Stéphane Lips

Ahlem Bensalem

Yves Bertin

Vincent Ayel

Cyril Romestant

Jocelyn Bonjour
email: jocelyn.bonjour@insa-lyon.fr

Experimental evidences of distinct heat transfer regimes in Pulsating Heat Pipes (PHP)

Keywords: Pulsating heat pipe, oscillating flow, two-phase flow, thin film evaporation, nucleation

Various experiments were conducted on two full-size Pulsating Heat Pipes (PHP) which differed from their diameter, number of turns, and working fluid. The analysis of the experimental results showed two kind of operating curves (overall thermal resistance vs. heat rate): for low heat fluxes, the curve is irregular and the PHP performance is sensitive to the orientation. For high heat fluxes, the operating curve is smooth and independent from the orientation. To contribute to the analysis of these results, experiments were conducted at the ACCEPTED MANUSCRIPT 2 scale of a single branch of a PHP. An oscillating motion was imposed to a single liquid plug surrounded by two vapour slugs in a capillary tube and high speed visualisations were performed. The test section was either adiabatic or heated. The adiabatic experiments brought to the fore the importance of dynamic contact angles in the flow and the dissymmetry between the advancing and receding contact angle. The non-adiabatic experiments showed that at low flux, the flow is disturbed by bubble nucleation, while at high heat flux, the main heat transfer mechanism is thin film evaporation, with a completely different thermal and hydrodynamic behaviour.

Nomenclature

Bo

Introduction

Since a few years, Pulsating Heat Pipes (PHPs) have been the subject of research in an increasing number of laboratories. As a matter of fact, their thermal performance and the simplicity of their design make them potential candidates for a great number of applications.

PHPs were presented in 1971 by Smyrnov in a Russian Patent, and in 2003 in a U.S. patent [START_REF] Smyrnov | Method of action of the pulsating heat pipe, its construction and the devices on its base[END_REF]. PHP in the form as they are investigated today have been first proposed by Akachi [START_REF] Akachi | Structure of a heat pipe[END_REF]. A PHP mainly consists of a meandering tube of capillary dimensions with many U-turns as shown on Fig. 1 [START_REF] Khandekar | Thermo-hydrodynamics of closed loop pulsating heat pipe[END_REF]. Contrarily to conventional heat pipes, a PHP does not include any capillary structure inside the tube. Because of the small size of the tube, the working fluid distributes itself naturally into the form of liquid slugs and vapour plugs. As in conventional heat pipes, heat is provided at one end of the PHP (evaporator) and dissipated to a heat sink at the other end (condenser), with an adiabatic zone between them. The heat transport is ensured by the oscillations of the slugs and plugs in the tube, which are believed to be due to thermally-induced pulsations of pressure and to a complex combination of two-phase flow instabilities. However, the understanding of the phenomena governing the PHPs behaviour is still limited and further studies are needed.

Khandekar [START_REF] Khandekar | Thermo-hydrodynamics of closed loop pulsating heat pipe[END_REF] conducted a large number of experiments for various PHP configurations.

He performed an analysis of the impact of several parameters on their behaviour (filling ratio, heat input, number of turns, orientation,…). He gained from his experiments an improved understanding of the heat and fluid flow mechanisms in PHPs. He discussed to effect of the tube diameter that should be small enough to ensure the flow oscillations, i.e. smaller than a critical value:

) .( g . C D D v l crit ρ - ρ σ = < (1)
with C being a constant of about 2.

Khandekar et al. [START_REF] Khandekar | Closed loop pulsating heat pipes, Part. B: visualization and semi-empirical modelling[END_REF] also performed some flow visualisations during the operation of a PHP. They distinguished four regimes that can be related to the PHP operating curve (overall thermal resistance of the heat pipe as a function of the input power): at low heat input, the oscillations amplitude remains limited, and the thermal resistance slightly decreases with the increase of the heat input. At higher heat input, the decrease of the thermal resistance is more noticeable, and this corresponds to a slug flow pattern. Nevertheless, when increasing the heat input, a preferred flow direction progressively appears. When this preferred flow direction is clearly established, and when the flow pattern is annular rather than of slug type, the thermal resistance is minimum and exhibits a plateau. Finally, at high heat flux, the thermal resistance strongly increases with the increase of the heat input; this regime corresponds to a dryout of the evaporator. Additional information was provided by Khandekar et al. [START_REF] Khandekar | Understanding operational regimes of pulsating heat pipes: an experimental study[END_REF] in their experiments with three different fluids (ethanol, water, R-123). For ethanol and water, the critical diameter was much larger than the tube diameter, while for the latter, the critical diameter was of the order of (or even slightly lower than) the tube diameter. According to their experiments, the effect of bubbles on the two-phase oscillating flow that develops out of the extreme operational limits of PHPs (i.e. PHP completely empty or completely filled with liquid) depends on the filling ratio and on the PHP orientation. At high filling ratio (e.g. 95%

of liquid) and favourable orientation (evaporator at bottom, condenser at top), the bubbles tend to limit the two-phase fluid motion. For a moderate filling ratio (about 20 % -70%, which actually gives birth to oscillations), gravity was shown to play a role even for water, i.e.

even for a critical diameter noticeably greater than the tube diameter (highly confined situation), while with R-123, the PHP was found to operate, in spite of a critical diameter slightly lower than the tube diameter. All these observations were interpreted through the effect of bubbles on the two-phase flow too.

Riehl [START_REF] Riehl | Characteristics of an open loop pulsating heat pipe[END_REF] used various working fluids and tested an open loop PHP in different orientations. He compared the thermal performance of a PHP working with water, ethanol, propanol, methanol and acetone. In his experimental conditions, the use of methanol and acetone led to the best thermal performance whereas the use of water led to the worst.

Moreover, he observed that the oscillations in the PHP are more important and more regular with methanol than with water. This was explained by the low latent heat of the methanol which promotes nucleation and boiling and, as a consequence, instabilities of the fluid flow.

Finally, he observed best thermal performance in horizontal orientation compared to vertical orientation. However, the importance of the effect of orientation depends on the fluid used:

with ethanol and methanol, the ratio of the thermal resistances in horizontal and vertical orientation is more than two, whereas with water, thermal performance is almost independent of the orientation.

Xu et al. [START_REF] Xu | High speed flow visualization of a closed loop pulsating heat pipe[END_REF] visualized the oscillating flow in a closed loop PHP with a high speed camera.

For methanol and water, various oscillation modes were observed. Moreover coalescence and vapor plug break-up phenomena were highlighted with water as the working fluid, particularly in the tubes U-bends. Their interpretation, based on an analytical model, was that the capillary pressure is not uniform in the bends, which results in a local accumulation of liquid. They also stated that, with methanol as the working fluid, the coalescence or break-up is limited because of the low surface tension of this fluid. In that case, the liquid plugs are longer than with water.

As regards numerical investigations, Khandekar et al. [START_REF] Khandekar | Closed loop pulsating heat pipes, Part. B: visualization and semi-empirical modelling[END_REF] developed semi-empirical models based on the experimental results obtained by Charoensawan et al. [START_REF] Charoensawan | Closed loop pulsating heat pipes-Part A: Parametric Experimental Investigations[END_REF]. Wong et al. [START_REF] Wong | Theoretical modeling of pulsating heat pipe[END_REF] proposed a PHP knowledge model that was improved by Shafii at al. [START_REF] Shafii | Thermal modeling of unlooped and looped pulsating heat pipes[END_REF]. Liang and Ma [START_REF] Liang | Oscillating motions of slug flow in capillary tubes[END_REF] studied the effect of different parameters thanks to a model close to that by Shafii et al. [START_REF] Shafii | Thermal modeling of unlooped and looped pulsating heat pipes[END_REF].

Dobson et al. [START_REF] Dobson | An open oscillatory heat pipe water pump[END_REF] presented a model of an open PHP. It consists of a U-shaped capillary tube whose extremities are placed in a liquid tank. The U-part of the capillary is heated. This numerical model takes into account the presence, or not, of the liquid film on the wall and predicts oscillations in the fluid flow.

Numerical results usually show a contradiction with experimental distribution of the vapour plugs, because the vapour plug break-up phenomena are not taken into account. In addition, the vapour plugs or liquid slugs are not symmetric [START_REF] Khandekar | Thermofluiddynamic study of flat plate closed loop pulsating heat pipe[END_REF], which induces a capillary pressure difference at their ends. This term is usually not included in the numerical models.

In this article, some experimental results obtained with a full-size PHP will first be analysed. We will particularly focus on the effect of the fluid and tube diameter, as well as the effect of orientation. Then, to help us analyse the results obtained at the system scale, we will discuss the results of an experimental study of the oscillating flow in a single tube of a single liquid plug, under adiabatic conditions (purely hydrodynamic aspect) and under non-adiabatic conditions (thermal effects due to heating of the test-section). PHPs are equipped with 24 K-type thermocouples distributed all-along their length. All the results presented in the following part have been obtained under steady-state conditions: the coolant temperature at the condenser and the heating power at the evaporator were first fixed to the desired value by the experimenter, and then the temperatures were recorded when they had reached a value independent from time. Both PHPs are filled up to a filling ratio (i.e. ratio of the volume of liquid measured at 20°C to the whole inner volume of the PHP) equal to 50%. It has been shown (e.g. in [START_REF] Khandekar | Thermo-hydrodynamics of closed loop pulsating heat pipe[END_REF]) that the filling ratio is a parameter of major effect on the PHP behaviour, and that filling ratios between 40% and 60% usually allow for minimizing the overall thermal resistance of the system.

Experiments with full-size PHPs

Experimental results

Experimental results for characterisation can be described from the temperatures at the evaporator and condenser and heat rate, or, as chosen in the following, in terms of overall thermal resistance, which is defined from the heat rate ( Q ), the average copper block temperature at the evaporator ( e T ) and the average coolant temperature at the cryostat cooling the condenser ( cryo T ) :

Q T T R cryo e - = (2)
A large number of other experimental results were obtained by Bensalem [START_REF] Bensalem | Contribution à l'analyse du comportement de caloducs oscillants à finalité spatiale par voies expérimentale et numérique[END_REF] on the same experimental bench in other operating conditions. and PHP2 (acetone, D = 2.5 mm) as a function of the heat rate and for a horizontal orientation (denoted 0°), a favourable vertical orientation (denoted +90°) and an unfavourable vertical orientation (denoted -90°). To a certain extend, the effect of orientation is a clue for understanding the effect of gravity on the system, which is of uttermost importance in the context of space applications that are often evoked as motivations of works on PHPs. In case of vertical orientation, gravity is expected to assist the flow of liquid from the condenser to the evaporator if the former is located above the latter. On the contrary, for a condenser below the evaporator, the flow of liquid is restricted because of the static head.

It is worth underlining that even in vertical unfavourable orientation, both PHP1 and PHP2, operating in open-loop configuration, were able to operate over a relatively large range of heat rate. In addition, even for the highest heat rate plotted on the graphs, the system would not deprime and stop. With such high heating powers, the evaporator temperature reached the maximum acceptable value for the safety of the test bench or of the instrumentation.

For both PHPs, and whatever the orientation, the overall thermal resistance decreases when increasing the heat rate. One should nevertheless observe that for PHP2 (Fig. 3), two behaviours can be detected. At low heat flux, the operating curves are relatively irregular, but beyond a certain threshold value of heat rate (about 1300 W), they become much smoother. In addition, at low heat flux, and in spite of the mentioned irregular behaviour, a favourable orientation can be viewed as roughly leading to lower thermal resistance than in unfavourable orientation. On the contrary, at high heat flux, the orientation has almost no impact on the thermal resistance. The behaviour of PHP1 is different in that sense that the orientation seems here to never affects the thermal resistance. One should first recall that the number of turns N is different for these PHPs. The number of turns is known to influence the performance of PHPs: especially, increasing the number of turns reduces the effect of gravity on their behaviour [START_REF] Charoensawan | Closed loop pulsating heat pipes-Part A: Parametric Experimental Investigations[END_REF][START_REF] Khandekar | Multiple quasi-steady states in a closed loop pulsating heat pipe[END_REF]. Several explanations were proposed for this observation. Firstly, increasing N increases the interconnexions in the PHP, which is thought to improve the heat transfer [START_REF] Charoensawan | Closed loop pulsating heat pipes-Part A: Parametric Experimental Investigations[END_REF][START_REF] Khandekar | Multiple quasi-steady states in a closed loop pulsating heat pipe[END_REF][START_REF] Tong | Closed-loop pulsating heat pipe[END_REF]. Secondly, for a constant tube diameter, increasing N increases the overall heat transfer area between the fluid and the wall. However, here, the higher number of turns is accompanied by a lower value of diameter. As a consequence, in the present case, the ratio of heat transfer area in the evaporator zone between PHP1 and PHP2 (N 1 .D 1 )/(N 2 .D 2 )is very close to unity. Lastly, the ratio between the total cross-section for the fluid flow in all tubes of PHP1 compared to PHP2 ((N 1 .D 1 ²)/(N 2 .D 2 ²)) is about 0.5. This means that the hydraulic resistance in PHP2 is lower than in PHP1. Moreover, as the diameter of PHP1 is low compared to the critical value (cf. eq. ( 1)) and as the operating curves of PHP2 are independent from orientation for high heat fluxes, we conclude that the impact of tube diameter on the independence of the thermal resistance against the orientation is higher than that of number of turns, a value which is in any case already relatively high (N 2 = 20) with respect to the observations of the literature on that issue. Finally, one can add that for PHP1, the operating curve is smooth over the whole range of heat rate, so that only one type of behaviour can be detected. In other words, the whole operating curve obtained for PHP1 is apparently in the "high heat flux" regime as it has been described for PHP2.

Nevertheless, for both PHPs, the maximum heat rate before the safety temperature is reached is lower in unfavourable orientation than in favourable or horizontal orientation.

Experiments at the scale of a single capillary tube

For better understanding some of the phenomena involved in PHPs, and particularly the existence of the two operating regimes described above, a specific experimental set-up dedicated to the analysis of oscillating two-phase flows in a single capillary tube was designed and built. The aim of such a study at the scale of a single tube, while real PHPs usually include a large number of meandering turns, is to focus on some effects that are discussed in the following while eliminating other effects. In other words, this experimental work will not explain all the phenomena involved in PHPs, but will reveal two of them. As a matter of fact, we will see that such a design will allow bringing to the fore the effect of the dissymmetry of the oscillating vapour plugs (i.e. the effect of the capillary pressure between the ends of the plus) and the impact of the phase-change on the oscillating two-phase flow.

These phenomena obviously exist in real PHPs, but they are mixed with other phenomena such as bubble break-up in U-bends, as reported by Xu et al. [START_REF] Xu | High speed flow visualization of a closed loop pulsating heat pipe[END_REF], cf. section 1.

Experimental set-up

A schematic of the test bench is shown in Fig. 4. It mainly consists of two tanks connected by a capillary tube. A variable frequency electric motor enables to impose a quasi-sinusoidal volume variation on the upstream tank (also called "variable pressure reservoir") through an eccentric wheel, and a flexible membrane mounted with a return spring. The capillary is a 2.4 mm inner and 4 mm outer diameter glass tube. Its length is 30 cm. It is connected to the tanks by means of Rilsan™ pipes. The downstream tank (also called "constant pressure reservoir") is filled with liquid pentane in equilibrium with its vapour, whereas the upstream tank contains only vapour pentane. Pentane was chosen for the convenience of experiments: its boiling point is 36°C, which makes the experiments easier than with water for example, and limits the risks of breaking the tubes or reservoir because of high pressures or high temperatures.

The amplitude of the pressure oscillation (about 0.1 bar) and the frequency (typically 4 Hz) are adjusted so as to represent typical measurements of these values on a branch of the fullscale PHPs [START_REF] Bensalem | Contribution à l'analyse du comportement de caloducs oscillants à finalité spatiale par voies expérimentale et numérique[END_REF]. This adjustment can be done practically by modifying the hydraulic resistance to the liquid flow in the capillary tube (i.e. by modifying the length of capillary tube and the length of liquid slug) and by choosing the appropriate strength of the return spring and eccentricity of the eccentric wheel.

Thermocouples and manometers are located in the tanks in order to check the absence of noncondensable gases: the temperature must be in agreement with the saturation temperature corresponding to the pressure prior to starting any experiment. The flow is visualized thanks to a high speed camera. The studied flow consists of only one liquid slug oscillating in the capillary. Tests and visualizations were first made under adiabatic conditions at ambient temperature, in order to reveal some features of the two-phase flow in the absence of phasechange and heat transfer phenomena. Then, experiments were made when the tube was inserted into a heated copper block in which an opening was pierced to allow for the flow visualisation in the heated tube section. These non-adiabatic experiments are useful for understanding the effect of phase-change (nucleation, evaporation) on the oscillating flow, which remains imposed by the motor and its action on the variable volume reservoir.

Adiabatic experiments: hydrodynamic aspects

The oscillation of the slug is illustrated in Fig. 5 A large number of experiments were done for various conditions: the liquid slug length ranged from 2.4 to 15.2 mm, the amplitude of the velocity oscillations could reach up to Another aspect of hydrodynamics lies in the existence of wavelets that can be observed at the advancing meniscus (Fig. 8). The liquid film does not smoothly get thicker and thicker from the contact line to the liquid core, but on the contrary, particularly at the moments when the velocity is high, it exhibits an instability which can probably be interpreted from the lubrication theory. Analogous observations and explanations were proposed by Bretherton [17].

This behaviour can also be interpreted from Moriyama and Inoue [START_REF] Moriyama | Thickness of the liquid film formed by a growing bubble in a narrow gap between two horizontal plates[END_REF] criterion that relates the film thickness to a Bond number written as:

dt du D Bo 2 l σ ρ = (3)
This number includes the fluid acceleration and represents the ratio of the inertial forces due to acceleration to surface tension forces. These authors stated that the film thickness is governed by hydrodynamics (development of a viscous boundary layer) at high Bo number (i.e. Bo > 2), and by capillary effects at low Bond number (Bo < 2).

In our experiments, the typical velocity amplitude is 0.5 m/s and the typical frequency if 4 Hz. With such values, and assuming a perfect sinusoidal evolution of the velocity, the critical velocity for the transition between low and high Bond numbers is about 0.3 m/s, which was found to be consistent with the development of wavelets in the liquid film. This can also be detected from Figure 7: the dispersion of the measurements around the linear fit is wider for m/s 3 . 0 u > or m/s 3 . 0 u - < : in these domains, the presence of wavelets disturbs the automatic image processing, and therefore reduces the accuracy of the measurement of the meniscus radius of curvature.

Thermal aspects: effect of phase-change on the two-phase flow

In order to study the effect of heat transfer and phase-change on the two-phase flow, the wall superheat ∆T sat = T w -T sat was fixed to 15 K or 35 K. The wall temperature was measured at the contact between the heating block in copper and the glass outer wall, but no accurate measurement of the inner glass wall temperature was possible. This is not expected to be major issue in that sense that these fundamental experiments do not aim at providing experimental values of thermal parameters, but rather to understand the processes occurring in the full-size PHPs that were described previously. Two typical video recording sequences are shown on Fig. 9a (∆T sat = 15 K) and Fig. 9b (∆T sat = 35 K). These both sequences are shorter (about 50 ms) than the oscillation half-period (125 ms). In other word, these sequences correspond to a portion of period of the imposed oscillation during which the overall pressure difference between the variable volume reservoir and the constant pressure reservoir is always oriented in the same direction.

For a 15 K superheat, nucleation takes place at the edge of the advancing meniscus, but the bubble detaches relatively quickly after its birth. Its diameter remains lower than the tube diameter and it follows the overall motion of the liquid plug. The bubble may merge into the advancing liquid plug, but this phenomenon does not affect much the overall motion and it just tends to flatten the advancing meniscus during the coalescence process.

For a 35 K superheat, in the early stage, nucleation also occurs at the edge of the advancing meniscus, which is comparable to what was observed for 15 K superheat. But one can see that nucleation also occurs in the middle of the liquid plug and that this second bubble starts growing. This second bubble merges with the vapour plug as it has grown and expanded so rapidly that it could reach the interface. But meanwhile, a third bubble was born and is expanding. Its expansion is relatively violent, so that the receding meniscus is deformed. The liquid plug is thus broken up into several smaller plugs. Finally, the expansion of the third bubble is so strong that it forces the remaining liquid plug to flow against the overall imposed pressure difference. In the very late stages of this process, the liquid is fully vaporized. The images show the evaporation of the remaining liquid film.

These two kinds of behaviour must certainly be related to the two kinds of behaviour of the full-scale PHPs described in section 2.2. Indeed, a lower heat input leads to relatively low wall superheat, for which small bubbles may be generated by a simple nucleation process.

Nucleation is well known to be a complex process, whose repeatability is relatively poor. This might explain the "chaotic" behaviour of the thermal resistance of the PHP vs. heat flux curves at low heat flux (lower than about 1300 W, cf. Figs. 2 and3). This only occurs for acetone (PHP2), because the typical diameter of the bubbles will remain lower than the tube diameter. Obviously, in such a case, and if the tubes are oriented vertically, the bubbles are likely to carry up some liquid with them, as suggested by Khandekar et al. [START_REF] Khandekar | Understanding operational regimes of pulsating heat pipes: an experimental study[END_REF]. On the contrary, at high heat flux (high superheat), the heat transfer is governed by thin film evaporation which is not sensitive to orientation, and this may be an explanation for the independence of thermal resistance to orientation. Nevertheless, one could notice that the maximum heat input rate allowed by the real PHPs is higher in favourable than in unfavourable orientation. In spite of the impact of the fast thermal expansion of bubbles on the flow, the bubbles may probably still carry up some liquid with them.

Conclusion

Two separate experiments have been built and used in this work in order to characterize two full-size open loop pulsating heat pipes (PHP) and to visualize oscillating two-phase flow phenomena occurring in a single capillary tube (typical of PHPs) subjected to controlled pressure oscillations and/or wall superheat.

Concerning the full-size PHPs, the experiments reveal that both tested PHPs can operate whatever the inclination, even in vertical unfavourable inclination. However, the performance in the latter case remains lower than in horizontal or favourable inclination. Moreover, it has been shown that the overall thermal resistance of the two PHPs decreases with increasing heat power, except for the large diameter PHP (filled with acetone) subjected to the lowest heat transfer rates: in such conditions, the operative curve (PHP overall thermal resistance vs. heat transfer rate) seems to be more chaotic.

The single tube experiments were done to highlight or reveal some phenomena that are usually mixed with other when they occur in real PHPs, but whose analysis is easier in a single tube. One can first conclude that the differences between the advancing and receding meniscus radii surrounding the liquid plug have to be taken into account in the models, because the induced capillary pressure difference is of the same order of magnitude as the friction losses. Visualisations also revealed the presence of instabilities (wavelets) at the advancing meniscus. These wavelets develop for velocities that were found in agreement with the critical velocity calculated with a theoretical model from the literature. Their impact on evaporation heat transfer remains to be studied. Finally, when the test section is heated, a 15

K wall superheat at the evaporator of the tube leads to nucleation of bubbles that are always lower than the tube diameter and that do not affect the overall motion of the plug. On the contrary, a 35 K wall superheat at the evaporator leads to a faster bubble expansion that deforms the receding meniscus and breaks up the liquid plug into several smaller plugs. This expansion is so strong that it can sometimes lead to make the liquid plug flow against the overall motion which results from the imposed pressure difference between the both ends of the tube. These two behaviours are consistent with the trends observed in full-size PHP, especially the two behaviours detected on the operating curves. Such kind of phenomena like the liquid plug motion and bubbles expansion have to be well understood and studied in order to precisely model the heat and mass transfers occurring in PHPs.

Figure Captions

2. 1 . 2 =

 12 Experimental set-up Two different open loop PHPs made of a single copper tube were built, as shown schematically on Fig. 1. A valve situated between the two ends of the tube of each PHP allows the operation either in open-or closed-loop configuration. Nevertheless, in the following, only results obtained in the open-loop configuration are presented. The inner diameter of the tube used for the first PHP (PHP1) is D 1 = 1.2 mm and D 2 = 2.5 mm for PHP2. The working fluid for PHP1 is water, and that for PHP2 is acetone. It is worth underlining that the critical diameter for water is about 5 mm in the investigated range of temperature, i.e. about 4 times the tube diameter for PHP1. The critical diameter for acetone is only slightly higher than the tube diameter (D crit = 3.2 mm for the considered range of operating temperature, i.e. about 1.3 times the tube diameter for PHP2).For each PHP, the capillary tube has been wound around (N 1 = 40 turns for PHP1, and N 20 turns for PHP2) two copper blocks which form the evaporator (length = 180 mm) and condenser (length = 200 mm) and which allow for thermal spreading. The adiabatic zone length is 190 mm. Heat is supplied to the evaporator by several cartridge heaters (max power 6 kW) and the condenser is cooled by a flow of a water-glycol mixture whose temperature is controlled by means of a cryostat. The PHPs can be operated either horizontally or vertically under adverse or favourable orientations (i.e. condenser above or below evaporator). Both

Figures 2 and 3

 3 Figures 2 and 3 respectively represent the thermal resistance of PHP1 (water, D = 1.2 mm)

  , which represents a set of photographs at different times. The time step between two frames is equal to 20 ms. Here, advancing and receding menisci refer to the liquid plug motion: advancing corresponds to the leading edge and receding to the tail of the liquid plug. Enlargements (a), (b) and (c) show a strong dissymmetry of left and right interfaces: the curvature radius of advancing menisci (right interface in (a) and left interface in (c)) is smaller than that of receding menisci. This dissymmetry is a view of the pressure difference between both sides of the liquid slug. In case (b), the interface velocity is equal to zero and the liquid slug is then symmetric.An image post-processing program has been developed to determine the position of the interfaces and the meniscus curvature radii for each visualization frame. The automation of this task allows having a lot of measurements and enables to verify their repeatability. During visualization, several periods are recorded. Figure6is an example of the time evolution of the curvature radii of the two interfaces. In that experiment, the liquid slug velocity oscillates between -0.2 and +0.3 m/s. A negative velocity relates to a receding meniscus, while a positive velocity relates to an advancing one. Eight periods of oscillation are recorded and superposed. The average evolution over these eight periods is represented as dotted line: a good repeatability is observed from one period to another.

Fig. 1 :Fig. 2 :Fig. 3 :Fig 4 :Fig. 5 :Fig. 6 :Fig. 7 :Fig. 8 :Fig. 9 :

 123456789 Fig. 1: Schematic of the PHP and experimental apparatus

0.65 m/s, and the frequency of the imposed sinusoidal evolution of the pressure ranged from 1.8 to 6.1 Hz, which is believed to be the correct order of magnitude in PHPs. The experimental results are presented as the superposition of curves showing the inverse of the dimensionless meniscus curvature radius r* vs. meniscus velocity u (Fig. 7). No clear effect of the slug length or frequency could be detected, and all the curves are roughly correlated by a linear fit as 88 .

. This correlation does not aim at being general: it is only valid for our experiment, but it will help us in the following for analysing the results.

Indeed, this correlation can be used to assess the impact of the slug capillary pressure difference between advancing and receding menisci on the two-phase flow, i.e. the importance of the slug dissymmetry. For that purpose, we consider as an example a liquid slug of length L = 1 cm whose maximum velocity is u = 0.5 m/s. According to the experimental results, the advancing dimensionless radius of curvature is 1/r* = 0.465 (i.e. for u = 0.5 m/s), and that of the receding meniscus is 1.295 (i.e. for u = -0.5 m/s). Therefore, the capillary pressure difference reaches 10.9 Pa for pentane at 20°C (σ = 15.2 mN/m) in the 2.4 mm inner diameter tube used in the experiments. Besides, the frictional pressure drop for the liquid slug is calculated as

kg/m 3 and µ l = 2.37.10 -4 Pa.s at 20°C, the frictional pressure drop for the considered 1 cm long slug reaches 13.5 Pa, which is of the same order of magnitude as the capillary pressure difference. This short numerical example demonstrates that the dynamic contact angle difference between receding and advancing menisci should definitely be taken into account when trying to model the oscillating process of a PHP, as the capillary pressure difference will be as important as the other main term in the momentum equation for liquid, namely the frictional pressure drop.


Dr Susanna Ranta
email: susanna.ranta@hus.fi

Heli Viljakainen

Anne Mäkipernaa

Outi Mäkitie

Hypercalciuria in children with haemophilia suggests primary skeletal pathology

Keywords: HAEMOPHILIA, CHILDREN, OSTEOPOROSIS hypercalciuria, bone mineral density, osteoporosis, children, haemophilia

Hypercalciuria in children with haemophilia suggests primary skeletal pathology

Introduction

Osteoporosis, decreased bone strength with increased fracture risk, affects mainly the elderly. However, the basis of osteoporosis can be established already during childhood if peak bone mass (PBM) attainment is compromised.

Genetic factors account for most of the variation in PBM. Other factors include inadequate dietary intake of calcium and vitamin D, reduced physical activity, chronic illnesses, hormonal deficiencies and some medications, especially glucocorticoids. In clinical practice, the diagnosis of osteoporosis is mostly based on dual-energy X-ray absorptiometry (DXA). Reduced bone mineral density (BMD) is associated with susceptibility to fractures in all age groups [START_REF] Boot | Peak bone mineral density, lean body mass and fractures[END_REF].

Haemophilia is an X-chromosomal inherited bleeding disorder characterized by bleeding tendency due to missing or deficient clotting factor VIII (FVIII) (haemophilia A) or IX (FIX) (haemophilia B). The clinical presentation and severity vary according to factor levels (in severe form, the FVIII or FIX is < 1 % of normal activity, moderate > 1 -4 % and mild > 5 -25 %). In severe haemophilia, the often spontaneous bleeds in joints and muscles lead to haemarthrosis and chronic arthropathy. Severe haemophilia is most often diagnosed within the first year of life. In moderate haemophilia bleeding episodes occur usually after small traumas or injuries. In the patient with mild disease, the spectrum of bleeding manifestations includes easy bruising and postsurgical bleeds. With modern prophylaxis (i.e. regular infusion of the missing coagulation factors) severe haemophilia can be rendered clinically to moderate disorder by preventing spontaneous joint bleeds. The children with prophylactic therapy are encouraged to participate in normal physical activities.

Several chronic illnesses predispose to osteoporosis. Also patients with severe haemophilia are at risk of osteoporosis [START_REF] Gallacher | Association of severe haemophilia A with osteoporosis: a densitometric and biochemical study[END_REF][START_REF] Wallny | Osteoporosis in haemophilia -an underestimated comorbidity?[END_REF][START_REF] Iorio | Bone mineral density in haemophilia patients. A meta-analysis[END_REF]. Increasing evidence suggests that even children with haemophilia are at risk for reduced BMD [START_REF] Barnes | Reduced Bone Density Among Children With Severe Haemophilia[END_REF][START_REF] Tlacuilo-Parra | Inactivity is a risk factor for low bone mineral density among haemophilic children[END_REF]. Several risk factors can contribute to these findings. First, children with haemophilia are likely to be less active in sports than their healthy peers. This may correlate to the severity of haemophilia, the number of previous joint bleeds and the availability of prophylactic treatment. Children who develop inhibitory antibodies against the coagulation factor needed are especially prone to spontaneous bleeds before successful immunotolerance induction (ITI). Secondly, sedentary lifestyle is associated with reduced sunlight exposure and vitamin D deficiency. Thirdly, chronic systemic inflammation due to hepatitis C or other blood-borne viruses may predispose to reduced BMD [START_REF] Masaki | Longitudinal changes of bone mineral content with age in patients with cirrhosis of the liver[END_REF][START_REF] Katsarou | Increased bone resorption is implicated in the pathogenesis of bone loss in haemophiliacs: correlations with haemophilic arthropathy and HIV infection[END_REF]. Furthermore, it has been speculated that chronic blood loss induces osteoporosis through functional interaction between haematopoietic and bone tissues [START_REF] Gurevitch | The hematological etiology of osteoporosis[END_REF].

Since the introduction and better availability of plasma-derived products of higher purity as well as recombinant concentrates in haemophilia treatment during the past 20 -30 years, the prognosis and life expectancy of patients with haemophilia have improved remarkably. As life expectancy approaches that of the general male population, we face new challenges with elderly haemophilia patients and their age-related illnesses. Osteoporosis in the advanced age can significantly complicate the treatment of haemophilic arthropathy. Achieving the optimal PBM is therefore especially important in patients with haemophilia or other severe bleeding disorder. showed that it is possible to preserve joint function by preventing bleedings with early prophylaxis. Recent studies [START_REF] Khawaji | Long-term prophylaxis in severe haemophilia seems to preserve bone mineral density[END_REF][START_REF] Zerwekh | Bone disease and hypercalciuria in children[END_REF] have indicated that early-onset prophylactic treatment can help to maintain mobility and preserve normal BMD. At our center the principal treatment regimen for children with severe haemophilia has been from the late 1990s regular continuous treatment as primary prophylaxis (PP), started before the age of 2 years without previous joint bleed or after the first joint bleed. Considering these protective circumstances their skeletal characteristics were expected to be similar to healthy population. This case-control study was undertaken to assess bone health and relevant life-style factors, including dietary intakes of calcium and vitamin D, and physical activity in children with haemophilia.

Patients and Methods

Patients and study protocol

This one-center case-control study included children with haemophilia who were aged > 4 years. Twenty two of them were on regular continuous treatment, defined as PP when started before age 2 years without previous joint bleed or after the first joint bleed [START_REF] Donadel-Claeyssens | Current coordinated activities of the PEDNET (European Paediatric Network for Haemophilia Management[END_REF] or as secondary prophylaxis (SP) when started after 2 years or after more than 2 joint bleeds. The subjects with SP include one boy with severe haemophilia and inhibitor, one with delayed diagnosis of haemophilia and one with moderate to severe haemophilia. The prophylactic clotting factor therapy in haemophilia A is typically 20 -40 IU/kg three times a week or every other day and in haemophilia B, 30 -50 IU/ kg twice weekly or every third day. The patients are encouraged to participate in sports activities. Contact sports such as ice hockey are allowed before 12 -14 years of age but not encouraged in the advanced series without tailoring of the treatment regimen. None of the children have hepatitis B, C or HIV infection.

Clinically the joint bleeds had not led to long term functional disability in any of the patients. None of the patients had a history of high blood pressure or haematuria.

An information letter and a consent form were sent to the parents of 33 patients with haemophilia followed at Hospital for Children and Adolescents, University of Helsinki, Finland. One patient with severe haemophilia A was from Vietnam and one with moderate haemophilia A from India, all others were of Caucasian origin. The Research Ethics Committee of Helsinki University Hospital approved the study and a written informed consent was obtained from all study participants and/or their guardian. With the exception of two patients with mild haemophilia, all consented to participate in the study (31/33, 94%).

Clinical data were collected from the patients' medical records. The patients were interviewed and clinically examined by SR or AM on their hospital follow-up visit. Anthropometry, bone mineral and body composition measurements and laboratory samples were obtained during the same visit. Two age and height-matched controls for each patient were selected from a representative cohort of apparently healthy Finnish school children from the same district [START_REF] Viljakainen | Dual effect of adipose tissue on bone health during growth[END_REF]. The original cohort comprised 202 children and adolescents from 7 to 19 years of age; 62% were girls. Background characteristics, lifestyle factors and medical history were collected with questionnaires and interview. Anthropometry, bone mineral and body composition measurements and laboratory samples were obtained during the study visit using the same methods as for the patients. With the exception of controls for two 4-year-old boys with haemophilia all the selected controls were male.

Bone densitometry and radiographic evaluation

Whole body (WB), lumbar spine (LS) (L1-L4) and total hip BMD (g/cm2), bone area and bone mineral content (BMC)

were measured with DXA (Hologic Discovery A, Waltham, USA). All the values were transformed into Z-scores (the difference of the measured value from the mean sex-and age-specific reference value in SD units) using the equipment-specific reference database for United States Caucasian Children. Body composition was analyzed with the manufacturer's software to separate lean body mass (LBM) and fat mass (FM). Percent fat was calculated as fat mass divided by total mass. For the detection of possible vertebral compression deformities, lateral images of the thoracic and lumbar spine were acquired using the DXA scanner (IVA, instant vertebral assessment). The vertebrae were compared with adjacent vertebrae and graded depending on percent reduction in anterior, mid-or posterior vertebral height. A 20% reduction in vertebral height was used as cut off to define abnormal morphology [START_REF] Mäkitie | Radiographic vertebral morphology: a diagnostic tool in pediatric osteoporosis[END_REF].

Bone age of the patients was determined from a plain radiograph of the left hand according to Greulich and Pyle (1959). Both the bone age and IVA radiographs were evaluated by an experienced paediatric endocrinologist (OM)

Growth assessment

Height was measured without shoes using a standard wall-mounted Harpenden stadiometer. Weight was measured using a standard electronic scale. The SD score for height and height-adjusted weight (a percentage of the mean ratio of normal population of the same gender and height) were obtained using the Finnish standards [START_REF] Sorva | Variation of growth in height and weight of children. II. After infancy[END_REF], Pere et al, 2000). Body mass index (BMI) was calculated as weight in kilograms divided by square of height in meters (kg/m2). Pubertal maturation of the patients was assessed clinically according to Tanner (1962), by sex steroid concentrations and the bone age. Assessment of pubertal maturation of the controls was based on serum gonadotrophin and sex steroid concentrations only. For data analysis the subjects were divided into three subgroups based on pubertal maturation (prepubertal, pubertal and postpubertal). For all the fractures, also the localization and trauma mechanism were recorded.

Lifestyle factors

The physical activity included regular every-day activities (e.g. walking to school), activity at school, and both guided and unguided leisure-time activities. Duration, frequency and intensity of activity sessions were recorded. A total physical activity score was obtained as a sum for a whole week. Physical activity scores of 13.5, 17 and 20 correspond to 1, 1.5 and 2 hours daily activity, respectively, which meet the recommendation for this age group (Nordic Council of Ministers, 2004). In addition, duration of each activity was multiplied with an intensity factor according to [START_REF] Neville | Relationship between physical activity and bone mineral status in young adults: the Northern Ireland Young Hearts Project[END_REF] to obtain scores for intensity of physical activity and strenuous exercise, expressed as a total for a whole week. Physical activity score was considered low when less than 13, corresponding to less than one hour of daily activity. Intensity of physical activity score included in addition the intensity of the sports activity (range 3.0-34.0). Dietary intakes of vitamin D and calcium were calculated using a food frequency questionnaire (FFQ), which is a validated semi quantitative questionnaire covering over 70 foods [START_REF] Outila | Vitamin D status affects serum parathyroid hormone concentrations during winter in female adolescents: associations with forearm bone mineral density[END_REF]. The nutrient contents of the foods were calculated using the Finnish National Food Composition Database (Fineli®, version 2001), which is maintained by the Nutrition Unit, National Public Health Institute of Finland. The calculated total intake of vitamin D included also intake from supplements. If FFQ was not returned (8 patients and 13 controls subjects), dietary intakes of vitamin D and calcium were calculated by a nutritionist either from 4-day food records or from a structured interview during the out-patient visit.

Laboratory measurements

Morning blood samples after overnight fast were assayed for testosterone, 25-hydroxy vitamin D (25-OHD), intact parathyroid hormone (PTH), calcium (Ca), phosphate (Pi), alkaline phosphatase (ALP), carboxyterminal telopeptide of type I collagen (ICTP), and procollagen type I aminoterminal propeptide (PINP). Concentration of calcium (u-Ca), phosphate (u-Pi) and creatinine (u-Crea) and type I collagen cross-linked N-telopeptide (INTP or NTx) was measured from second void urine samples. All laboratory samples for patients and controls were obtained between October and April when the amount of sunlight is minimal except in three patients with severe B haemophilia whose samples were obtained in late summer and therefore excluded from the analyses. The measurements were in all study subjects based on a single sample, obtained similarly in the patients and the controls. All blood and urine measurements were analyzed in the Central Laboratory of Helsinki University Hospital.

Statistical analyses

Statistical analyses were performed with SPSS for Windows, Version 17.0 for Windows (SPSS Inc. Chicago, IL).

Comparison of two groups was performed with Independent samples t-test and in case of categorical variables with performed to investigate differences in BMD after adjusting for confounder e.g. intensity of physical activity. If variables were not normally distributed corresponding non-parametric tests were applied. Results are expressed as means with 95% confidence limits or SDs. P values <0.05 were considered significant.

Results

Demographic and anthropometric data

The final study group included 31 haemophilic boys aged between 4 and 16 years. The clinical characteristics of the patients are presented in Table I. The median age at the start of the prophylactic treatment was 0.97 years (mean 1.47). Seven of the total cohort (24%) and 6 (29%) of the patients with severe haemophilia had a history of no joint bleeds. For the remaining 22 patients, the number of joint bleeds (either self-reported or documented) varied between 1 and 11 (mean 3.1, median 2). The anthropometric characteristics of the patients and controls were similar (Table II). The number of overweight or obese patients was slightly higher in patient group. Also, the mean fat% Z-scores were somewhat higher in cases compared to controls. Neither of these figures was statistically significant and there were no differences in BMI or LBM between the groups.

Lifestyle factors

Data for vitamin D intake and calcium were available for 29 patients and 52 controls. Fourteen of the patients (48 %) and 32 of the controls (55%) received vitamin D supplements. The daily total intakes of vitamin D ranged from 4.3 to 34.3 µg/day for the patients and from 4.3 to 15 µg /d for the controls, and were lower than recommended in 8 patients (28%) and 13 controls (28%). The daily intakes of calcium were below the recommendation for 2 patients and 1 control. There were no significant differences in vitamin D or calcium intakes between the groups (Table III).

Altogether 3/28 of the patients with bleeding disorder and 4/43 of the controls were considered physically inactive (score <13). The intensity for physical activity score was significantly lower in the patient group (p=0.002). (Table III).

Bone density and fractures

The z-scores for BMD are shown in Table IV. The BMDs for LS, femur and WB were lower in the patients but this reached statistical significance only in the WB z scores (p=0.007). As the intensity of physical activity was lower in the patient group we hypothesized that this could be the factor behind the difference. However, the difference persisted even after adjusting the WB z-scores for physical activity (p= 0.044). When evaluated separately in subgroups according to pubertal maturation (pre-, mid-and postpubertal) the difference between patients and controls remained in the older groups but not in prepubertal children. There were no significant differences in lean mass and fat mass between the two groups indicating that the trend for lower physical activity had not resulted in altered body composition in the patients (Table II). The fat% Z-scores correlated inversely with BMD Z-score in femur and (Chi-Square; p=0.135). Three patients had one vertebral wedge fracture (anterior vertebral height loss >20%) and one had one crush fracture (vertebral height loss of 20%) with normal bone densities, whereas all the three control patients had one vertebral wedge deformity. Three (10% ) patients had had one or more traumatic fractures and one had clavicular fracture at birth. Of the controls, 12/52 (23%) had a history of at least one traumatic fracture (Chi-Square; p= 0.26).

Biochemical findings

Urine calcium secretion (U-Ca/Crea) was significantly higher in the patient group (p=0.001) (Figure 1; Table V) and 3 patients (12.5 %) with haemophilia had hypercalciuria (U-Ca/Crea >0.70 mmol/mmol) while hypercalciuria was observed in only 2 (3.5%) of the controls. Calcium excretion was not associated with age, pubertal stage, severity of the haemophilia, number of joint bleeds, prophylaxis, history of inhibitors, or BMD in our relatively small cohort.

Urinary calcium excretion correlated inversely with PTH (r = -0.314, p = 0.004) and the bone resoprtion marker ICTP (r= -0.276, p=0.012) and positively with 25-OHD (r= 0.289, p= 0.008) and urinary Pi (r= 0.290, p =0.008). There were no differences in mean serum 25-OH-D concentrations between the study group and the controls (Table V), the median concentrations being 47 (range 18-90) and 45 (range 17-77) nmol/L for the patients and the controls, respectively. The concentration was suggestive of vitamin D insufficiency (<50 nmol/L) in 13 patients (50%) and 37 (66%) controls and of severe D vitamin deficiency (below 25 nmol/L) in 2 (8%) patients and 1 (2%) control. No significant differences were detected in the serum bone turnover markers PINP, ICTP, or ALP between the two groups but the bone resorption marker U-INTP was significantly lower in the patient group (p=0.002). Consequently, the ratio S-PINP/U-INTP was significantly higher in the patients as compared with the controls (p=0.009).

Interestingly, the patients also had higher serum Ca (p=0.006) concentration.

Discussion

Several previous studies have suggested a connection between haemophilia and osteoporosis. A recent metaanalysis, which summarized the results of seven case-control studies, comprising 111 paediatric and 101 adult haemophilia patients, confirmed an association between haemophilia and low BMD [START_REF] Iorio | Bone mineral density in haemophilia patients. A meta-analysis[END_REF]. Most of the studies have involved adults and since the number of paediatric studies is limited it has remained unknown whether compromised bone health is secondary to joint bleeds and physical inactivity. The findings of the present study, involving children with early-onset prophylactic coagulation factor treatment, suggest that in addition to life-style factors also primary factors, especially hypercalciuria, contribute to impairment of bone health and development of osteoporosis over time even in prophylactically treated patients. In this study we examined 29 Finnish children with haemophilia and 58 matched controls. The study group did not differ significantly from the control group regarding calcium or vitamin D intake or other lifestyle factors, although there was a trend towards lower physical activity. Approximately half of the study subjects received vitamin D supplements, which are presently recommended to all Finnish children. Although the BMD in the haemophilia patients was within normal range, their whole body BMD was significantly lower than in the healthy controls. Our results indicate that in spite of prophylaxis, haemophilia patients are at risk of lower BMD compared to their peers. This finding becomes more apparent after puberty. While lower physical activity seems a possible explanation, it does not alone account for the difference. Low BMI or lean body mass, but also overweight and obesity may compromise bone mass accrual [START_REF] Dimitri | Fat and bone in children: differential effects of obesity on bone size and mass according to fracture history[END_REF][START_REF] Viljakainen | Dual effect of adipose tissue on bone health during growth[END_REF]. No significant differences in lean mass and fat mass were observed between our patients and control subjects.

Previous paediatric studies have involved variable patient populations and assessment of confounding factors has often been inadequate. One of the largest paediatric studies consisting of 62 haemophilic children (35% severe) was reported by Tlacuilo-Parra in 2008. None of the patients were on prophylactic therapy and all had a history of one or more joint bleeds. There were no differences in dietary calcium intake whereas, as expected with on-demand therapy, there was a significant difference in physical activity between the patients and controls. The lumbar spine BMD in patients was significantly lower than in controls; the authors proposed that this was due to the lower physical activity. Recently the same group compared serum bone turnover markers in 69 children with haemophilia and normal vs. low BMD. They found that children with haemophilia and low LS BMD had lower osteocalcin levels indicating diminished osteoblastic activity but found no differences in bone resorption markers (Tlacuilo-Parra 2010).

An Australian study reported reduced areal BMD z-scores in 19 children with severe haemophilia A compared with healthy controls [START_REF] Barnes | Reduced Bone Density Among Children With Severe Haemophilia[END_REF]. The mean age of the patients was 12.2 years; all had severe haemophilia. Most (79%) of the participants were on prophylactic treatment. The reduction in BMD correlated with the severity of haemophilic arthropathy. However, diet, other lifestyle factors or laboratory tests were not taken into account.

Furthermore, neither the age of prophylaxis onset nor the number of joint bleeds was reported. Another recent study [START_REF] Christoforidis | Bone status of children with haemophilia A assessed with quantitative ultrasound sonography (QUS) and dual energy X-ray absorptiometry (DXA)[END_REF] used both quantitative ultrasound sonography (QUS) and DXA to assess bone status in 27 children or young adults (mean age 12,3 years) with moderate or severe haemophilia A, most of whom were on either primary or secondary prophylaxis. Their bone properties were within normal limits. This study also lacked a normal control group and lifestyle factors or laboratory tests were not evaluated. In line with these previous studies, our findings suggest that early-onset prophylactic treatment is likely to have a favorable effect on BMD and allow nearly normal physical activity. However, despite successful prevention of joint bleeds and inactivity, our study subjects tended to have lower BMD than age-and height-matched controls suggesting that the compromised bone health in haemophilia is not caused by lifestyle factors only.

Importantly we observed significantly increased urine calcium secretion in our study cohort as compared with the controls, and 3 patients with haemophilia had pathological hypercalciuria (U-Ca/Crea >0.70 mmol/mmol).

Hypercalciuria is a known risk factor for osteoporosis and fractures; the pathophysiology is multifactorial (Srivastava (1997) showed that one third of 73 children with idiopathic hypercalciuria had osteopenia. [START_REF] Penido | Bone alterations in children with idiopathic hypercalciuria at the time of the diagnosis[END_REF] reported reduction in BMD among 88 children with idiopathic hypercalciuria. They suggested altered bone metabolism as a mechanisms for lower BMD in these patients. Our finding of significantly higher urinary calcium in haemophilia patients than in their healthy peers suggests primary pathology in bone metabolism, which in turn may result in impaired bone mass accrual and low BMD in haemophilia patients. The cumulative effects of hypercalciuria over years may become fully evident only in an older patient group.

The mechanisms behind the high urine calcium excretion in children with haemophilia remain unclear. None of the patients were immobilized, their calcium and vitamin D intakes were comparable to the controls and none had S-25-OHD values exceeding the normal range. Considering the higher p-Ca and consequently lower PTH, one can presume that the hypercalciuria is a result of increased renal filtration load of calcium possibly secondary to reduced bone metabolism and mineralization and not due to increased bone resorption, as high urinary calcium was associated with lower PTH and lower bone resorption markers ICTP and INTP. This finding is in contrast with a recent report on adult Greek haemophiliacs who had increased levels of bone resorption markers as compared to their controls [START_REF] Katsarou | Increased bone resorption is implicated in the pathogenesis of bone loss in haemophiliacs: correlations with haemophilic arthropathy and HIV infection[END_REF]. However, their study group differed from ours on several other aspects as well, including high number of HIV and HCV seropositivity and arthropathy. As our study group was relatively small and the observations were based on single samples only the biochemical findings need to be confirmed in future studies involving larger numbers of patients.

Although hypercalciuria has not been evaluated or reported in previous studies, other findings have suggested primary skeletal pathology in haemophilia. [START_REF] Manco-Johnson | Prophylaxis versus Episodic Treatment to Prevent Joint Disease in Boys with Severe Haemophilia[END_REF] studied the effect of prophylactic treatment compared with enhanced on demand therapy on the joints of 65 boys with severe haemophilia A. Joint preservation was evaluated by magnetic resonance imagining (MRI) and plain-film radiography. A subset of patients with abnormal MRI had no history joint bleeds. The authors proposed that deterioration of joints evidenced by MRI is caused by microhaemorrhage into the joints or subchondral bone. The same mechanism may also underlie the altered bone metabolism and hypercalciuria seen in our patients. Gurevitch and Slavin hypothized in 2006 that chronic blood loss resulting in overexertion of haematopoietic system plays an important role in the etiology of osteoporosis. They speculated that blood loss creates pressure on the haematopoietic system leading to both osteogenic activity through stimulation of haematopoietic system including osteoclasts and enhancement of bone development though increased production and maturation of osteoblasts.

Chronic microhaemorrhage could also induce chronic inflammation, which has been associated with low BMD. As inflammatory cytokines were not evaluated we can not rule out this possibility. However, assuming that the number of joint bleeds correlates with the level of inflammatory cytokines one would expect reduced BMD to become more evident with higher number of joint bleeds. This was not observed in our study. [START_REF] Avgeri | Assessment of Bone Mineral Density and Markers of Bone Turnover in Children Under Long-term Oral Anticoagulant Therapy[END_REF]. As expected, the patient group had considerably lower vitamin K dependant coagulation factors (F II, VII, IX, X, protein S, protein C) than the controls due to anticoagulation. There were several lines of evidence supporting the association between osteopenia and long-term oral anticoagulation: signs of osteopenia in majority of the patients in DXA scanning, higher osteoclast activity markers, lower osteoblast activity markers and higher PTH.

Nevertheless, hypercalciuria was not connected to oral anticoagulation and suggests that impaired coagulation cascade does not per se lead to high urinary calcium excretion.

The age-specific rates of chronic renal disease in persons with haemophilia were higher in every age group than among US males (Kulkarni et al, 2003). The reasons underlying chronic renal disease in haemophilia patients are multifactorial. However, in the light of our findings, the role of hypercalciuria needs to be investigated in future studies.

In conclusion, the findings of our study indicate that even though BMD in boys with haemophilia is within normal range, the BMD values tend to be lower than in healthy peers. Importantly, our study identified hypercalciuria as a novel risk factor for osteoporosis. This finding suggests that in addition to life-style factors, such as reduced physical activity, primary skeletal pathology contributes to the development of osteoporosis in patients with haemophilia.

The clinical implications of high urinary calcium excretion and its causal relationship with low bone mass and renal function impairment remains unclear in patients with haemophilia. Further studies are needed to elucidate the mechanisms behind hypercalciuria and its long-term skeletal consequences.

6

  Together with their parents the subjects filled in a questionnaire on background characteristics, lifestyle factors and medical history; information was reviewed and completed together with a paediatrician during the follow-up visit.

8

  Asymptomatic vertebral fractures assessed by IVA were present in 4 (15 %) of the patients and 3 (4.8 %) controls.

  2007; Zerwekh 2010). Garcia-Nieto et al.

  antagonists are used as anticoagulants by patients with thrombosis or risk of thrombosis. A possible known side effect is reduction in BMD. A Greece group assessed the BMD and bone metabolism markers of 23 children with long-term oral anticoagulant therapy and the bone metabolism markers of 25 age matched controls

Figure 1 .

 1 Figure legends

F

Table I .

 I Clinical characteristics of the patients with haemophilia.

									16
	Age	Diagnosis	Age at diagnosis	Treatment	Age at start of prophylaxis	History of inhibitor	Joint bleeds
									0	1-3	>4
	16.4 mild haemophilia A	0		D			no	x
	16.7 mild haemophilia A	0.5		D			no	x
	8.3	moderate haemophilia A	1.8		D			no	x
	11.3 moderate haemophilia A	1.1		P		2.8	no	x
	16.1 moderate haemophilia A	1.1		D			no	x
	8.0 10.7 moderate haemophilia B moderate haemophilia B 10.8 moderate haemophilia B 4.2 severe haemophilia A 4.8 severe haemophilia A F 0 0.5 0 0.1 0.6 o r	D D P P P		8.7 1 0.9	no no no no no	x x	x	x x
	8.5 8.7	severe haemophilia A severe haemophilia A	0 0.3	P P P		0.9 0.6	no no	x	x
	8.8	severe haemophilia A	1	P e		1.3	yes	x
	9.2 10.1 severe haemophilia A severe haemophilia A 10.5 severe haemophilia A	0 0 0		P P P e r	0.7 0.6 0.9	no yes yes	x	x x
	11.2 severe haemophilia A 12.2 severe haemophilia A	0 1		P P	0.6 1.2 R	no no	x	x
	12.6 severe haemophilia A 14.6 severe haemophilia A 14.6 severe haemophilia A 14.9 severe haemophilia A 15.1 severe haemophilia A 16.0 severe haemophilia A 6.4 severe haemophilia B	0 0.2 1.1 0.5 0.2 5.5 0		P P P ITI P P P	2.3 0.9 e 3.6 3.9 1.1 v i yes no no yes* no e 5.5 no 1 no w	x	x	x x x x x
	6.4	severe haemophilia B	0		P		1	yes*, LT	x
	8.6	severe haemophilia B	0.5		P		0.6	no	x
	9.3	severe haemophilia B	0.6		P		0.7	no	x
	13.7 severe haemophilia B	0.6		P		1	no	x
	P=prophylaxis, D= on demand, ITI= immunotolerancy induction. Prophylaxis for haemophilia A was given every other
	day and for haemophilia B every 3rd -5th day, *inhibitor at the study time, LT=low titer

Table II .

 II Age and anthropometrical data of the 29 patients with haemophilia and the 58 control subjects.

			mean ± SD (n)
		patient (29)	control (58)	p value
	Age (years)	11.0 ± 3.6	11.6 ± 2.8	0.32
	Weight (kg)	44.2 ± 21.6	45.3 ± 18.1	0.80
	Height (cm)	147 ± 21	150 ± 18	0.39
	Height z-score	0.3 ± 1.1	0.5 ± 1.0	0.46
	Relative weight (%)*	13.9 ± 21.6	8.0 ± 18.8	0.21
	Lean mass (kg)	31.4 ± 12.6	33.1 ± 12.1	0.54
	Fat mass (kg)	13.2 ± 11.3	11.5 ± 7.4	0.44
	Overweight/obese (%)	35.5		24.6	0.28**
	BMI (kg/m2) Fat % z score Puberty prepubertal	19.3 ± 4.5 -0.039 ± 1.41 19 F o r	19.2 ± 4.0 -0.266 ± 1.17 38	0.92 0.44
	midpubertal	2		6	0.84**
	postpubertal * height adjusted weight ** Chi-Square test	8	P e	14
			e r
				R
				e
				v i e
				w

Table III .

 III Dietary intakes of vitamin D and calcium and physical activity among patients and controls (mean ± SD)

				19
		(mean ± SD)
	Variable	patients (29)	controls (45)	p value
	Calcium intake (mg)	1570 ± 540	1550 ± 490	0.86
	Dietary Vitamin D intake (mg)	8.2 ± 3.1	8.4 ± 3.3	0.79
	Total vitamin D intake(mg)	12.8 ± 7.3	11.9 ± 5.8	0.58
	Physical activity score	17.6 ± 5.1	19.6 ± 3.2	0.064*
	Intensity of physical activity	17.3 ± 7.8	23.0 ± 7.0	0.002
	* Mann-Whitney test
	F o r F o r
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
			w w

Table V .

 V Biochemical findings in the patients with haemophilia and the controls. The values are given as mean ± SD. Serum; U, Urinary; 25-OHD, 25-hydroxy vitamin D; PTH, intact parathyroid hormone; ALP, alkaline phosphatase; Ca, calcium; Pi, phosphate; PINP, procollagen type I aminoterminal propeptide; ICTP, carboxyterminal telopeptide of type I collagen; Crea, Creatinine; INTP, type I collagen cross-linked N-telopeptide *The differences remained even after adjusting for age.

	Variable	Patients (26)	Controls (57)	p value
	S-25-OH-D	47 ± 17	44 ± 12	0.351
	S-PTH	32 ± 22	42 ± 23	0.073
	S-ALP	230 ± 58	228 ± 65	0.857
	S-Ca	2.40 ± 0.08	2.34 ± 0.09	0.006*
	S-Pi	1.37 ± 0.19	1.46 ± 0.18	0.042*
	S-PINP	547 ± 172	564 ± 197	0.700
	S-ICTP	14.6 ± 4.0	15.6 ± 4.5	0.348
	U-Ca/U-Crea	0.361 ± 0.29	0.181 ± 0.18	0.001*
	U-Pi/U-Crea	2.62 ± 1.31	2.35 ± 2.53	0.616
	U-INTP	448 ± 231	604 ± 300	0.021*
	S-PINP/U-INTP	1.144 ± 0.72	1.05 ± 0.40	0.009*
	S,

British Journal of Haematology