MD Eddy Robberecht
email: eddy.robberecht@uzgent.be

PhDa Sara Vandewalle

MD a, Charline Wehlou

MD Jean- Marc Kaufman

PhDb Jean De Schepper

Sunlight is an important determinant of vitamin D serum concentrations in cystic fibrosis

Keywords: Cystic fibrosis, 25-OH cholecalciferol serum concentrations, sunshine, oral vitamin D supplements, children and adolescents, adults

Background/Objectives:

The increase of bone disease in adult CF patients is partly attributed to inadequate serum concentrations of 25-OH cholecalciferol (25 (OH) D) blamed on fat malabsorption. Based on physiological, clinical and biochemical observations this pathogenesis is debatable. The objective was to ascertain the relative importance of different 25 (OH) D sources. Subjects/Methods: Over four consecutive years, 474 annual 25 (OH) D serum concentrations from 141 CF patients of all ages were compared to values of healthy peers and weighed against annual UVB exposure. Results: Ranked per month, 25 (OH) D concentrations depicted a curve strikingly parallel to the amount of UVB exposure in the preceding months. A significant difference exists between 25 (OH) D concentrations in the "Months with high UVB exposure" (May-October) and the "Months with low UVB exposure" (November-April) but not with healthy controls in the same period. Conclusions: 25 (OH) D concentrations clearly respond to the amount of sunshine in preceding months. They are not clearly influenced by daily oral supplements of 800 IU of cholecalciferol.

Sun exposure should be encouraged, and the recommended dosage of oral supplements increased.

Introduction

Changes in the understanding and in the clinical management of cystic fibrosis (CF) have increased life expectancy from single digits in the 1970's to more than 35 years at present (CF foundation, 2006) . As a consequence, new clinical problems have emerged, such as the increased bone fracture rate in adolescents and adults. These bone fractures are the result of a multitude of contributing factors such as lack of physical exercise, elevated cytokine production caused by chronic inflammation, delayed pubertal maturation, corticosteroid therapy, vitamin D and calcium deficiency and a poor nutritional status [START_REF] Aris | Consensus statement: Guide to bone health and disease in cystic fibrosis[END_REF]. Remarkably, because of the fact that vitamin D is fat-soluble and most CF patients suffer from fat malabsorption, vitamin D deficiency is primarily blamed [START_REF] Lark | Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients[END_REF] [START_REF] Aris | Consensus statement: Guide to bone health and disease in cystic fibrosis[END_REF]. As a natural consequence, all nutritional guidelines insist on preventing this deficiency by means of a daily oral supplement [START_REF] Borowitz | Consensus report on nutrition for pediatric patients with cystic fibrosis[END_REF] [START_REF] Sinaasappel | Nutrition in patients with cystic fibrosis: a European Consensus[END_REF] [START_REF] Aris | Consensus statement: Guide to bone health and disease in cystic fibrosis[END_REF]. Data on serum concentrations of 25-OH cholecalciferol (25 (OH) D) in CF are, however, not unequivocal, since they have been reported as too low by some authors [START_REF] Donovan | Bone mass and vitamin D deficiency in adults with advanced cystic fibrosis lung disease[END_REF] [START_REF] Mortensen | Bone mineral status in prepubertal children with cystic fibrosis[END_REF] [START_REF] Rovner | Vitamin D insufficiency in children, adolescents and young adults with cystic fibrosis despite routine oral supplementation[END_REF] , while others disagree [START_REF] Chavasse | Serum vitamin D levels in children with cystic fibrosis[END_REF]) [START_REF] Buntain | Bone mineral density in Australian children, adolescents and adults with cystic fibrosis: a controlled cross sectional study[END_REF]. Moreover, the failure of oral supplement treatments is common and has been widely published [START_REF] Donovan | Bone mass and vitamin D deficiency in adults with advanced cystic fibrosis lung disease[END_REF] [START_REF] Boyle | Failure of high-dose ergocalciferol to correct vitamin D deficiency in adults with cystic fibrosis[END_REF] [START_REF] Green | Current treatment recommendations for correcting vitamin D deficiency in pediatric patients with cystic fibrosis are inadequate[END_REF] [START_REF] Green | Transient effectiveness of vitamin D2 therapy in pediatric cystic fibrosis patients[END_REF]. Holding fat malabsorption solely responsible for vitamin D deficiency overlooks the importance of the dermal supply, which in healthy people determines up to 85% of 25 (OH) D concentrations in conditions of sunshine [START_REF] Heaney | Human serum 25hydroxcholecalciferol response to extended oral dosing with cholecalciferol[END_REF]. In less sunny regions, dermal vitamin D production is expected to be high during the sunnier months and low for the rest of the year [START_REF] Rapuri | Seasonal changes in calciotropic hormones, bone markers, and bone mineral density in elderly women[END_REF] . Annual determination of 25 (OH) D concentrations, as is usually performed [START_REF] Carr | Annual review or continuous assessment?[END_REF], will thus only provide information about a single limited and recent period of time. In an attempt to understand the relative importance of different 25 (OH) D sources, more specifically the influence of sunlight, a retrospective study was undertaken which looked at all available 25 (OH) D concentrations from the patients' annual follow-up visits, collected over four consecutive years. They were compared to values of healthy controls of the same age group and similar geographical latitude (latitude 50° N). We hypothesize that sunlight will prove to be an important determinant of 25 (OH) D concentrations in cystic fibrosis patients.

Materials and methods

In a retrospective study design, a total of 474 values of 25 (OH) D serum concentrations from 141 CF patients was gathered. The values had been measured at the systematic annual follow-up visits between October 2001 and December 2005. For each patient the annual follow-up visit had taken place around the same time of the year. The group included all CF patients above the age of one year that were followed at the CF centre of the Ghent University Hospital (latitude 50°N), without any selection. The group therefore contains every degree of disease severity. No transplanted patients were included. The median age was 15.6 years, ranging from 1 to 42 years; 56% of the patients were male. Based on faecal elastase 1 measurement, [START_REF] Borowitz | Use of fecal elastase-1 to classify pancreatic status in patients with cystic fibrosis[END_REF] 91 % of the patients (n=128) had pancreatic insufficiency (<15μg/g) and were being treated with pancreatic enzyme replacement, in accordance with guidelines [START_REF] Sinaasappel | Nutrition in patients with cystic fibrosis: a European Consensus[END_REF]. Patients with faecal elastase 1 concentrations above 400 μg/g were regarded as pancreatic sufficient (7%).

Faecal elastase 1 concentrations between 15 and 400 μg/g were considered equivocal and not taken into consideration (3%). Although a daily vitamin D supplement was prescribed to all patients regardless of exocrine pancreatic status, just only 93% reported taking it regularly

Results

Seasonal variations.

The 474 values of 25 (OH) D serum concentrations from the annual follow-up visits (over the four years) were pooled and plotted per month (figure 1). These values depicted an S-shaped curve which was convex from May to October ("Months with high UVB exposure") and concave in the subsequent period from November to April ("Months with low UVB exposure"). The lowest values were seen in February.

The median 25 (OH) D serum concentrations during the" Months with high UVB exposure" were significantly higher than those in the "Months with low UVB exposure" (table 1).

There is an important fraction of the CF population with 25 (OH) D concentrations below 20 ng/ml (table 2). However there is an important influence of the varying exposure to UVB light from the sun over the different years.

The 25 (OH) D serum concentrations were compared to reference values from healthy peers. No significant difference was found in comparison to local controls during the "Months with low UVB exposure": CF patients: 18.0 ng/ml (IQR: 10.2 -24.5 ng/ml); control group: 17.2 ng/ml (IQR: 12.7-19.3 ng/ml); p = 0.60. During the "Months with high UVB exposure", 25 (OH) D serumconcentrations were not statistically inferior to values from a reported group of comparable age and geographical location [START_REF] Guillemant | Wintertime vitamin D deficiency in male adolescents: effect on parathyroid function and response to vitamin D3 supplements[END_REF].

Relation to sunshine hours

The comparison between the variation in 25 (OH) D serum concentrations in the CF group over the four years and the amount of UVB exposure in the preceding months showed a remarkably manifest correlation. It was clear that median 25(OH) D serum concentrations ran parallel to the amount of UVB exposure in the two or three preceding months (p < 0.001). It was also evident that in a year with more UVB exposure during the summer -such as 2003-25(OH) D serum concentrations were higher than those recorded in a year with a summer with less UVB exposure (figure 2).

Discussion

The past decade a great amount of studies are published concerning vitamin D in CF. These with CF are significantly different from those in healthy people. We find no statistical difference in 25(OH) D serum levels between people with CF and healthy controls. This seems to confirm results from other studies [START_REF] Buntain | Bone mineral density in Australian children, adolescents and adults with cystic fibrosis: a controlled cross sectional study[END_REF]). These findings have important implications for clinical practice as they allow comparison of 25(OH) D serum concentrations from CF patients with values from healthy controls, always taking into account the season of the year. It should be kept in mind that these conclusions are based on data from CF patients who were systematically taking a daily oral vitamin D supplement and from a healthy control population taking none. We found a significant difference in 25 (OH) D concentrations when we compared "Months with high UVB exposure" to "Months with low UVB exposure". We found those differences as well in the patient group as in the healthy controls. The total amount of UV light during the period May-October at our location is approximately 5889 J/cm² (estimated amount of UVB light 589J /cm²) and the total amount of UV light during the period November-April is approximately 1761 J/cm² (estimated amount of UVB light 176 J/cm²). The curve of 25(OH) D serum concentrations and that of varying intensity of UVB light from the sun run a parallel course with a time-lag of approximately two months, as was also described in healthy people [START_REF] Need | Effects of skin thickness, age, body fat and sunlight on serum 25-hydroxyvitamin D[END_REF]. A maximum is reached in late summer and a minimum at the end of the winter. Higher 25 (OH) D serum concentrations in September correlate with higher 25 (OH) D serum concentrations in March of the subsequent year. This is explained by the storage of 25-OH cholecalciferol from dermal sun exposure during the months with high UVB exposure and consumption during the months with less UVB exposure, resulting in a progressive depletion of stocks [START_REF] Rapuri | Seasonal changes in calciotropic hormones, bone markers, and bone mineral density in elderly women[END_REF]) [START_REF] Guillemant | Wintertime vitamin D deficiency in male adolescents: effect on parathyroid function and response to vitamin D3 supplements[END_REF]. It is generally accepted that the skin is the major source of vitamin D, probably more than 85% of 25 (OH) D is obtained through exposure to sunlight [START_REF] Heaney | Human serum 25hydroxcholecalciferol response to extended oral dosing with cholecalciferol[END_REF]. Exposure of 6% of the body surface to one minimal erythemal dose of sunlight is equivalent to the oral administration of 600 to 1000 IU of vitamin D. This means that in conditions of sunshine, mild sun exposure of hands, arms, face or back, 2 to 3 times per week, would comply with vitamin D recommendations [START_REF] Holick | Vitamin D in health and prevention of metabolic disease[END_REF] (Hollick, 1999). This is certainly feasible in sunny climates, whereas in regions with less sunny climates, it is only possible during some months of the year, and oral supplements thus remain imperative.

The data obtained in the present study suggest that the current practice of supplementing CF patients can result in 25 (OH) D serum concentrations similar to those found in healthy controls. However, if concentrations constantly above 30 ng/ml are to be reached, as recommended, [START_REF] Aris | Consensus statement: Guide to bone health and disease in cystic fibrosis[END_REF]

(

  median dose: 800 IU cholecalciferol per day). The 25 (OH) D serum concentrations of 160 local (latitude 50° N) healthy individuals, with a median age of 20 years (age range: 10 y -45 y), served as controls for the comparison with the values of CF patients measured during the "Months with low UVB exposure", from October to December. Analysis Venous blood samples were obtained. 25 (OH) D serum concentrations were determined after extraction by RIA (DiaSorin, Stillwater, Minnesota, USA). This assay shows 100% crossreactivity between 25-OH-D2 and 25-OH-D3. In order to ascertain the importance of sunlight, 25 (OH) D serum concentrations were checked against the varying intensity of UV-B light from the sun over the four year period. This information was retrieved from records at the Royal Meteorological Institute at the University Observatory Armand Pien, which is located within a 50 km distance from the place of residence of all patients in the study. Based on these records of varying intensity of UVB light, one year can be divided into two distinct periods: a period with less UVB exposure between November and April and a period with higher UVB exposure from May to October. Statistical methods SPSS 12.0 (Chicago, Illinois USA) was used in the statistical processing of the study data. Since the data did not show a normal distribution, non-parametric tests were chosen. The correlation between 25 (OH) D serum concentrations and the varying intensity of UVB light was examined using the Spearman correlation coefficient. For the comparison between two groups, the Mann-Whitney U test was used. The study was approved by the Medical Ethics board of the University Hospital at Ghent.

  publications mainly discuss the 25 (OH) D serum concentrations in CF patients. As the incidence of spontaneous bone fractures rises along with patient longevity, there is also a growing interest in the role of vitamin D in CF. The results presented in the different studies however are not unequivocal. The main source of confusion is disagreement on reference values to determine which values are too low. In most studies 25 (OH) D serum concentrations are considered normal when above 20 ng/ml (Malabanan et al, 1998), while more recently values above 30 ng/ml at any time of the year are recommended (Aris et al, 2005) (Green et al, 2008). It is not entirely clear on what basis those recommendations are made, while at the same time, in most surveys, these levels are not met by large groups of healthy people. Depending on geographical location, age and season, between 22 % and 97 % of 25 (OH) D serum concentrations in healthy controls are below 20 ng/ml (Cashman, 2007) (Andersen et al, 2005). It is therefore to be expected that at least as many people with CF will also have 25(OH) D serum concentrations under the recommended values. Just as it occurs in healthy people, the percentages of 25(OH) D serum concentrations below 20 ng/ml observed in CF patients can vary considerably from one study to another due to disparity in disease severity, age, geographical location and season (Buntain et al, 2004) (Boyle et al, 2005) (Gronowitz et al, 2004) (Rovner et al, 2007). In the present study the difference between the percentages of 25 (OH) D serum concentrations below 20 ng/ml in the four successive years amounted to 58 % exclusively because of sunshine variations. Since vitamin D insufficiency (i.e.: 25(OH) D serum concentrations below the theoretically recommended 30 ng/ml (Rovner et al, 2007)) is widespread even in healthy controls, preference was given to a comparison of actual serum concentrations in order to determine whether 25(OH) D serum concentrations in patients

  phototherapy could be a promising alternative still awaiting the development of adequate methods

Table 1 :Figure 1 :

 11 Figure 1: Median 25 (OH) D serum concentrations in the period 2002-2005 per month

Figure 2 :

 2 Figure 2: Median 25 (OH) D serum concentration and UVB exposure per month through the

Acknowledgments

The authors' contributions are as follows: ER, SV participated in all stages of the research project. ER, CW and SV conceived the study and the collection of data. JMK and JDS provided data on control patients. ER, CW and SV carried out the data analysis and ER and SV wrote the manuscript. All authors read and approved the final manuscript. We would like to thank Nele Iserentant and Santiago Grau for linguistic help in the drawing of the manuscript.

Conflict of interest

None of the authors had a personal or financial conflict of interest.


L Cabaret

C Drag

Highly scannable injection seeded nanosecond Ti:Sapphire ring laser

Keywords: . 42.55.-f Lasers -42.60.-v Laser optical systems: design and operation -42.60.Lh Efficiency, stability, gain, and other operational parameters

A widely tunable injection seeded Ti:Sa ring cavity has been developped and characterized. The seeding source is a single mode quasi continuous wave Ti:Sa laser, continually tunable over more than 2.5 cm -1 (75 GHz). Its short term (pulse to pulse) stability is on the order of ± 3.2 MHz at a repetition rate of 20 Hz. The long term absolute stability is limited up to now to about ± 10 MHz. These frequency charactristics are transferred to the pulsed emission with minor degradations. The frequency chirps and shifts during the pulses are also studied using an optical heterodyne technique.

Introduction

High resolution laser spectroscopy, particularly molecular spectroscopy, requires single mode laser sources that cover a wide wavelength range and moreover that are continuously tunable over large frequency domains in a single run.

A great part of the visible and near IR spectrum is easily covered by single mode CW lasers, such as dye lasers, Titanium:Sapphire (Ti:Sa) lasers, laser diodes, and more recently optical parametric oscillators (OPOs). Even the Send offprint requests to: C. Drag : cyril.drag@lac.u-psud.fr UV side of the spectrum is attained by the use of highly efficient frequency doubling cavities, up to a wavelength of around 200 nm. However, solid state tunable CW lasers do not cover the entire domain (UV to near IR); large 'holes' remain, particularly between 500 and 680 nm, and even OPOs are not able up to now to replace completely visible dye lasers. Most favorable configurations associate OPO and frequency doubling, with an external cavity [1] or without [2]. Due to the high peak power of the laser emission, the pulsed mode allows us to have access to virtually any wavelength, from VUV to far IR, by the use of nonlinear effects in various media. In fact, starting from a wide band laser medium such as Ti:Sa, this spectrum can be almost completely covered using more or less complicated mixing schemes. The oscillator linewidth is reduced if one inserts dispersing components inside the cavity. In a single mode CW cavity, the thinner selecting filter is generally a Fabry-Perot etalon. Obtaining a single mode emission from a pulsed cavity is less easy since the existence of a non homogeneous broadening behavior in the high gain transient regime permits the coexistence of many axial modes. Therefore, rather than stacking filters, it is by far more efficient and cleaner to seed a pulsed cavity with a single mode, well frequency controlled, CW beam. Provided that the seeded frequency is matched (in phase) with one resonant mode of the pulsed cavity and that its amplitude greatly exceeds the spontaneous emission noise, all the fluorescence can be quenched inside this mode. In the optimum matching conditions, up to 99.8% of the pulsed emission intensity has been measured at the seeder frequency [3]. In return, for the conceptual simplicity of the configuration, several precautions must be taken concerning the beam alignment, the parameters adjustment (waist position and dimension) and the cavity length control. In the case of Ti:Sa pumped by a nanosecond pulse light, the cavity works in the gain switched regime so that the seeding is operated in its simplest form since there is no need for a Q-switch assembly.

Application domains of single mode nanosecond lasers are various and they are those that need highly coher-ent beams. One of the most demanding experiment, in terms of spatial and temporal coherence, is population transfer in molecules by the use of a STIRAP scheme [4,5]. The efficiency of the transfer to a selectively chosen level is directly linked to the quality of the beams coherence. In practice, one measures the spatial coherence for instance by propagating the laser beam through the focal zone of a lens and evaluating the quality factor M 2 . This factor gives how many times the beam is from the diffraction limit. Measuring the temporal coherence is a more tedious task: the linewidth must be evaluated in the MHz range which corresponds to a resolution limit for most of the measuring methods. Global linewidth measurement methods sum in time all the physical broadening contributions, they include for instance the use of a high finesse Fabry-Perot interferometer for observing the Airy pattern broadening and make a deconvolution from the apparatus function. This method has been recently used by P. Dupré and T. A. Miller to measure the frequency pulse shape of a high energy injection seeded Ti:Sa laser [6]. Another powerful method makes use of a heterodyne technique to analyze the frequency drift during the pulse [7]. In an injection seeded scheme, the CW laser injector usually serves as a reference local oscillator to achieve the signal beating after frequency shifting by an acoustooptical device. With nanosecond duration pulses, the AO crystal needs to be high frequency modulated (typically at more than 200 MHz) in order to obtain enough sampling definition inside the pulse. Such a method has been extensively studied by R. T. White et al. to characterize frequency chirps in OPOs injection seeded cavities [8][9][10] and by S. Hannamann et al. in a Ti:Sa oscillator-amplifier system [11].

We describe here a new ring cavity designed for an injection seeded Ti:Sa laser that is optimized for high resolution pulsed laser spectroscopy. The seeding laser is a tunable single-longitudinal-mode Quasi-Continuous-Wave (QCW) Ti:Sa with a large continuous scanning capability. Typically, 2.5 cm -1 (75 GHz) scans without mode hop are obtained and up to 6 cm -1 (180 GHz) scans can be attained in the most favorable spectral regions of the Lyot filter. The pulsed ring cavity is designed to reproduce the main spectral characteristics of the seeder, except the line-width that is nearly Fourier-transform limited. In particular, we demonstrate that the frequency stability and the scanning properties are transferred without appreciable degradation. The frequency chirp has been measured at different wavelengths thanks to an optical-heterodyne analysis technique and a direct fit method. mm, thickness 15 mm). The front face is anti-reflection coated in the range 700-1000 nm and the back face has a multilayer coating that is calculated to optimize the output coupling reflection with respect to the gain in the same spectral range; this function is more easily realized in a reflection mode than in a transmission mode which explains our choice. The seeded cavity is therefore a "in-line" setup for the Ti:Sa beam. The crystal is laser pumped by a home made master-oscillator power-amplifier frequency-doubled Nd:YAG laser delivering an energy of up to 30 mJ per pulse at a wavelength of 532 nm and at a repetition rate of 20 Hz with a pulse duration adjustable between 20 and 50 ns [12]; it was originally configured for the pumping of single mode optical parametric oscillators, and the output beam has a nearly Gaussian profile after amplification and frequency doubling.

Experimental setup 2.1 injection seeded pulsed ring cavity

The pumping beam is introduced through one of the Brewster prisms so that the Ti:Sa crystal is pumped in an almost collinear configuration with respect to the cavity axis taking account of the small dispersion angle at the crystal input. Generally, that situation is not a real drawback since it only decreases a little bit the output energy.

However, we will see that in our case the non-collinear pumping limits the tunability towards the infrared part of the spectrum. The Ti:Sa crystal is a Brewster cut slab of cross section 5 mm x 7 mm and 15 mm length (from Saint Gobain / Roditi); its doping concentration is 0.15% which leads to a nominal absorption coefficient of 2.1 cm -1 at 532 nm.

Overall setup

Figure 2 illustrates the setup of the laser system with its locking and tuning electronics. The apparatus for frequency characterization is also shown. The injection seeding laser is composed of a quasi-continuous-wave (QCW) frequency-doubled Yb:YAG pumping laser (λ = 515 nm) [13] followed by a QCW Ti:Sa laser, tunable in the range 725-960 nm. This laser emits single-axial-mode gain-switched pulses of 150-200 μs duration at a repetition rate of 20 Hz; the laser cavity and the locking system are described in reference [14]. In the present version, the intracavity LiNbO 3 electro-optic Fabry-Perot etalon has been replaced by a RTP (RbTiOPO 4 ) solid etalon that is less subject to piezo-acoustic perturbations. After a short period of relaxation oscillations (20 μs) at the beginning of the pulse, the laser power increases smoothly up to the steady-state regime that is generally reached at the end of the pulse (figure 3). The pulse building time depends mainly on the pumping power and on the output wavelength. Short time stabilization is obtained by servo-locking the QCW Ti:Sa laser frequency on a static sigmameter (phase quadrature Michelson interferometer) [16]. We modified the electronic setup to use this interferometer in quasicontinuous regime. For the long term stabilization, the sigmameter itself has its optical path difference servo-locked

Frequency characterization

Quasi-continuous lasers (QCW)

The frequency stability and the tuning characteristics of the seeding laser are of major importance to manage properly the frequency of the pulsed laser. To this end, we have fully characterized its emission. The single axial mode nature of the laser beam is monitored by the use of an electro-optically scanned confocal Fabry-Perot interferometer [14]. When the laser is in a stable configuration, i.e. when the transmission maxima of the wavelength selection filters are exactly superimposed, we verified that the emission is always single mode over the full pulse duration. Spurious mode hops appear only if the cavity is unlocked or at the end of long scans. The main drawback is probably the transient frequency shift during the pulse that was previously underlined and measured in Nd:YAG and Yb:YAG QCW lasers [13,14]; the shift was identified as an effect of thermal deposition inside the pumped volume. In the case of Ti:Sa, two parameters tend to increase the shift: first, the large quantum defect between the energies of pumping and laser photons so that more power is lost in the crystal, and second, the high power density of the pumping beam. However, since the thermal diffusivity of the Sapphire host is higher than YAG's one, the measured frequency shift is no longer linear. An accurate analysis of the transient frequency shift in the case of a Ti:Sa QCW laser will be published elsewhere. The residual measured frequency chirp is around 10 MHz in our experimental conditions.

Fortunately, the pulse to pulse repetability of the chirp evolution is excellent so that the seeded (nanosecond) laser will be only sensitive to the natural global frequency jitter of the QCW laser, provided the Q-switched pumping laser is triggered at a constant delay with respect to the front edge of the QCW Yb:YAG pumping pulse (see Fig. 3).

Using the sigmameter, the frequency fluctuations of the QCW Ti:Sa laser are reduced but we observe an ap- A fine frequency tuning of the laser is realized through the piezo mounted mirror by applying a slow voltage ramp.

Large scans are obtained if rapid returns of exactly an integer of the cavity free spectral range are induced on the piezo. The ramp returns are triggered just after a laser pulse in such a way that the locking loop does not "see" the cavity mode change. Since the reset time is short (≈2ms) compared to the shot period (50 ms), the laser frequency is not perturbed and the scan is quite linear. One can produce scans as large as 6 cm -1 (180 GHz) for some favourable positions of the Lyot filter; however, scans of 2.5 cm -1 are more routinely obtained as shown on figure 6 where a single mode hop can be clearly seen. The measurement is done with the pulsed seed laser.

The power needed to seed the pulsed cavity with a high efficiency is low, typically in the mW range [3]. However, injection locking can be effective even very far from the free running wavelength if a high power seeding signal is employed. In our case we measured a peak power of 1 watt at the output of the QCW Ti:Sa laser (λ = 810 nm) when it is pumped by 10 watts peak power from the Yb:YAG QCW laser. As we shall see later, this will ensure a large locking range without the need of additional wavelength selection filters. The cavity has also to be locked in phase with the injected beam.

Pulsed laser

The frequency characterization of the pulsed singlemode emission has also been realized in two ways: a global absolute frequency measurement (with the lambdameter for Fig. 6. Linear frequency tunig of the pulsed injection seeded cavity (2.5 cm -1 ). A single mode hope is clearly seen. exemple) and a chirp analysis during the pulse. The wavemeter is able to give an absolute value of the laser frequency (at peak intensity) with a precision of +/-10 Mhz in pulsed mode as recorded in Fig. 6. However, this apparatus cannot give indications about the frequency evolution during the pulse. To analyse the chirp, we employed an optical heterodyne method largely developed by several goups, in particular by R. T. White et al. [8]. In our setup, the QCW Ti:Sa laser serves as a local oscillator; the beam passes (one way) through an acousto-optic Bragg modulator that is modulated at a frequency f m of 200 MHz. The frequency shifted QCW beam and the unshifted pulsed beam are superimposed on a fast detector (EOT model ET-2020) and the beating signal is acquired by a TDS5104 Tektronics scope having a 1 GHz analogic bandpass and a 5 Gsample/s numeric sampling rate. Figure 7.a gives a typical beating signal at a wavelength corresponding to the center of the free running cavity emission band. The "instantaneous" frequency chirp is extracted from the de-tected signal V (t) in the following manner. Let E QCW be the amplitude (constant over the pulse duration) of the QCW laser field and E P (t) be the amplitude of the pulsed laser field; the beating signal satisfies to the proportionality relation:

V (t) ∝ 2E QCW E P (t) cos(ω m t + φ(t)) ( 1
)
Where ω m = 2πf m is the angular frequency. Multiplying V (t) by cos(ω m t) and then sin(ω m t) gives the two relations:

V (t) cos(ω m t) ∝ E P (t)[cosφ(t) + cos(2ω m t + φ(t))] (2) V (t) sin(ω m t) ∝ E P (t)[sin(2ω m t + φ(t)) -sin φ(t)] (3)
This allows us to get back to the amplitude E P (t) and to the "instantaneous" phase φ(t) after elimination of the terms at the modulation frequency. For this purpose a numerical filter at a frequency f c = f m /4 is applied to the functions (2) and (3) so that the remaining terms are proportional to E P (t) cos φ(t) and -E P (t) sin φ(t). The "instantaneous" frequency shift f inst (t) is directly obtained using:

f inst (t) = 1/2π × dφ(t)/dt.
Figure 7.b shows the reconstructed intensity and the calculated instantaneous frequency shift. In this particular situation (near the maximum of the gain curve), the total frequency chirp is negligible. On the contrary, when the seeded wavelength is far from this maximum, an important chirp is measured (cf. Fig. 8). This kind of frequency shift was analysed in dye amplifiers [17], in OPOs [9] and Ti:Sa [6,11]. While in a non-linear medium the shift is primarily due to the phase mismatch between the interacting waves, in fluorescent media it is the gain dependence of the refractive index, because of the varying population inversion density that is responsible for the frequency distorsions. The elimination (or at least the minimization)

of the frequency shift is straightforward in the case of an OPO since one must only take care to adjust the phase matching condition in the crystal for the exact injection seeded wavelength. In the case of a fluorescent medium, the "instantaneous" frequency f inst can be related to the population density N 1 (t) of the excited state. In the case of dye amplifier, an analytical model is developped by Melikechi et al. [17]. For an amplifier of length L, the evolution of the "instantaneous" frequency is given in low gain approximation by:

f inst (L, t) = f inst (0, t) - L 2n(ν)λ α (ν) d dt N 1 (t) (4)
where n(ν) and α (ν) are the refractive index and polar- For the comparaison with our experiment, it is necessary to note that, due to the presence of the output coupler, the free running wavelength (770 nm) does not coincide with the peak of the gain curve (790 nm). However, the null chirp is rather localized at 790 nm which confirms the predominance of the electric susceptibility parameter.

We present a record of the chirp in the figure 8 where the wavelength is around 810 nm, i.e. 40 nm redder than the free running wavelength.

Thanks to the high power of the QCW seeding source, the injection seeding is achieved with a high efficiency far outside the free running bandwidth of the laser. The total seeding range of the cavity is therefore nearly 100 nm without any wavelength selection filter inside the cavity. To enlarge the single mode spectral range, the use of a coarse selector is necessary in order to force the free running laser emission to reach the desired wavelength. We have tried a one plate (0.5 mm thick) Lyot filter that allows us to cover the range 700-810 nm. However, the red part of the spectrum (810 -1000 nm) has never been reached although the cavity was originally designed to cover the whole spectral emission band of the Ti:Sa. Actually, when the Lyot filter is inserted in the cavity, the scanned range is reduced to the blue part of the spectrum as the result of three combined effects: the shape of the gain spectral curve, the Moody [20] where simulations show clearly that the output pulse frequency is determined by the pulse laser oscillator. Note that the emitted frequency is the nearest axial mode of the resonator from the seeder frequency.

Conclusion

We described a novel scheme for an injection seeded Ti:Sa ring cavity widely tunable over 2.

Figure 1

 1 Figure 1 displays a scheme of the injection seeded cavity. The original ring cavity arrangement is composed of four folding prisms in a X configuration, a Ti:Sa crystal and an output coupler. The prisms are BK7 rectangular parallelepipeds (12 mm x 18.25 mm thickness 6 mm) oriented in such a manner that the intra-cavity beam is incident at Brewster's angle on the lateral faces and in total internal

Fig. 1 .

 1 Fig. 1. Scheme of the injection seeded ring cavity.

Fig. 2 .

 2 Fig. 2. Overall setup of the tunable injection seeded Ti:Sa laser chain. The picture includes the sigmameter and the singlemode laser for long term stabilization. The wavemeter and the Rb saturated absorption setup are used for the frequency measurements.

Fig. 3 .

 3 Fig. 3. Temporal evolution of the pulse shape of the QCW Ti:Sa laser. Inset : relaxation oscillations the beginning of the pulse.

Fig. 4 .

 4 Fig. 4. Long term frequency fluctuations of the QCW Ti:Sa laser when locked to a sigmameter. The absolute wavelength during the aquisition is around 810 nm.

Fig. 5 .

 5 Fig. 5. Pulse to pulse frequency jitter distribution over a sequence of 1000 pulses. The QCW Ti:Sa laser is locked to the sigmameter.

Fig. 7 .

 7 Fig. 7. a) Typical beating signal between the shifted QCW Ti:Sa and the pulsed seeded Ti:Sa. b) Recalculated intensity and "instantaneous" frequency in the case of a seeding at the maximum of the gain curve. The noise at the rising and falling edges of the pulse is due to the filtering process used in the calculation. Vertical bars indicate approximatively the validity domain of the measurement.

  isability. The source of frequency shifts in the amplifier is linked to the rate of change of population density, or , equivalently, the rate change of the gain. So the frequency chirp may be null only at the maximum of the gain curve where the real part of the polarizability α (ν) is zero. However, the dynamical behavior of Ti:Sa is somewhat different from dye's one due to its much longer fluorescent life time, almost three orders of magnitude. In a dye solution, for typical pulse durations of 10 to 20 ns, the time evolutions of the pumping pulse and of the gain are almost synchronized leading to a dispersion-like curve for f inst . In a Ti:Sa amplifier working in the gain switched regime, the gain is already at its maximum when the laser pulse starts growing so that the gain decreases all along the pulse duration. Consequently, the chirp seen by the detection system corresponds only to the central part of the dispersion curve. The polarizability α (ν) is positive on the blue side of the gain curve, and it is negative on the red side. As just mentioned, the term dN1(t) dt is always negative so that the chirp is positive if the injected wavelength is in the blue part of the gain curve and negative in the red part.

Fig. 8 .

 8 Fig. 8. Measured intensity and "instantaneous" frequency for an injected beam at 810 nm. The chirp is about 30 MHz during 20 ns.

Fig. 9 .

 9 Fig. 9. Measured intensity and "instantaneous" frequency for an injected beam at 793 nm. The chirp is negligible because the emitted wavelength is around the peak of the gain curve (790 nm). The cavity pulling effect is observed in both cases : when the cavity red (a) or blue detuned (b) from the seeder frequency.

  5 cm -1 . The QCW seeding Ti:Sa and pulsed lasers have been characterized. The long term (± 10 MHz) and short term (± 3,2 MHz) stabilities of the QCW Ti:Sa are transferred to the pulsed cavity. An optical heterodyne technique allows us to measure frequency chirps and shifts during the pulses. We have clearly shown that in a Ti:Sa amplifier, as already in lasers, the frequency chirp is caused by the transient time evolution of the gain during the pulse. The instantaneous frequency increases when the injected wavelength is in the blue part of the gain curve, decreases in the red part and is almost stationary at the maximum of the gain curve. Cavity pulling effects have also been observed.


PhD Marika Ferrari
email: ferrari@inran.it

Msc Lorenza Mistura

PhD Emma Patterson

PhD Michael Sjöström

PhD Ligia Esperanza Díaz

PhD Peter Stehle

PhD Marcela Gonzalez- Gross

PhD Mathilde Kersting

MD Kurt Widhalm

PhD Dénes Molnár

PhD Frederic Gottrand

PhD Stefaan De Henauw

PhD Yannis Manios

President Anthony Kafatos

PhD Luis A Moreno

PhD Catherine Leclercq

Fréderic Gottrand

Marcela González-Gross

Chantal Gilbert

Christian Libersa

Jackie Sáchez

Fréderic Gottrand

Mathilde Kesting

Michael Sjostrom

Jean Dallongeville

Gunnar Hall

Lea Maes

Luca Scalfi

Pilar Meléndez

Jesús Fleta

José A Casajús

Gerardo Rodríguez

Concepción Tomás

María I Mesana

Germán Vicente-Rodríguez

Adoración Villarroya

Carlos M Gil

Ignacio Ara

Juan Revenga

Carmen Lachen

Juan Fernández

Gloria Bueno

Aurora Lázaro

Olga Bueno

Juan F León

Jesús Mª Garagorri

Manuel Bueno

Juan Pablo

Rey López

Iris Iglesia

Paula Velasco

Ascensión Marcos

Julia Wärnberg

Esther Nova

Sonia Gómez

Esperanza Ligia Díaz

Javier Romeo

Ana Veses

Mari Angeles Puertollano

Belén Zapatera

Tamara Pozo

Laurent Béghin

Frédéric Gottrand

Catalina Iliescu

Wolfgang Sichert-Hellert

Ellen Koeppen

Dénes Molnar

Eva Erhardt

Katalin Csernus

Katalin Török

Szilvia Bokor

Mrs Angster

Enikö Nagy

Orsolya Kovács

Caroline Codrington

María Plada

Angeliki Papadaki

Katerina Sarri

Anna Viskadourou

Christos Hatzis

Michael Kiriakakis

George Tsibinos

Constantine Vardavas

Manolis Sbokos

Eva Protoyeraki

Maria Fasoulaki

Klaus Pietrzik

Christina Breidenassel

Andre Spinneker

Jasmin Al-Tahan

Miriam Segoviano

Anke Berchtold

Christine Bierschbach

Erika Blatzheim

Adelheid Schuch

Petra Pickert

Manuel J Castillo

Ángel Gutiérrez

Francisco B Ortega

Jonatan R Ruiz

Enrique G Artero

Vanesa España-Romero

David Jiménez-Pavón

Davide Arcella

Elena Azzini

Emma Barrison

Noemi Bevilacqua

Pasquale Buonocore

Giovina Catasta

Laura Censi

Donatella Ciarapica

Paola D ' Acapito

Myriam Galfo

Cinzia Le Donne

Giuseppe Maiani

Beatrice Mauro

Antonella Pasquali

Raffaela Piccinelli

Angela Polito

Raffaella Spada

Stefania Sette

Maria Zaccaria

Paola Vitaglione

Ilse De Bourdeaudhuij

Tineke De Vriendt

Christophe Matthys

Carine Vereecken

Mieke De Maeyer

Evaluation of iron status in European adolescents through biochemical iron indicators: the HELENA Study

Keywords: Anaemia, iron deficiency, adolescent, Europe

Objectives: To assess the iron status among European adolescents through selected biochemical parameters in a cross sectional study performed in 10 European cities.

Methods:

Iron status was defined utilizing biochemical indicators. Iron depletion was defined as low serum ferritin (SF<15µg/l). Iron Deficiency (ID) was defined as high soluble Transferrin Receptor (sTfR >8.5 mg/l) plus iron depletion. Iron Deficiency Anaemia (IDA) was defined as ID with haemoglobin (Hb) below the WHO cut-off for age and sex: 12.0 g/dl for girls and for boys aged 12.5-14.99 years and 13.0 g/dl for boys aged ≥15 years. Enzyme Linked ImmunoSorbent Assay (ELISA) was used as analytical method for SF, sTfR and CRP.

Subjects with indication of inflammation (C-Reactive Protein, CRP > 5 mg/l) were excluded from the analyses. 940 adolescents aged 12.5 to 17.49 years (438 boys and 502 girls) were involved.

Results:

The percentage of iron depletion was 17.6%, significantly higher in girls (21.0%) compared to boys (13.8%). The overall percentage of ID and IDA was 4.7% and 1.3% respectively, with no significant differences between boys and girls. A correlation was observed between Log(SF) and Hb (r=0.36, p<0.01) and between Log(sTfR) and Mean Corpuscular Haemoglobin, MCH (r=-0.30, P<0.01). Iron body stores were estimated on the basis of Log(sTfR/SF). A higher percentage of negative values of body iron was recorded in girls (16.5%) respect to boys (8.3%) and body iron values tended to increase with age in boys while the values remained stable in girls.

Conclusion:

In order to ensure adequate iron stores, specific attention should be given to girls at European level to ensure that their dietary intake of iron is adequate.

Introduction

Iron Deficiency (ID) continues to be one of the most prevalent nutritional deficiencies in the world, particularly in developing countries (World Health Organization (WHO), 2003). WHO estimated that ID occurs in about 66-80% of the world's population [START_REF] Who | Micronutrient deficiencies: Battling iron deficiency anaemia[END_REF]. ID has many negative effects on health, including changes in immune function, cognitive development, temperature regulation, energy metabolism and work performance [START_REF] Dallman | Biochemical basis for the manifestations of iron deficiency[END_REF]. On the other hand, iron is a pro-oxidant and thus a possible risk factor for coronary heart disease (CHD). However, a recent review suggests that there is no a strong association between body iron stores and coronary heart disease in adults (Zegrean, 2009).

The diagnosis of ID is based primarily on laboratory measurements of biochemical iron

indicators. There is no full international consensus on the indicators to be used for assessing iron status since each indicator has its own limitations due to either poor sensitivity or specificity, or because it is modified by conditions other than ID [START_REF] Dallman | Iron deficiency and related nutritional anemias[END_REF]. There are three general approaches to assessing ID in populations. In the first one, used in studies conducted by Hallberg et al [START_REF] Hallberg | Screening for iron deficiency: an analysis based on bone-marrow examinations and serum ferritin determinations in a population sample of woman[END_REF] and Amstrong (Amstrong, 1989) ID is defined as a low level of serum ferritin (SF). In the second one, used in the US in the National Health and Nutrition Examination Survey (NHANES) II and III, the multiple-criteria model is used to classify ID; it consists of two or three abnormal values for transferrin saturation (TS), SF and erythrocyte protoporphyrin (EP) [START_REF] Cogswell | Assessment of iron deficiency in US preschool children and nonpregnant females of childbearing age: National Health and Nutrition Examination Survey 2003-2006[END_REF]. The main limitation of these two approaches is that inflammation leads to high ferritin values even though iron store are low. The third approach proposed by WHO (2004) and adopted by [START_REF] Zimmermann | Methods to assess iron and iodine status[END_REF] is based on the measurements of SF and soluble Transferrin Receptor (sTfR) in combination with C-Reactive Protein (CRP), in order to eliminate false negative cases in presence of inflammation. The third approach was considered more appropriate and was therefore used in the present study. It is well known that SF concentrations reflect the size of the iron stores [START_REF] Cook | Defining optimal body iron?[END_REF] and that low SF levels identify "iron depletion" [START_REF] Who | Iron Deficiency Anaemia. Assessment, Prevention and Control. A Guide for Programme Managers[END_REF]. On the other hand, the measurement of sTfR has been reported to be useful in the diagnosis of "tissue ID" [START_REF] Skikne | Serum transferrin receptor: a quantitative measure of tissue iron deficiency[END_REF] and its values are not influenced by acute or chronic inflammatory conditions [START_REF] Ferguson | Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia[END_REF][START_REF] Olivares | Effect of acute infection on measurement of iron status: usefulness of the serum transferrin receptor[END_REF]. An approach to estimate body iron was developed by Cook et al [START_REF] Cook | The quantitative assessment of body iron[END_REF] using the formula: body iron (mg/kg) = -[Log(sTfR/SF)-2.8229]/0.1207 to allow the full range of the iron status of a population to be evaluated with negative values representing a deficit of iron respect to the quantity required to maintain a normal haemoglobin concentration.

Adolescents are at risk of ID because of their high iron requirements, especially during the growth spurt period [START_REF] Moreno | Adolescence[END_REF]. In boys, increased muscular growth leads to increased demand for iron. In girls, in addition to the fast growth, the onset of menstruation leads to iron losses [START_REF] Wharton | Nutrition in adolescents[END_REF]. As a consequence, ID occurs in adolescents also in developed countries, if their additional iron needs are not met [START_REF] Dallman | Influence of age on laboratory criteria for the diagnosis of iron deficiency anemia and iron deficiency in infants and children[END_REF]. Some studies suggested that ID can have detrimental effects on memory and learning processes in preadolescents and adolescents [START_REF] Beard | Iron status and neural functioning[END_REF].

Few data are available on iron status in European adolescents and these data derive from studies utilizing different methodologies, different indicators of iron status and varying criteria to define ID [START_REF] Hercberg | Iron deficiency in Europe[END_REF]. There was, therefore, a need to investigate the situation of ID among European adolescents with a harmonized methodology.

Within the framework of the HELENA Study (Healthy Lifestyle in Europe by Nutrition in Adolescents), funded within the 6 th EU framework program, a biochemical evaluation of iron status was performed with a cross sectional approach. The objective of the present paper is to report the iron status of European adolescents.

Materials and methods

Study design and sample selection

A detailed description of HELENA-cross sectional study (HELENA-CSS) in terms of sampling and recruitment approaches, standardization and harmonization processes, data collection, analysis strategies and quality control activities has been published elsewhere [START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF]. The study was approved by the Research Ethics Committees of each country involved according to the Declaration of Helsinki and International Conferences on Harmonization for Good Clinical Practice. Written informed consent was obtained from the parents of the adolescents and the adolescents themselves [START_REF] Béghin | Quality assurance of ethical issues and regulatory aspects relating to good practices in the HELENA Cross-Sectional Study[END_REF]. Briefly, subjects with an age range of 12.5-17.49 years were recruited in ten cities across Europe.

Selection of cities was based on two criteria: regional distribution and presence of an active research group assuring sufficient expertise and resources to successfully perform epidemiological studies. Within the study, Stockholm (Sweden) represented Northern Europe.

Athens, Heraklion (Greece), Rome (Italy) and Zaragoza (Spain) represented Southern Europe. Pecs (Hungary) represented Eastern Europe. Ghent (Belgium) and Lille (France) represented Western Europe whereas Dortmund (Germany) and Vienna (Austria) represented Central Europe. As the overall aim of the HELENA study was to assess the nutritional status of the adolescent population in Europe, during the design phase the variability of a large set of nutritional status indices obtained in previous studies was checked. Body Mass Index (BMI) was the indicator that showed the largest variability. Therefore, the sample size was estimated according to the mean BMI and variance values for each sex and age strata. This procedure ensured to adequately describe all the variables included in the study. For the sample size calculation, a confidence level of 95% and an error of ± 0.3 kg/m 2 were assumed leading to a sample size of 3000 adolescents. More details on the procedure are provided by [START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF].

Blood sampling was performed in one third of the adolescents recruited. Therefore, the total expected sub-sample was 1,000 adolescents (100 subjects for each city). The size of subsample was chosen so as to assess the immunological parameters with sufficient precision. Such parameters were those with the highest variability within the blood measurements that were included in the study as discussed by [START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF]. The selection procedure for schools and classes, including the selection of a subset of classes for blood sampling has been applied centrally by one partner (Ghent) for all study centers.

Blood sampling and laboratory measures

The blood sampling procedure and sample logistics have been described in detail elsewhere (Gonzáles et al., 2008). Briefly, fasting blood samples were collected by venipuncture at school between eight and ten o' clock in the morning. Whole blood samples for the red blood ImmunoSorbent assay (ELISA) [START_REF] Erhardt | Combined Measurement of Ferritin, Soluble Transferrin Receptor, Retinol Binding Protein, and C-Reactive Protein by an Inexpensive, Sensitive, and Simple Sandwich Enzyme-Linked Immunosorbent Assay Technique[END_REF]. A commercially available control sample from Bio-Rad (Liquichek Immunology Control, Level 3) was used to obtain a calibration curve on each plate.

A quality-control serum was produced in-house by pooling human serum from four healthy volunteers in order to monitor accuracy and precision of the determinations. The coefficient of variation (CV) from 10 replicates of the quality control serum pool was 5.3%, 6.4% and 12.4% for SF, sTfR and CRP respectively. Additionally, the inter-assay CV of the pool samples analysed in a total of 32 different analytical runs (4 replicates) was 10.8%, 14.8% and 23.4% for SF, sTfR and CRP respectively.

INRAN laboratory is involved in an external quality assurance program of CDC Vital-Eqa for SF, sTfR and CRP analyses [START_REF] Haynes | The CDC VITAL-EQA program, external quality assurance for serum retinol, 2003-2006[END_REF]. In Spring 2007, the measures of iron indicators of this study were within the Acceptable Range (AR), except for CRP which was just below the inferior limit (2.42 mg/l versus an AR of 2.70-5.38 mg/l) and sTfR which was at the superior limit (7.3 mg/l versus an AR of 1.7 mg/l and 7.3 mg/l).

Anaemia and iron classification

Anaemia was defined as haemoglobin (Hb) concentration below the cut-off set by WHO (WHO, 2004) 12.0 g/dl for girls aged 12 years and above and for boys aged 12 to 14 and 13.0 g/dl for boys aged 15 years or above. Abnormal values for iron indicators were defined as concentration <15 µg/l for low SF (WHO, 2004), >200 µg/l and >150 µg/l (boys and girls respectively) for high SF (WHO, 2004) (severe risk of iron overload) and >8.5 mg/l for high sTfR [START_REF] Cook | Serum Transferrin Receptor[END_REF]. A participant was considered to have active inflammation if the CRP concentration exceeded 5 mg/l [START_REF] Woodruff | Anaemia, iron status and vitamin A deficiency among adolescent refugees in Kenya and Nepal[END_REF]. The methodology to classify the iron status is described in Table 1. The classification of iron status is considered in presence of normal CRP concentration.

Total body iron was estimated with the use of the formula: body iron (mg/kg) = -[Log(sTfR/SF)-2.8229]/0.1207 [START_REF] Cook | The quantitative assessment of body iron[END_REF].

Statistical analyses

The Kruskal Wallis test was used for comparing haematological parameters among iron sufficiency, iron depletion and ID. The student t test was used to evaluate the significance of differences between mean values of iron indicators between gender. Pearson correlation was used to test the linear relation between haematological variables. The χ 2 test was used for comparing proportions. Iron status indicators such as sTfR and SF were transformed to logarithms before performing statistical analyses. The results in the tables are shown using the original units without any log-transformation.

As already performed by Ortega et al [START_REF] Ortega | Physical fitness levels among European adolescents: the HELENA study[END_REF] all the analyses conducted on the HELENA data were adjusted by a weighing factor to balance the sample according to the age and sex distribution of the theoretical sample, in order to guarantee representation of each of the stratified groups. The counts of subjects (n) are reported in the tables as unweighted values. All the analyses were performed using SPSS 12.00 for Windows.

Results

Iron indicators and iron status

The HELENA-CSS consists of 1,089 adolescent subjects with blood measurements. A total of 102 subjects were excluded because at least one of the iron indicators (SF n= 80; sTfR n= 84) or haemoglobin (Hb n =16) was missing. The final sample of 987 adolescents included 465 boys and 522 girls i.e. 90.7% of the adolescents who participated in the blood sampling.

Forty-seven subjects had high levels of CRP (27 boys and 20 girls) and were excluded.

Therefore, the present study of iron status was performed on a total of 940 adolescents (438 boys and 502 girls) with no indication of the presence of inflammation.

The mean age (standard deviation, SD) of adolescents was 14.7 (1.2) years both in boys and in girls and the mean BMI was 21.2 (3.8) and 21.1 (3.3) kg/m 2 respectively for boys and girls.

The reported age of menarche in the present study ranged from 9 to 16 years of age. Fiftyeight (12%) girls reported not to have experienced menarche at the time of the study. 39% and 43% of the boys and girls respectively declared to have smoked cigarettes at least once.

Mean, median (Me) and percentiles (25 th -75 th ) values of red blood parameters, SF and sTfR are summarized in Table 2. Percentage of iron sufficiency, abnormal values of iron indicators, anaemia, iron depletion, ID and IDA are presented in Table 3.

In boys, the mean (SD) of Hb was 14.7 (1.2) g/dl (14.3 (1.0) g/dl aged 12.5-14.99 years and 15.2 (1.2) g/dl aged 15 years or above). In girls it was 13.8 (1.2) g/dl. The mean values of SF suggest a gender-related significant difference (p<0.05) with a lower mean SF value in girls (27.9 µg/l) than in boys (36.6 µg/l). The percentage of low SF values was also higher in girls (26.3%) compared to boys (17.9%) (p<0.01) (Table 3). No single case of high level of SF was identified (SF>200 µg/l for boys and >150 µg/l for girls) indicating no risk of iron overload (WHO, 2004) in the study sample.

Twenty three subjects had high values (> 8.5 mg/l) of sTfR (that could have indicated iron deficiency) but with normal values of SF (indicating no iron depletion) and could therefore not be categorized in one of the iron status classification (Table 1). They were excluded from the analyses in terms of the classification of ID.

The percentage of iron depletion was 17.6%, significantly higher in girls (21.0%) than in boys (13.8%) (p<0.05) (Table 3).

The overall prevalence of anaemia was 4.4% and it ranged from 1.1% (Stockholm) to 10.1% (Heraklion) among cities. Intermediate prevalences were 1.4% for Zaragoza, 3.2% for Pecs, 3.6% for Dortmund, 4.0% for Rome, 4.6% for Ghent, 5.5% for Lille, 5.6% for Vienna and 5.7% for Athens. The overall prevalence of anaemia without ID was 3.1% (4.6% in girls and 1.4% in boys) and ranged from 0% (Stockholm and Zaragoza) to 7.6% (Heraklion). The second highest prevalence was observed in Athens (5.7%). Intermediate prevalences were 1.8% for Dortmund, 1.9% for Vienna, 3.0% for Rome, 3.2% for Pecs, 4.1% for Lille and 4.6% for Ghent. Percentage of iron depletion ranged from 9.6% (Zaragoza) to 23.0% (Pecs).

Percentage of ID ranged from 0% (Lille) to 11.3% (Athens). Percentage of IDA ranged from 0% (Athens, Gent, and Pecs) to 2.8 % (Vienna).

Among adolescents with anaemia (n=40), 27.5% (n=11) were classified as IDA, with abnormal SF and sTfR values whereas 24.3% (n=10) had abnormal SF values but with normal sTfR values (iron depletion without ID) and 47.5% (n=19) had anaemia without ID, with normal SF and sTfR values. Log(SF) and Log(sTfR) were significant correlated with the red cell parameters (MCV, MCH, MCHC and RDW) (Table 4). In the total sample, a significant correlation was observed between Log(SF) and haemoglobin concentration (r=0.36, p<0.01) and between Log (SF) and Log(sTfR) (r=0.33, p<0.01). For Log(sTfR) a significant correlation was observed with Log (SF), MCH (r=-0.30, p<0.01) and MCV (r=-0.26, p<0.01) No significant correlation was observed between RBC and iron indicators [Log(SF) and Log (sTfR; r =0.13 and r=0.07 respectively].

The mean of red cell parameters in the different classification of iron status is reported in Table 5. Statistically significant differences between iron sufficiency, iron depletion and ID were observed for red cell parameters for boys and girls. There were significant differences (p<0.001) for Hb, MCV, MCH, MCHC and RDW between iron sufficiency group and the iron depletion and ID groups. No significant difference was observed between iron sufficiency group and ID group for any of the red cell parameters considered.

Estimation of body iron

Body iron was calculated according to the formula of Cook et al [START_REF] Cook | The quantitative assessment of body iron[END_REF] and the cumulative percentage distributions by gender is shown in Figure 1. Mean (SD) body iron was significantly higher (p<0.001; Student t test) in boys [4.4 (3.2) mg/kg] than in girls [3.3 (4.1) mg/kg]. Girls showed a light shift of the body iron distribution curve to the left in comparison to boys in which higher positive values were observed. The reference line indicates the cut-off point for tissue iron deficiency (body iron < 0 mg/kg). The percentage of negative values was significant higher (p<0.001; χ 2 ) in girls (16.5%) respect to boys (8.3%).

The distribution of body iron stores by age was also examined. Figure 2 shows that the linear relationship between body iron and the age of the adolescents is not very strong. Body iron increased slightly with age (r=0.28) but this trend did not reach statistical significance. On the other hand, it tended to be stable (r=0.025) in girls.

Discussion

The present study is the first report evaluating iron status in adolescents across multiple European countries. As discussed by [START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF], the selected cities were equivalent and comparable between countries but the samples were representative for the cities and not for the countries. Therefore, the HELENA Study does not allow for statistical comparison of differences in iron status between regions. However the prevalence of iron status stages according to geographical location of the cities adopted for this study are provided underneath for descriptive purpose.

In the present study, the overall proportion of iron depletion was 17.6%; it was significantly higher in girls (21.0%) than in boys (13.8%). The prevalence of iron depletion by geographical location was: 23% in Eastern Europe (Pecs), 19% in Northern Europe (Stockholm), 18% in Western Europe (17% in Ghent and 19% in Lille),17% in Central Europe (16% in Dortmund and 19% in Vienna) and 15% in Southern Europe (14% in Athens,17% in Heraklion,19% in Rome and 10% in Zaragoza). These data are in the range of findings from a recent review on iron status in European adolescents [START_REF] Hercberg | Iron deficiency in Europe[END_REF] where the highest prevalence of iron depletion was observed in girls in Ireland (43%) and in boys in Denmark (16%) while the lowest prevalence was observed in girls in Finland (4.7%) and in boys in France where no cases were detected.

For ID, in the present study, there was a tendency for a higher percentage in girls (5.4%) respect to boys (3.9%) but the difference was not significant. The percentage of ID in girls in our study was slightly lower than the 8.7% prevalence observed in girls aged 12-16 years in the US [START_REF] Halterman | Iron deficiency and cognitive achievement among school-aged children and adolescents in the United States[END_REF] but in line with the 6% prevalence observed in girls aged 13-19 years in Canada [START_REF] Deegan | Assessment of iron status in adolescents: dietary, biochemical and lifestyle determinants[END_REF]. Both the prevalence observed in our study and in the US are below the cut-off established by WHO to determine "no prevalence" of ID, i.e. 10% (WHO, 2004). Both are also far lower than that observed in other parts of the world as in Nepal with an ID prevalence of 56% observed in adolescents girls [START_REF] Woodruff | Anaemia, iron status and vitamin A deficiency among adolescent refugees in Kenya and Nepal[END_REF].

The slightly lower prevalence in our study may in part result from a different approach to define ID. The impact of ID on the cognitive functioning of adolescent girls who are at highest risk of ID requires major clarification. In fact there is only limited evidence that iron supplementation may improve attention, concentration and Intelligent Quotient (IQ) [START_REF] Falkingham | The effects of oral iron supplementation on cognition in older children and adults: a systematic review and meta-analyses[END_REF]. The prevalence of ID by geographical location was: 6% in both Northern Europe (Stockholm) and Southern Europe (11% in Athens, 6% in Heraklion, 3% in Rome and Zaragoza) plus Eastern Europe (Pecs), 5% in Central Europe (3% in Dortmund and 7% in Vienna) and 2% in Western Europe (no cases in Ghent and 4% in Lille). No comparable data are available in the literature in relation to ID in adolescents in the European context for comparison with the results of the present study.

The percentage of IDA was 1.3% (0.5% in boys and 2.0% in girls), with a (non-significantly) higher value in girls. The prevalence of IDA by geographical location was: 2% in Central Europe (2% in Dortmund and 3% in Vienna), 1% in both Northern Europe (Stockholm) and Southern Europe (no cases in Athens, 2% in Heraklion, 1% in both Rome and Zaragoza), 0,6% in Western Europe (no cases in Ghent and 1% in Lille) and no cases in Eastern Europe (Pecs). These data are similar to the estimates of prevalence in adolescents in Canada [START_REF] Deegan | Assessment of iron status in adolescents: dietary, biochemical and lifestyle determinants[END_REF], the United States [START_REF] Looker | Iron deficiency-United States, 1999-2000[END_REF] and Sweden [START_REF] Samulson | Dietary iron intakes and iron status in adolescents[END_REF], ranging from 0% to 3%. In the present study, the proportion of anaemia was 4.4%, consistent with the estimates of the worldwide prevalence of anaemia during adolescence: 27% in developing countries and 6% in developed countries [START_REF] Kara | The prevalence of anemia in adolescents: a study from Turkey[END_REF]. The observed prevalence of anaemia not due to ID (3.1%) is probably overestimated due to unavoidable misclassifications which are inherent to the definition of limits. In fact, the limits set to define anaemia are based on 95% reference ranges and therefore it is assumed that 2.5% of healthy individuals would be classified as anaemic. On the other hand, a proportion of anaemia without ID may be originated from genetic haemoglobin disorders since subjects with such disorders were not excluded. In our study the highest prevalence of anaemia without ID among cities was observed in two cities of Greece (7.6% in Heraklion and 6.3% in Athens).

Greece is known to be heavily affected by haemoglobinopathies (thalassemia and sickle-cell disease). In fact, according to [START_REF] Angastiniotis | Prevention and control of haemoglobinopathies[END_REF], the highest percentage of carriers of thalassemia and sickle-cell disorders is observed in Greece (9.0%).

In our study, no single case of iron overload was identified through serum ferritin, a stored body iron marker.

Results of the present study on correlations between iron status parameters are all plausible.

SF and sTfR which were chosen in this study as iron indicators showed a correlation with red cell parameters. The observed correlation coefficients between Log(sTfR) and red cell parameters (MCV and MCH) are not surprising since sTfR is a measure of iron deficiency erytropoiesis [START_REF] Metzgeroth | Soluble transferrin receptor and zinc protoporphyrin -competitors or efficient partners?[END_REF] as MCV and MCH. The present data are in general agreement with the result of a previous study showing a correlation between sTfR and red cell parameters in iron-deficient adolescents [START_REF] Choi | Serum transferrin receptor concentrations correlate more strongly with red cell indices than with iron parameters in iron-deficient adolescents[END_REF]. These findings are confirmed since the concentrations of the mean of red cell parameters decreased gradually (except for RDW that increased) with increasingly negative iron balance, as iron deficiency appeared (sTfR > 8.5 mg/l) (Table 5). The results of the present study document a positive correlation between Log(SF) level and Hb concentration (r=0.39, p<0.01) (Table 3) comparable with those observed in other studies among children: r=0.24 [START_REF] Jeremiah | Iron deficiency anaemia and evaluation of the utility of iron deficiency indicators among helthy Nigerian children[END_REF] and r=0.26 [START_REF] Ayoya | α 1 -Acis glycoprotein, hepicidin, C-reactive protein and serum ferritin are correlated in anemic schoolchildren with Schistosoma haematobium[END_REF]. The low correlation between these two indicators is due to the fact that they are related to differing stages of ID. The correlation between the indicators of iron status and red cell parameters may be considered as a confirmation of the reliability of these indicators (WHO, 2004;[START_REF] Zimmermann | Methods to assess iron and iodine status[END_REF] in the assessment of iron status in adolescents.

The correlation of SF with Hb and the higher percentage of iron depletion in girls respect to boys suggest that the risk of developing iron deficiency anaemia is higher in girls. If iron depletion is not corrected, it may indeed progress to iron deficiency. In addition, the calculated measure of body iron was lower in girls than in boys. The distribution of body iron stores by age (Figure 2) suggests that in boys body iron stores may have slightly increased during and after their rapid growth period, whereas in girls iron stores remain low during the whole pubertal period. One possible explanation for the lower iron status in girls, besides growth, is the loss of iron with menstruation. In previous studies, it was observed that the growth spurt associated with menstrual status, blood loss and a low iron intake may have adverse effects on iron stores in adolescents girls [START_REF] Ilich-Ernst | Iron status, menarche and calcium supplementation in adolescents girls[END_REF].

Conclusion

Our study confirms SF and sTfR as biochemical indicators that could be used in the assessment of iron status in adolescents, a problematic group of population for the period of rapid growth. On the basis of WHO guidelines (2004) for the interpretation of the iron status data at population level, the results indicate that, in the European context, ID is not prevalent neither in boys nor in girls and that iron depletion is prevalent in girls but not in boys.

Adolescent girls therefore constitute a group at risk of ID and specific attention should be given to them during adolescence to ensure that their dietary intake of iron is adequate to their requirements.

  parameters (Haemoglobin, Hb, Red Blood Cell, RBC, Mean Corpuscular Volume, MCV, Mean Corpuscular Haemoglobin, MCH, Mean Corpuscular Haemoglobin Concentration, MCHC and Red Cell Distribution Width, RDW) were sent directly to the local laboratory of each country to be analysed. Samples for iron status analyses were clotted at room temperature for at least 30 minutes and then centrifuged at 3,500 rpm for 10 minutes. Serum samples were shipped to the central laboratory in Bonn University (IEL, Germany) Department of Nutrition and Food Science, to be stored at -80°C before being shipped to Italy in May 2007, within six months to the collection. The aliquots of serum samples were analysed in the Human Nutrition Laboratory of the National Research Institute on Food and Nutrition (INRAN). SF, sTfR and CRP were measured by using an Enzyme Linked

Figure 2 .

 2 Figure 2. Distribution of body iron stores by age in boys and girls in the HELENA Study.

Table 1

 1 Iron status a as defined in the present paper

	Classification	Values of iron indicators
		SF	sTfR
		(µg/l)	(mg/l)
	Iron sufficiency b	≥ 15	≤ 8.5
	Iron depletion c	< 15	≤ 8.5
	Iron Deficiency (ID) b	< 15	> 8.5
	Iron Deficiency with Anaemia (IDA)	< 15	> 8.5
	a Iron status is considered in presence of CRP>5 mg/l

b With or without anaemia c Without ID and with or without anaemia

Table 3

 3 Proportion of European adolescents having anaemia, abnormal values of iron indicators , iron sufficiency, iron depletion, ID and IDA a All data were adjusted by a weighing factor to balance the sample according to the theoretical age distribution of the Indicators and cut-off values used to identify abnormal values of iron indicators and iron status are reported in Table 1 d 23 subjects (13 boys and 10 girls) were excluded because of with high values of sTfR and normal values of SF . * P<0.01 statistically significant between girls and boys Figure 1. Cumulative percentage distributions of estimated body iron 1 in boys and girls from the HELENA Study . 1 Total body iron was estimated using the formula: body iron (mg/kg) = -[log(sTfR:ferritin ratio)-2.8229]/0.1207 developed by Cook 16 . Positive values indicate the presence of storage iron, whereas negative values indicate tissue iron deficiency.

		Total		Boys	Girls
	Iron status c	(n =940) b	(n = 438) b	(n =502) b
		n	%	n	%	n	%
	Low SF	210	22.4	78 17.9 **	132 26.3
	High sTfR	66	7.0	30	6.9	36	7.2
	Anaemia	41	4.4	8	1.8 **	33	6.6
	Classification d
	Iron sufficiency	705	75.2	344 79.3 *	361 71.7
	Iron depletion	167	17.6	63 13.8 *	104 21.0
	ID	45	4.7	18	3.9	27	5.4
	IDA	11	1.3	2	0.5	9	2.0
	European adolescent population

a b The count of subjects (n) are reported in the table as unweighted values c * P<0.05; *

Acknowledgements

We gratefully acknowledge all participating adolescents, and their parents and teachers for their collaboration. We also acknowledge Dr. Eugenia Venneria for her contribution for the ELISA analyses of serum samples.

Funding

The HELENA Study takes place with the financial support of the European Community Sixth RTD Framework Programme (Contract FOOD-CT-2005-007034). The content of this article reflects only the author's view and the European Community is not liable for any use that may be made of the information contained therein.

Competing interests

None of the authors had any conflict of interests. Mean Hb, g/dl (s.d.)

14.9 (1.2) 13.6 (0.9)