurses of the Department of Pediatrics, University of Chieti, in particular Di Santo D, Di Luca D, Diomedi A, for their daily support in clinical research and care of children.

Acknowledgements

We thank all nurses of the Department of Pediatrics, University of Chieti, in particular Di Santo D, Di Luca D, Diomedi A, for their daily support in clinical research and care of children.

Age (years) 7.03 [6.05 -8.02] 7.06 [6.77 -8.05] 8.04