B Andriyevsky
email: bohdan.andriyevskyy@tu.koszalin.pl

M Romanyuk

V Stadnyk

Simulation of elastic-and-optical properties of K 2 SO 4 crystals

Keywords: A. Inorganic compounds, C. Ab initio calculations, D. Electronic structure, D. Optical properties

The electronic optical spectra of the mechanically free and stressed crystals of potassium sulfate, K 2 SO 4 , in the orthorhombic phase Pnma have been calculated by the CASTEP (CAmbridge Serial Total Energy Package) code. On the basis of these calculations the components of the stress elastic-and-optical tensors based on the changes of refractive index n (π im ) and birefringence Δn ( * km π ) (i,k,m = 1, 2, …, 6) have been obtained for the indices i,k,m = 1, 2, 3. Absolute magnitudes of the calculated tensor π im are probably underestimated because the magnitudes of the calculated elastic stiffness tensor c rm are found to be near two times overestimated. Features of the spectral dependences n(E) and k(E) of refractive and absorption indices of the mechanically free and stressed potassium sulfate crystal have been analyzed.

partially occupied [2]. The hexagonal high temperature phase of the crystal is known as α-K 2 SO 4 and the orthorhombic phase as β-K 2 SO 4 . Analogous phase transformations (PT's) from αto β-type phase are shown for the other K 2 SO 4 -family crystals (e.g. Na 2 SO 4 , LiKSO 4 , K 2 CrO 4 , and K 2 SeO 4 ) [3,4]. Another PT of second order at the temperature of 56 K was detected in K 2 SO 4 crystal. The crystal symmetry of this low-temperature phase (γ-K 2 SO 4 ) is assumed to be of monoclinic [5].

A c c e p t e d m a n u s c r i p t 2

Valuable information on the structural and physical properties of a crystal can be provided by optical investigations of its refractive and birefringence characteristics. Several investigations of such type were done for PS crystal earlier in the photon energy range 1 -5 eV of the crystal's transparency [6,7]. It is known that PS crystal possesses two isotropic points in the spectral range 250 -850 nm as well as demonstrates the property of isospectrality [7]. An influence of the uniaxial stress on these characteristics of PS crystals has been studied experimentally in [8].

Because of the experimental studies of optical properties of PS crystals mentioned, it is interesting to perform theoretical first-principles calculations of the tensor of refractive index, n p = n p + ik p , in the photon energy range of electronic excitations, which determines the dispersions of refractive indices n p (λ) and birefringence Δn pq (λ) in the range of crystal's transparency. Here, p, q = 1, 2, 3, are principal directions of optical indicatrix of a crystal.

First calculations of the band structure and optical properties of K 2 SO 4 crystals were performed in the paper [9] using a norm-conserving pseudopotential method. Experimental spectra of the imaginary part of dielectric permittivity ε 2 (E) of the crystal obtained in this paper showed some influence of the electron-phonon broadening. Because of known experimental difficulties of the investigations of absolute elastic-and-optical tensors of materials, the calculations of this tensor for K 2 SO 4 crystals by the first-principles code, based on the density functional theory (DFT), presents an interest. Such type of ab-initio study of the pressure influence on the refractive indices of PS crystals gives possibility to obtain new respective data for the relatively big magnitudes of mechanical stresses, which are inaccessible in the experiment because of the crack of crystalline samples under the uniaxial stresses.

Method of calculations

Calculations of the electronic energy structure of potassium sulfate crystal have been performed within a framework of the density functional theory (DFT) using the pseudopotentials-plane-wave-basis-set CASTEP code (Cambridge Serial Total Energy Package) [10]. The calculations were performed within a framework of the generalized gradient approximation (GGA) with PW91 functional for the exchange and correlation effects [11] with the ultra-soft pseudopotentials [12]. These pseudopotentials require a quite low energy cutoff and guarantee good transferability, that is, the same potential correctly reproduces the valence electron scattering by the ionic core in different chemical Optimization (relaxation) of the atomic positions and crystal's cell parameters was performed before the main calculations of electronic characteristic: total electronic energy E, band energy dispersion E(k), density of electronic states (DOS), and dielectric functions ε(E). Band structure of the crystal was calculated for 27 k-points of the Brillouin zone (BZ). The SCF convergence criterion of electronic energy per atom was 2⋅10 -6 eV and the tolerance for the electronic total energy during optimization was 1⋅10 -5 eV.

The CASTEP program permits to apply unlimited mechanical stress to the crystal studied, perform full structure optimization and then to calculate the respected photon energy dependences of the real and imaginary parts of dielectric permittivity, ε 1 (E) and ε 2 (E). These results can be presented in the form of the photon energy dependences of the refractive and absorption indices, n(E) and k(E), using the known relation ε = n 2 . According to the reference data [START_REF] Choy | Landolt-Bornstein: numerical data and functional relationships in science and technology new series[END_REF][START_REF] Bhalla | Landolt-Bornstein: numerical data and functional relationships in science and technology new series[END_REF], the bulk modulus B and the Young's modulus E of PS crystal are equal to 31.9 GPa and 44.2 GPa, respectively. In the present calculation, the uniaxial stress σ = 1 GPa was applied along three Cartesian axes of the crystal matching the principal directions of optical indicatrix. Such stress still falls under the range of elastic stiffness of the crystal, if taking into account the above mentioned value of the Young's modulus E. In this range, the pressure induced change of refractive index δn is probably proportional to the stress σ.

Results and discussion

Spectra of the calculated refractive and absorption indices n(E) and k(E) of the PS are

shown in figure 1 for the scissor factor of 1.0 eV. Scissor factor is often used in the DFTbased calculations of dielectrics and semiconductors to agree magnitudes of the calculated and experimental low-energy edge of fundamental absorption. However, even in this case the calculated principal refractive indices obtained for the photon energy E = 0.01 eV, n x = 1.5600, n y = 1.5572, n z = 1.5538, are substantially greater than the experimental ones, n x = 1.5016, n y = 1.4992, n z = 1.4977 [7], for the photon energy E = 2.48 eV. This discrepancy may be caused by the negative input from the absorption of crystal caused by atomic oscillations (E < 0.4 eV) into the experimental refractive index in the range of transparency 0.4 -7.0 eV, which is not being calculated by the CASTEP code. This negative input derives from the known Kramers-Kronig's relation between the spectra of refractive (n) and absorption (k) indices of material [START_REF] Reitz | Foundation of Electromagnetic Theory[END_REF][START_REF] Hilgevood | Dispersion Relation and Causal Description[END_REF],

A c c e p t e d m a n u s c r i p t

4 2 2 0 2 ( ) ( ) 1 tk t dt n E t E π ∞ = + - ∫ . ( 1
)
When the average difference of the calculated and experimental refractive indices at the photon energy E = 2.48 eV is regarded to be Δn osc = 0.06, then rough calculations of the average absorption index k osc in the range of atomic oscillations, E < 0.4 eV, by the relation (1) gives the reliable magnitude of this index, k osc ≈ 6.

(Figure 1 here)

The calculated refractive indices of PS crystals (n x = 1.5600, n y = 1.5572, n z = 1.5538) are a bit smaller than the corresponding values for the rubidium ammonium sulfate (RAS) crystals (n x = 1.5848, n y = 1.5690, n z = 1.5617), which were calculated by the CASTEP code recently [START_REF] Andriyevsky | [END_REF]. Thus, substitution NH 4 + → K + causes a decrease of refractive index by 0.008 -0.025. This result is in good agreement with experimental data [7].

There are no crossing of the refractive index dispersions n i (E) in the range of 0.01 -7.0 eV, this fact is in agreement with the corresponding experimental data for the temperature range T < 617 K [7]. In this connection, one has to remember, that PS crystal is characterized by two temperature points, 617 K and 700 K, in which the crystal becomes optically uniaxial for all photon energies in the range of crystal transparency. So, the phenomenon of the socalled "isospectrality" takes place at these temperatures: n x = n y at 617 K, and n y = n z at 700 K [7].

Analysis of main features of the band structure of the PS crystals reveals close similarity to the band structure of the rubidium ammonium sulfate (RAS) crystal obtained recently [START_REF] Andriyevsky | [END_REF]. The main difference of the band structures of PS and RAS crystals, which can affect difference in the spectra of optical properties n(E) and k(E), is a presence (absence) of the electronic states of hydrogen in the bottom of conduction band [START_REF] Andriyevsky | [END_REF]. Really, this difference is revealing in the low photon energy region of the spectra of absorption index k(E)

of PS an RAS crystals (Fig. 2). Namely, essential band of the absorption index spectrum k(E) in RAS crystals observed in the region 7 -9 eV is absent in the corresponding spectrum of PS crystal. There are no other essential differences in the absorption spectra k(E) of PS and RAS crystals. This is also an evidence for the close similarity of band structures of these crystals.

The magnitude of the experimental long-wavelength edge of the fundamental absorption of PS crystal, E edge = 7.2 eV [18], is close to the theoretical one obtained in the paper with using a scissor factor of 1.0 eV. There is no possibility to compare the theoretical and experimental (Figure 2 here)

Refractive indices and birefringences of the mechanically free and uniaxially stressed PS crystals are presented in Table 1. For every direction of mechanical stress, the refractive index of stressed PS crystal is greater than the corresponding refractive index of free sample.

This result agrees with the experimental data [8]. The main reason of this result is probably the pressure induced increase of the crystal density ρ and, at the same time, the relative stability of the refraction R of the crystal. This is followed from the known Lorentz-Lorenz

relation [19], 2 2 ( 1)
( 2) n R n M ρ -= + , ( 2
)
where M is a molar weight.

(Table 1 here)

When analyzing the dispersion of birefringence Δn(E) of PS crystals for different stresses σ (Fig. 3), one can notice that the absolute value of birefringence |Δn| increases with increase of photon energy E in the range of transparency and therefore no "isotropic points" caused by the anisotropy of electronic excitations are expected here. Second significant feature of the results presented in figure 3 is that a birefringence Δn, corresponding to the chosen indicatrix cut, decreases when the uniaxial stress is directed along the greater refractive index of this indicatrix cut of the mechanically free crystal. At the same time, absolute refractive indices n i (i = x, y, z) increase with increase of the corresponding uniaxial stresses σ i (see Table 1).

On the basis of the results in the figure 3 one can also suggest a receiving of the "isotropic points", Δn xy = 0 and Δn yz = 0, for the corresponding uniaxial stresses σ x and σ y close to the magnitude of 2 GPa.

(Figure 3

i i i i n a n n δ δ δ ⎛ ⎞ = = ⎜ ⎟ ⎝ ⎠ , ( 4
)
one can calculate several coefficients π im for PS crystals using the data in Table 1. The components of the tensor of stress elastic-and-optical coefficients π im , corresponding to only uniaxial stresses (k = 1, 2, 3) are presented in Table 2. Positive sign of π im means an increase of refractive index under compression of a crystal. The characteristic feature of the elasticand-optical tensor π im of K 2 SO 4 crystal obtained is that the diagonal components π 11 , π 22 and π 33 are the smallest comparing with non diagonal components π im (i,m = 1, 2, 3 and i ≠ m).

One can estimate the validity of results on the coefficients π im taking into account the experimental data on the Young's modulus E of the mineral arcanite (K 2 SO 4 ), E = 4.42⋅10 10 Pa [START_REF] Choy | Landolt-Bornstein: numerical data and functional relationships in science and technology new series[END_REF][START_REF] Bhalla | Landolt-Bornstein: numerical data and functional relationships in science and technology new series[END_REF], and the calculated components of elastic stiffness c rm of PS crystal:

k km m n δ π σ Δ = , (5)
and results presented in Table 1, the components of * km π tensor corresponding to only uniaxial stresses (m = 1, 2, 3) have been calculated and presented in Table 3. Here, the tensor * km π can be expressed by the following relation:

* 3 3 0 0 km im i jm j n n π π π = - , (6)
where k is the direction of light propagation, m is the direction uniaxial stress, i and j are (Tablee 3 here)

The figure 4 illustrates that birefringence of the mechanically free PS crystal is a complicated function of the photon energy E in the range of fundamental absorption (E > 7 eV). The change of sign of the birefringence Δn in the range of fundamental absorption is rather a characteristic feature of every crystal. It is also seen in figure 4 that the influence of uniaxial mechanical stress also leads to the complicated changes of the dependence Δn zx (E) in the range of E > 7 eV. However, the uniaxial stress of 1 GPa does not change essentially characteristic structure of the dependence Δn zx (E), therefore this magnitude of pressure can be applying to similar crystals for determination of their elastic and associated properties.

(Figure 4 here)

Conclusions

The density functional theory based CASTEP code was applied successfully to the calculation of electronic optical spectra of the potassium sulfate crystals K 2 SO 4 in the orthorhombic phase Pnma. On the basis of these calculations the components of elastic-and-

A c c e p t e d m a n u s c r i p t 1 .

 1 Introduction Potassium sulfate (PS) K 2 SO 4 belongs at room temperature T = 25 °C to the orthorhombic space group D 2h 16 = Pnma and has four formula units per unit cell, with lattice parameters a = 7.476 Å, b = 5.763 Å, and c = 10.071 Å [1]. The substance transforms upon heating at T t ≈ 587 °C into a hexagonal structure D 6h 4 = P6 3 /mmc with a = 5.92 Å and c = 8.182 Å (measured at 640 °C) where the oxygen positions of the SO 4 2-tetrahedra are only

A c c e p t e d m a n u s c r i p t 3 environments.

 3 The cutoff energy of 340 eV was assumed in the plane-wave basis set.

A c c e p t e d m a n u s c r i p t 5 dielectric

 5 functions n(E) and k(E) of PS crystal in the range of E > 7 eV because of absence of the reliable corresponding experimental data.

  here)If taking into account the following definition of the tensor of stress elastic-and-optical coefficients π im ,

c 11 =

 11 10.57⋅10 10 Pa, c 22 = 9.69⋅10 10 Pa, c 33 = 12.5⋅10 10 Pa. This comparison reveals that the calculated components of elastic stiffness c rm are overestimated in our calculations approximately two times. This probably leads to the corresponding underestimation of the strain ε m and the change of polarizability Δa i of the crystal under an action of the definite stress σ m . Then, taking into consideration the relation (3) one arrives to the conclusion of underestimation of the calculated components of the tensor of stress elastic-and-optical coefficients π im .(Table2 here)Using the know relation for the components of the tensor of stress elastic-and-optical coefficients regarding to birefringence *

A c c e p t e d m a n u s c r i p t 7 the

 7 directions of light polarization and i, j, k, m = 1, 2, 3. Positive sign of the elastic-and-optical coefficients * km π in Table3corresponds to the increase of absolute value of corresponding birefringence and negative sign corresponds to the decrease of absolute birefringence. One of characteristic peculiarity of the results obtained is the smallest magnitudes of diagonal components feature however can be as a general characteristic of wide class of crystals. It is also obvious that the conclusion about underestimation of the components of tensor of stress elastic-and-optical coefficients π im are also valid to the components of tensor of stress elastic-and-optical coefficients * km π based on birefringence.

  optical tensor π im (i,m = 1, 2, …, 6) have been obtained for the indices i,m = 1, 2, 3. Calculated principal refractive indices of K 2 SO 4 crystal in the range of transparency, 0.4 -7.3 eV, agree satisfactorily with experimental data when taking into account the input from the atomic oscillations in the range of 0 -0.4 eV.An increase of refractive indices of K 2 SO 4 crystal under the compressive stress of 1 GPa observed agrees with the pressure induced increase of the crystal density ρ when a relative stability of the refraction R of the crystal is supposed. This conclusion is in agreement with the known Lorentz-Lorenz relation.On the basis of present study one can suggest a receiving of the "isotropic points" in the range of transparency of K 2 SO 4 crystal, Δn xy = 0 and Δn yz = 0, for the corresponding uniaxial compressive stresses σ x and σ y close to the magnitude of 2 GPa.

A c c e p t e d m a n u s c r i p t 8 TheA c c e p t e d m a n u s c r i p t 11 FigureFigure 1 .

 8111 Figure captions

Figure 2 .

 2 Figure 2. Spectra of absorption index k(E) of potassium sulfate (PS) and rubidium ammonium sulfate (RAS) crystals for principal directions x, y, and z of optical indicatrix.

Figure 3 .

 3 Figure 3. Dispersion of birefringence Δn(E) of PS crystal in the range of transparency for three principal cuts of optical indicatrix Δn xy , Δn yz , Δn zx and different uniaxial stresses σ x or σ y , σ y or σ z , and σ z or σ x , in the plane of corresponding cut.

Figure 4 .

 4 Figure 4. Dispersions of the birefringence Δn zx (E) of PS crystal in the range of transparency and fundamental absorption for the mechanically free sample (σ = 0) and for the axially stressed samples along principal axes of optical indicatrix (σ x = 1 GPa, σ y = 1 GPa, σ z = 1 GPa).

A c c e p t e d m a n u s c r i p t 12 Table 1 .

 121 Refractive indices n x , n y , and n z of potassium sulfate: experimental (at photon energy E = 2.48 eV)[7] and calculated (at photon energy E = 0.01 eV) for mechanically free (σ = 0) and uniaxially stressed crystal (σ x = σ y = σ z = 1 GPa).

A c c e p t e d m a n u s c r i p t 13 Table 2 .A c c e p t e d m a n u s c r i p t 14 Table 3 . 2 A c c e p t e d m a n u s c r i p t 15 Fig 1 A c c e p t e d m a n u s c r i p t 16 Fig 2 A c c e p t e d m a n u s c r i p t 17 Fig 3 A c c e p t e d m a n u s c r i p t 18 Fig 4

 1321432151162173184 Fig 1

Acknowledgment

The calculations presented were performed in the supercomputer centers of ICM (Warsaw University) and WCSS (Technical University of Wroclaw).

A c c e p t e d m a n u s c r i p t


Wiebke Later

PhD Anja Bosy-Westphal

PhD Elke Kossel

PhD Claus-C Glüer

MD Martin Heller
email: mmueller@nutrfoodsc.uni-kiel.de

MD Manfred J Müller

Prof. Dr. med James Manfred

Müller

Is the 1975 Reference Man still a suitable reference?

Keywords: Reference Man, body composition, organ mass, magnetic resonance imaging

Background: In 1975 a Reference Man for the estimation of radiation doses without adverse health effects was created. However, during the past decades considerable changes in body weight and body composition were observed and new in vivo technologies of body composition analysis are available. Thus, the Reference Man might be outdated as adequate standard to assess medication and radiation doses.

Objective: To compare body composition of an adult population with 1975

Reference Man data questioning its value as a suitable reference.

Methods

: Body composition was assessed in 208 healthy, Caucasian subjects (105 males, 103 females) aged 18-78 years with a BMI range of 16.8-35.0 kg/m 2 . Fat mass (FM) and muscle mass (MM) were assessed by Dual X-ray Absorptiometry (DXA), organ masses (OM) were measured by Magnetic Resonance Imaging (MRI).

Results: There was a considerable variance in body weight and body composition.

When compared with Reference Man, great differences in body composition were found. Men and women of the study population were heavier, taller and had more FM, MM and higher masses of brain, heart and spleen. These differences did not depend on age. Relationships between body weight and body composition were investigated by general linear regression models whereby deviations in FM, MM and heart mass disappeared, while differences in brain and spleen mass persisted.

Conclusions:

Our data indicate the need of a modern Reference Man and thus a recalculation of medical radiation doses and medication.

Introduction

Based on the increasing exposure of humans to radiation due to occupational, public and medical reasons and procedures the "Task Group on Reference Man" created a Reference Man and a Reference Woman in 1975. This was based on analyses of anatomic databases. Considering this Reference Man lowest radiation doses were estimated for the planning and the application of medical radiation that do not cause harm in humans [START_REF] Snyder | Report of the Task Group on Reference Man[END_REF]. The Reference Man established quantified constraints, or limits, on individual doses from medical sources. The limitation of this approach is obvious with regard to the combination of data sets from a multitude of several studies from different countries and geographic zones all over the world, which included results of hundreds of patients at different times. A representativeness of a certain population or population group is therefore not given for the Reference Man. However, the statistically precise definition as average men was not the aim of the "Task Group on Reference Man" [START_REF] Snyder | Report of the Task Group on Reference Man[END_REF].

It is well known that body composition of men and women had changed since 1975 and that obesity has reached epidemic proportions [START_REF] Lahti-Koski | Twenty-year changes in the prevalence of obesity among Finnish adults[END_REF][START_REF] Ogden | Prevalence of overweight and obesity in the United States, 1999-2004[END_REF][START_REF] Roche | Secular trends in human growth, maturation, and development[END_REF][START_REF] Wardle | Changes in the distributions of body mass index and waist circumference in English adults, 1993/1994 to 2002/2003[END_REF], World Health Organisation 2000). This is especially true in rich countries due to changes in life style, eating behaviour, living conditions and working demands. Thus, we hypothesize that the estimated radiation or medication doses based on the Reference Man and the Reference Woman, respectively, might no longer be reasonable and appropriate for a current population.

The aim of our study was to compare body composition data of the Reference Man from 1975 with recent data measured by state of the art in vivo methods in a representative healthy Caucasian population with a normal distribution of age and BMI. Based on this comparison a re-evaluation of the Reference Man is intended.

Subjects and Methods

The study population consisted of 208 healthy, Caucasian volunteers (103 females and 105 males) aged 18 to 78 years with a BMI range of 16.8 to 35.0 kg/m 2 .

Participants were recruited from students and staff at the University of Kiel and by notice board postings in local supermarkets and pharmacies. All subjects were nonsmokers and took no medication known to influence body composition. Subjects with splenomegaly (enlargement of the spleen > 350g) were excluded from analyses. The study protocol was approved by the local ethical committee of the Christian-Albrechts-Universität zu Kiel. Each subject provided informed written consent before participation.

Study protocol

All participants arrived at the metabolic unit of the Institute of Human Nutrition and Food Science in the morning at 0730h after an over night fast of >8h.

Body composition analysis

Anthropometrics

Body height was measured to the nearest 0.5 cm with subjects in underwear and without shoes (stadiometer Seca, Vogel & Halke, Germany). Weight was assessed by an electronic scale (TANITA, Japan).

Dual-energy X-ray absorptiometry (DXA)

Whole body measurement by DXA was performed using a Hologic absorptiometer (QDR 4500A, Hologic Inc., MA, USA). Scans were carried out by a licensed radiological technican. Manufactures´ software (version V8.26a:3) was used for the analyses of whole body and regional bone mineral content (BMC), lean soft tissue (LST) and percentage fat mass (FM). Skeletal muscle mass (MM) was calculated from the sum of appending LST (e.g. LST arms + LST legs ), using the formula of Kim et al. [START_REF] Kim | Total-body skeletal muscle mass: estimation by a new dual-energy X-ray absorptiometry method[END_REF].

Magnetic resonance imaging (MRI)

The volumes of 5 internal organs (brain, heart, liver, kidneys and spleen) were measured by transversal MRI images. Scans were obtained by a 1.5T scanner (Magnetom Vision Siemens, Erlangen, Germany). Brain and abdominal organs were examined by a T1-weighted sequence (FLASH) (TR: 177.8ms (abdominal organs); TR: 170.0ms (brain); TE: 4.1ms/echo). ECG-triggered, T2-weighted turbo spin-echo ultrashot scans (HASTE) (TR: 800.0ms; TE: 43ms/echo) were used to examine the heart. The slice thickness ranged from 6mm for brain (1.2mm interslice gap) to 7mm for the heart (2.1mm interslice gap) and 8mm the internal organs (2.4mm interslice gap). Cross-selectional organ areas were determined manually using a segmentation software (SliceOmatic,version 4.3,TomoVision Inc. Montreal,Canada). Volume data were transformed into organ masses using the following densities: 1.036g/cm 3 for brain, 1.06 g/cm 3 for heart and liver, 1.05 g/cm 3 for kidneys and 1.054 g/cm 3 for spleen [START_REF] Duck | Physical properties of tissue Academic Press[END_REF]).

Data analysis

Descriptive subject data are given as means ± SDs and range. Statistical analyses were performed using SPSS© for Windows 13.0 (SPSS Inc., Chicago, IL, USA).

Deviations between means are given as percent (Δ mean [%]) and a cut-off of <10% differences was accepted as data agreement. Influences of varying body height were analysed by comparison of within group height-tertiles in men (Group 1: <1.74m; Group 2: 1.74 -1.83m; Group 3: >1.83m) and women (Group 1: <1.64m; Group 2: 1.64 -1.72m; Group 3: >1.72m). Multiple stepwise regression analyses was used to estimate the explained variances in body composition parameters given as R 2 .

Values of standardized beta coefficients and SEE are presented for each of the developed regression models. Relationships between body composition and body mass or age are shown as general linear models and linear regression equation were used to calculate body composition parameters. Differences between sexes were analysed using the independent t-test. All tests were 2-tailed and a P-value <0.05 was accepted as the limit of significance.

Results

Comparison of body composition between Reference Man and study population

In Table 1 body composition of the study population is compared with mean data of Reference Man and Reference Woman. When compared with the reference subjects of the study population were heavier, taller and had more FM, MM and organ masses (brain, heart (in women only), spleen). No differences were found for liver and kidney masses (Δ<10%). When compared with women men had significantly higher BMI, MM and OM and less FM (P<0.01) but sex had no effect on the difference between measured values and the reference. Considering the influence of age on body composition a subgroup of young subjects (20 -30 years) was compared to Reference Man and Reference Woman (Table 1). This approach revealed similar results, i.e. higher weight, FM, MM and OM (except liver and kidney mass). Also significant sex differences in FM, MM and OM were found (P<0.01).

Impact of age on variance in body composition

In Figure 1 A-G age is plotted against organ and tissue masses for men (closed circles) and women (open circles). The mean age of men and women is given as continuous vertical line, while the age range of Reference Man and Woman is presented by shaded areas. Mean organ and tissue masses are given as continuous (study population) or dashed horizontal (Reference Man and Woman) line. Results of organ and tissue masses showed only small differences between the "younger" reference subjects and the "older" study population. Thus, there was no significant influence of age on the variance of the data.

Influence of body weight, height and age on variances on body composition

Using a stepwise multiple regression analyses explained variance in body composition parameters is shown in Table 2. Weight, height and age were used as independent variables within different models. Except for brain mass variance in organ and tissue masses was mainly explained by body weight alone in men (FM: 77%; MM: 54%; heart: 9%; liver: 43%; kidneys: 16%) and in women (FM: 81%; MM: 35%; heart: 14%; liver: 36%; kidneys: 24%; spleen: 18%). In addition, body height explained further variance in FM (men: 2%; women: 7%), MM (women: 6%), liver (women: 4%) and kidneys (men: 4%). Furthermore, age contributed significantly to explained variance in MM (5%) and spleen mass (8%) in men, and in variance in brain (4%) and spleen mass (5%) in women. No significant correlation were found between body weight and age, both in men and women (data not shown). Based on the significant contribution of body height to the variance in body composition (Table 2) the study population was categorized into body height-tertiles to analyse differences between actual and reference data (Table 3). When compared to tall subjects deviations between measured data and Reference Man and Reference Woman, respectively, in weight, height, MM (and FM in men) were lower for smaller people (Group 1). In addition, brain, heart and spleen mass (and kidney mass in men) of smaller subjects showed the highest agreement with the Reference Man and Reference Woman. In general, most body composition data were consistent to reference data (Δ<10%) in small men and women when compared with taller subjects (Group 2 and 3) showing a higher difference in weight, height, FM, MM, brain, heart and spleen mass (and liver and kidney mass in men) (Table 3). Within different height-tertiles these findings were also true in young subjects (20 -30 years) (data not shown).

Relationship between body mass and organ / tissue masses

The relationship between body weight and organ / tissue masses is given in Figure 2 A-G. The mean body weight of Reference Man (70 kg) and Reference Woman (58 kg) is shown as dashed vertical line within the figures. A horizontal line is presenting the calculated tissue mass for the reference subjects using the regression equations given in Table 4. For both sexes highest R 2 were found between fat mass and body mass (men R 2 = 0.88; women R 2 = 0.89; p<0.01) while a weak or no relationship was seen between body mass and brain mass (men R 2 = 0.21, P<0.05; women R 2 = ns).

Calculation of organ and muscle mass based on body mass

Linear regression equations calculated from the relationship between body mass and body composition (Figure 2 A-G) are presented in Table 4. Based on these regression equations the masses of brain, heart, liver, kidneys and spleen, fat and muscle were calculated for a man (with a body weight of 70 kg ≈ body weight of the Reference Man) and a woman (with a body weight of 58 kg ≈ body weight of the Reference Woman), respectively. The estimated tissue masses of the study population (measured by MRI and DXA) and the reference subjects (autopsy data) with identical body weights were compared. There were considerable differences in brain and spleen mass (and MM in women) with an overestimation of these masses in reference subjects. By contrast, FM and liver mass in men and kidney mass in women were underestimated in Reference Man and Reference Woman, respectively.

Discussion

The primary purpose of this study was to compare reference data from 1975 with recent database based on in vivo measurements of body composition in a greater group of healthy subjects. Considerable differences in body composition were found, with todays men and women being heavier, taller and having more FM and MM when compared with Reference Man and Reference Woman, respectively. Furthermore, organ masses of brain, heart and spleen of the study population differed. These finding were independent of age and gender. Accounting for differences in body weight deviations in FM, MM (for men only) and heart mass disappeared whereas differences in brain and spleen mass remained. Comparing different height groups revealed highest agreement in body composition for small people while taller subjects showed higher percentage deviations. The latter finding is in line with data of Heymsfield et al. [START_REF] Heymsfield | Scaling of human body composition to stature: new insights into body mass index[END_REF].

Differences in body composition between actual data and the 1975 reference subjects may be partly caused by methodical issues. While in the present study masses of internal organs have been measured in vivo using MRI, data of the Reference Man were based on autopsy post-mortem analyses and organ weighing, i.e. the organs were removed from the body followed by exclusion of remaining tissue before weighing. It is well known that during the first 15 minutes after extraction from the body the organ looses significant weight. On the other hand considerable differences in in vivo organ weight estimates might be due to segmentation techniques. E.g., analysing brain mass cerebrospinal fluid has been excluded by manual slice segmentation. In accordance, gallbladder, portal vein and other big blood vessels were excluded from the liver mass which were included within postmortem organ weighing. Thus, organ masses of Reference Man und Reference Woman might not resemble metabolic active organ mass, but remaining fluid within the organ and thus add to systematic differences between results of the two measurement procedures.

Comparing MM as assessed in autopsy studies with MM measured by DXA also implicates method-based inaccuracies. DXA has a great precision of soft tissue composition measurement although it includes some assumptions which should be taken into account, e.g. constant attenuation of fat mass [START_REF] Lohman | Dual-Energy X-Ray Absorpiometry[END_REF].

Another assumption is that DXA-measurements are not affected by the anteroposterior thickness of the human body. However, previous studies found slightly overestimated fat and lean masses due to thickness less than 20cm [START_REF] Laskey | The influence of tissue depth and composition on the performance of the Lunar dual-energy X-ray absorptiometer whole-body scanning mode[END_REF]. In addition, the accuracy of DXA may differ with tissue. E.g., in the thorax DXA has limits to distinguish between bone and soft tissue, thus, estimations of thoracic composition tend to be imprecise [START_REF] Roubenoff | Use of dual-energy xray absorptiometry in body-composition studies: not yet a "gold standard[END_REF]. However, advances of the DXA technique prevail. In research and clinical settings DXA is a non-invasive, accurate and reproducible tool for assessing body composition with minimal radiation doses superior to many other method [START_REF] Brownbill | Measuring body composition in overweight individuals by dual energy x-ray absorptiometry[END_REF][START_REF] Gately | Comparison of body composition methods in overweight and obese children[END_REF][START_REF] Slosman | Assessment of whole-body composition with dual-energy x-ray absorptiometry[END_REF]. There are high correlations between DXA and Computer Tomography (CT) estimates of lean mass and MM [START_REF] Visser | Validity of fan-beam dual-energy X-ray absorptiometry for measuring fat-free mass and leg muscle mass. Health, Aging, and Body Composition Study--Dual-Energy X-ray Absorptiometry and Body Composition Working Group[END_REF].

However, there may be a small influence of different measurement techniques on deviations found in in vivo body composition and the Reference Man.

We hereby present data of a large homogeneous Caucasian study population with a wide range in age and BMI (18-78 years, 16.8-35.0 kg/m 2 ). Due to the limited recruitment area of our sample we do not consider our data as representative. To get an idea we compared our data to the dataset of the second national nutrition survey (NVSII) conducted by the Federal Research Centre for Nutrition and Food in Germany (Max Rubner Institute 2008). High agreements were found in BMI (men: 26.9kg/m 2 vs. 26.4kg/m 2 ; women: 26.1kg/m 2 vs. 24.4kg/m 2) and body weight (men: 84.6kg vs. vs. 84.3kg; women: 69.9kg vs. 68.7kg) (Max Rubner Institute 2008). Thus, with respect to BMI our study population was similar to the representative NVSII population.

Our body composition data were also compared with previous detailed in vivo body composition studies on smaller populations. In these studies, FM, MM and OM were measured using the same in vivo methods, e.g. DXA, MRI or CT (Bosy-Westphal et al 2004, [START_REF] Gallagher | Organ-tissue mass measurement allows modeling of REE and metabolically active tissue mass[END_REF][START_REF] Sparti | Relationship between resting metabolic rate and the composition of the fat-free mass[END_REF]. When compared with these previous BCA data, men and women of our study population were older, had slightly higher body weights and FM compared to other populations [START_REF] Gallagher | Organ-tissue mass measurement allows modeling of REE and metabolically active tissue mass[END_REF][START_REF] Sparti | Relationship between resting metabolic rate and the composition of the fat-free mass[END_REF]. Excluding subjects >50 years from our present analysis, weight, BMI, FM and MM were in good agreement with previous data [START_REF] Bosy-Westphal | Effect of organ and tissue masses on resting energy expenditure in underweight, normal weight and obese adults[END_REF][START_REF] Gallagher | Organ-tissue mass measurement allows modeling of REE and metabolically active tissue mass[END_REF][START_REF] Sparti | Relationship between resting metabolic rate and the composition of the fat-free mass[END_REF]. In addition, differences in liver and kidneys masses [START_REF] Gallagher | Organ-tissue mass measurement allows modeling of REE and metabolically active tissue mass[END_REF] might be explained by methodical differences in different segmentation procedures. Contrary to previous data in the present study renal pelvis and portal vein were not included within the calculation of organ volume.

Taken as a whole we found good agreements between our estimates of body composition and the results of previous studies.

When compared with data observed in Caucasians, ethnic differences in body composition have been reported [START_REF] Gasperino | Ethnic differences in body composition and their relation to health and disease in women[END_REF][START_REF] Rahman | Racial differences in body fat distribution among reproductive-aged women[END_REF]: Afro-Americans have more bone mass and MM, but less OM and FM than Caucasians [START_REF] Aloia | Body composition by dual-energy X-ray absorptiometry in black compared with white women[END_REF][START_REF] Gallagher | Small organs with a high metabolic rate explain lower resting energy expenditure in African American than in white adults[END_REF][START_REF] Weinsier | Body fat distribution in white and black women: different patterns of intraabdominal and subcutaneous abdominal adipose tissue utilization with weight loss[END_REF][START_REF] Wu | Truncal fat in relation to total body fat: influences of age, sex, ethnicity and fatness[END_REF]. These differences remained after controlling for differences in age, weight, and height [START_REF] Gasperino | Ethnic differences in body composition and their relation to health and disease in women[END_REF]. When compared with the Reference Man from 1975, Afro-Americans had higher FM (+0.6kg -+10.8kg) [START_REF] Aloia | Body composition by dual-energy X-ray absorptiometry in black compared with white women[END_REF][START_REF] Gallagher | Small organs with a high metabolic rate explain lower resting energy expenditure in African American than in white adults[END_REF][START_REF] Weinsier | Body fat distribution in white and black women: different patterns of intraabdominal and subcutaneous abdominal adipose tissue utilization with weight loss[END_REF]. Concomitantly, MM was considerably higher (+7kg) in black women when compared with the Reference Woman [START_REF] Aloia | Body composition by dual-energy X-ray absorptiometry in black compared with white women[END_REF]. Racedependent differences in body composition argue in favour to develop a Reference Men and a Reference Women for various ethnic groups.

In conclusion, we found considerable differences in current in vivo estimates of body composition and Reference Man and Reference Woman, with present men and women being heavier, taller and having higher FM and MM. Substantial differences were also found for OM of brain, heart and spleen, whereas no difference occurred for liver and kidney mass in both gender. Comparing subjects with identical body weight deviations in FM, MM (only in men) and heart mass disappeared whereas differences in brain and spleen mass persisted. Considering different height groups revealed lowest deviations to reference values for small people (men < 1.74m; women < 1.64m).

Based on considerable differences in body composition between the present results and the 1975 Reference Man a modern Reference Man is needed as a basis to estimate accurate medical radiation doses and to calculate medication application (e.g. doses of drugs).

Figure legends

Figure 1 A

 1 Figure 1 A-E Age-dependent changes in masses of brain (A), heart (B), liver (C), kidneys (D), spleen (E), skeletal muscle (F) and fat mass (G) for women (open circles) and men (closed circles). Mean tissue and organ masses are shown as horizontal line for reference subjects (dashed) and for men and women of the study population (continuous).

Figure 2 A

 2 Figure 2 A-E Weight-dependent changes in masses of brain (A), heart (B), liver (C), kidneys (D), spleen (E), skeletal muscle (F) and fat mass (G) for women (open circles) and men (closed circles). Weight of reference woman (58kg) and reference man (70kg) is shown as dashed vertical line and the calculated organ mass as dashed horizontal line.

Figure 1 A

 1 Figure 1 A-E Age-dependent changes in masses of brain (A), heart (B), liver (C), kidneys (D), spleen (E), skeletal muscle (F) and fat mass (G) for women (open circles) and men (closed circles). Mean tissue and organ masses are shown as horizontal line for reference subjects (dashed) and for men and women of the study population (continuous).

Table 1

 1 Comparison of body composition between the study population (n = 208) and younger subjects (n= 63) [mean ± SD, range] and the reference man and sexes (t-test), * study population, † younger subjects (20 -30 years), P < 0.01, 1 Snyder et al., Report of the Task Group on Reference Man, 1975; 2 body mass index; 3 fat mass; 4 muscle mass

	reference woman from 1975 [mean]
			men	(n = 105)	young men	(n = 26)	reference man 1	vs. men	vs. young
										men
			mean ± SD	range	mean ± SD	range	mean	Δ mean [%]	Δ mean [%]
	age [years]	45.4 ± 15.3	18 -72	26.7 ± 2.4	20 -30	20 -30
	weight [kg]	84.3 ± 13.0 *	58.2 -116.8	78.1 ± 13.2 †	58.2 -107.8	70	+ 20.4	+ 11.6
	height [m]	1.79 ± 0.06 *	1.61 -1.95	1.78 ± 0.06 †	1.68 -1.89	1.70	+ 5.3	+ 4.8
	BMI [kg/m 2 ] 2	26.4 ± 3.7 *	18.3 -34.9	24.5 ± 3.3	19.0 -34.3	24.2	+ 9.1	+ 1.4
	FM [kg] 3	18.8 ± 8.2 *	4.3 -43.7	14.5 ± 7.7	†	4.3 -29.5	13.5	+ 39.3	+ 8.0
	MM [kg] 4	31.9 ± 3.7 *	22.1 -39.6	31.6 ± 3.7 †	26.1 -39.6	28	+ 13.9	+ 12.7
	brain [g]	1606 ± 100 *	1343 -1872	1613 ± 94 †	1469 -1872	1400	+ 14.7	+ 15.2
	heart [g]	357 ± 76 *	211 -631	366 ± 82 †	283 -567	330	+ 8.4	+ 11.2
	liver [g]		1708 ± 291 *	1048 -2466	1602 ± 261 †	1161 -2290	1800	-6.2	-11.0
	kidneys [g]	326 ± 58 *	202 -488	312 ± 60 †	202 -443	310	+ 5.2	+ 0.9
	spleen [g]	24 ± 67 *	86 -347	263 ± 55 †	174 -347	180	+ 34.4	+ 46.3
			women	(n = 103)	young women	(n = 37)	reference woman 1	vs. women	vs. young
										women
			mean ± SD	range	mean ± SD	range	mean	Δ mean [%]	Δ mean [%]
	age [years]	41.2 ± 15.4	22 -78	25.5 ± 1.8	20 -30	20 -30
	weight [kg]	68.7 ± 11.3	44.0 -97.1	67.4 ± 11.3	44.7 -97.1	58	+ 18.4	+ 16.2
	height [m]	1.68 ± 0.06	1.48 -1.83	1.69 ± 0.06	1.54 -1.83	1.60	+ 5.0	+ 6.2
	BMI [kg/m 2 ] 2	24.4 ± 3.9	16.8 -34.3	23.3 ± 3.7	16.8 -33.3	22.7	+ 7.5	+ 2.8
	FM [kg] 3	22.9 ± 8.8	4.2 -50.8	20.8 ± 8.6	8.5 -50.9	16	+ 43.1	+ 30.2
	MM [kg]	4	21.2 ± 2.9	15.2 -29.7	21.7 ± 2.9	16.2 -28.2	17	+ 24.1	+ 27.5
	brain [g]	1428 ± 95	1239 -1689	1456 ± 98	1248 -1689	1200	+ 19.0	+ 21.4
	heart [g]	267 ± 60	172 -437	261 ± 60		172 -401	240	+ 11.5	+ 8.7
	liver [g]		1422 ± 236	944 -2165	1433 ± 219	944 -1918	1400	+ 1.6	+ 2.3
	kidneys [g]	255 ± 47	159 -366	254 ± 49		161 -350	275	-8.2	-8.5
	spleen [g]	193 ± 58	82 -334	194 ± 56		103 -334	150	+ 28.8	+ 29.8

Table 2

 2 Explained variance in body composition of men and women (n = 208) using

	a multiple stepwise regression analyses
		men (n = 105)	R 2	ß-Coeff.	SEE
	FM [kg] 1	1	weight	0.77	0.936	3.951
		2	height	0.79	-0.124	3.863
	MM [kg] 2	1	weight	0.54	0.757	2.514
		2	age	0.59	-0.196	2.420
	brain [g]	1	height	0.10	0.310	0.096
	heart [g]	1	weight	0.09	0.302	0.072
	liver [g]	1	weight	0.43	0.654	0.222
	kidneys [g]	1	weight	0.16	0.494	0.053
		2	height	0.20	-0.213	0.052
	spleen [g]	1	age	0.08	-0.319	0.065
		2	weight	0.15	0.256	0.063
		women (n = 103)	R 2	ß-Coeff.	SEE
	FM [kg] 1	1	weight	0.81	0.979	3.872
		2	height	0.88	-0.272	3.133
	MM [kg] 2	1	weight	0.35	0.474	2.338
		2	height	0.49	0.389	2.087
	brain [g]	1	age	0.14	-0.376	0.088
	heart [g]	1	weight	0.14	0.372	0.056
	liver [g]	1	weight	0.36	0.528	0.191
		2	height	0.40	0.230	0.184
	kidneys [g]	1	weight	0.24	0.493	0.041
	spleen [g]	1	weight	0.18	0.434	0.053
		2	age	0.23	-0.227	0.052

Table 3

 3 Comparison of body composition between the study population (n = 208) [mean ± SD (range)] and the reference man and reference woman from 1975[mean]

	considering different body heights
			men (n = 105)		reference man 1	men -reference man 1
		Group 1	Group 2	Group 3		Group 1	Group 2	Group 3
		< 1.74m	1.74 -1.83m	> 1.83m		Δ mean [%]	Δ mean [%]	Δ mean [%]
	age [years]	51.6 ± 17.8 (22 -72)	40.5 ± 14.4 (21 -70)	40.7 ± 12.5 (18 -65)	20 -30

Table 4

 4 Linear regression equations between body mass and body composition for men and women(n = 208). Deviations between calculated mean values (based on the linear regression equation) and data for Reference Man and Reference Woman, respectively. Body composition was compared for subjects with identical body weight.

	men (n = 105)
	linear regression equation	man, 70 kg		reference man 1 , 70 kg	Δ mean [%]
			(mean calculated	2 )	(mean)
	FM [kg] 3	0.556 x -28.11	10.8		13.5	-20.0
	MM [kg] 4	0.208 x + 14.38	28.9		28	+ 3.2
	brain [g]	0.0018 x + 1.45	1576		1400	+ 12.6
	heart [g]	0.0018 x + 0.20	326		330	-1.2
	liver [g]	0.0152 x + 0.44	1504		1800	-16.4
	kidneys [g] 0.0019 x + 0.17	303		310	-2.3
	spleen [g]	0.0013 x + 0.138	229		180	+ 27.2
	women (n = 103)
	linear regression equation	woman, 58 kg		reference woman 1 , 58 kg	Δ mean [%]
			(mean calculated 2 )	(mean)
	FM [kg] 3	0.699 x -25.07	15.5		16	-3.1
	MM [kg] 4	0.1502 x + 10.86	19.6		17	+ 15.3
	brain [g]	0.001 x + 1.36	1418		1200	+ 18.2
	heart [g]	0.002 x + 0.13	246		240	+ 2.5
	liver [g]	0.0125 x + 0.56	1285		1400	-8.2
	kidneys [g] 0.002 x + 0.11	226		275	-17.8
	spleen [g]	0.0022 x + 0.0391	166		150	+ 10.7

fat mass;

muscle mass; independent variables: weight [kg], height [m], age[years]

Snyder et al., Report of the Task Group on Reference Man, 1975;

body mass index;

fat mass;

muscle mass

Snyder et al., Report of the Task Group on Reference Man, 1975;

mean calculated by linear regression equation;

fat mass;

muscle mass

Acknowledgements

Authors contributions: Data collection (WL, ABW), data analysis (WL, ABW), writing of the manuscript (WL, ABW, MJM), study design (ABW, MJM), DXA and MRI protocol (EK, CCG, MH). There are no conflicts of interest.

weight [kg] 77. 5 ± 11.7 (63.7 -98.2) 84.5 ± 12.3 (58.2-116.8) 91.5 ± 12.5 (68.9 -111.6


Dr Birgit-Christiane Zyriax
email: bzyriax@uke.de

Katharina Lau

Tilman Klähn

Heiner Boeing

Henry Völzke

Eberhard Windler

à la diffusion de documents scientifiques de niveau

Keywords: alcohol, intima-media-thickness (IMT), risk factors, lifestyle, nutrition

recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Association between Alcohol Consumption and Carotid Intima-Media Thickness in a Healthy Population -Data of the STRATEGY Study (Stress, Atherosclerosis, and ECG Study) -

INTRODUCTION

Epidemiologic evidence indicates a J-shaped or U-shaped association between alcohol consumption and cardiovascular morbidity and mortality [START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF][START_REF] Fagrell | The effects of light to moderate drinking on cardiovascular diseases[END_REF][START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF]1997;[START_REF] Corrao | Alcohol and coronary heart disease: a meta-analysis[END_REF]. Light to moderate drinking seems to reduce the risk of cardiovascular events compared to abstention or heavier consumption of alcohol [START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF][START_REF] O'keefe | Alcohol and cardiovascular health: the razorsharp double-edged sword[END_REF][START_REF] Roerecke | Irregular heavy drinking occasions and risk of ischemic heart disease: a systematic review and meta-analysis[END_REF]. Also among individuals with previous cardiovascular disease, a protective effect of alcohol has been reported [START_REF] Kaski | Secondary prevention after acute myocardial infarction and coronary revascularisation: focus on Angiotensin converting enzyme inhibitors[END_REF].

As yet, the mechanisms of cardiovascular protection of alcohol are not fully elucidated. It has been suggested that cardiovascular benefits of moderate alcohol consumption are causal and may be explained in part by its influence on serum lipids and insulin sensitivity [START_REF] Rimm | Alcohol and cardiovascular disease[END_REF][START_REF] Djoussé | Alcohol consumption and risk of cardiovascular disease and death in women: potential mediating mechanisms[END_REF]. However, clinical advice to abstainers to initiate daily alcohol consumption for cardiovascular prevention has been given with caution, because of competing risks such as that of cancer (O' Keefe et al., 2007).

Previous studies focused on the association between alcohol intake and clinical endpoints rather than atherosclerosis. Carotid intima-media thickness (IMT) is an established strong predictor of future coronary and cerebrovascular events and presumed to reflect subclinical atherosclerosis, however data in young or healthy individuals are limited and have yielded inconsistent results [START_REF] Bots | Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam Study[END_REF][START_REF] Chambless | Association of coronary heart disease incidence with carotid arterial wall thickness and major risk factors: the Atherosclerosis Risk in Communities (ARIC) Study,1987-1993[END_REF][START_REF] O'leary | Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group[END_REF][START_REF] Lorenz | Prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and metaanalysis[END_REF]. Some investigators found an inverse association in men but not in women, while others failed to show a relationship at all [START_REF] Lee | Alcohol consumption and carotid artery structure in Korean adults aged 50 years and older[END_REF][START_REF] Schminke | Association between alcohol consumption and subclinical carotid atherosclerosis. the Study of Health in Pomerania[END_REF][START_REF] Zureik | Alcohol consumption and carotid artery structure in older French adults: the Three-City Study[END_REF][START_REF] Kauhanen | Pattern of alcohol drinking and progression of atherosclerosis[END_REF]. Previous studies have mainly been performed in middleaged or elderly subjects with established risk factors for cardiovascular disease (CVD).

Recently, a positive relationship has been shown in young adults [START_REF] Juonala | Alcohol consumption is directly associated with carotid intima-media tickness in Finnish young adults. The Cardiovascular Risk in Young Finns Study[END_REF].

Thus, more studies are needed to provide a larger database on alcohol and IMT in different subpopulations, particularly in young or healthy subjects using IMT-measurements as a predictor of individual cardiovascular risk.

The aim of the present study was to investigate the association between alcohol consumption and IMT in healthy men and women aged 30 to 70 years without evident risk factors taking account of possible confounding dietary habits.

METHODS

Design and recruitment

The

Data collection

Assessment of lifestyle and alcohol consumption

All interviews and physical examinations were performed by the same trained investigator.

Data on lifestyle, nutrition, sociodemographic characteristics and family history of cardiovascular disease were obtained using validated questionnaires developed for the EPIC study (European Prospective Investigation into Cancer and Nutrition) (Bohlscheid et al., 1997a;Bohlscheid et al., 1997b). A self-administered food questionnaire recorded the frequency and portion size of 146 food items eaten during the preceding year. Daily alcohol intake was calculated from the individual portion size and frequency of the consumption of various alcoholic beverages (beer, wine, champagne, spirits, liqueur and mixed alcohol beverages). One portion size was defined as 0.5 l beer or 0.25 l wine or 0.1 l of sparkling wine, 8 cl of liquor, 2 cl of spirits or 0.2 l of mixed alcohol beverages. Non-beverage sources of alcohol such as filled chocolates, cake, fruit juice or milk products (yoghurt, kefir) were also taken into account.

Physical activity in hours per week was estimated from the kind, time and frequency of sports in summer and winter by a validated lifestyle questionnaire. With regard to smoking status, participants were categorized as current smokers, former smokers and never smokers.

Education level characterized by years of schooling comprised the following categories: less than 10 years (low level), 10 years (intermediate level) and more than ten years (higher level).

Anthropometric and laboratory data

Height and weight were measured to the nearest 0,5 cm or 0,1 kg, respectively and body mass index (BMI) was calculated as BMI = (weight, kg)/(height, m) 2 . Waist circumference was measured in the middle between the lower rib margin and the iliac crest. Central obesity was defined by a waist circumference ≥ 80 cm in women and ≥ 94 cm in men. A fasting morning blood sample was taken, immediately centrifuged at 4°C, and stored at 4°C in tubes containing 1 mg fluoride per ml blood. Fasting plasma glucose, plasma glucose two hours after 75 g glucose (OGTT), high-density lipoprotein (HDL) cholesterol, triglycerides, lipoprotein(a) and serum enzyme levels of ASAT, ALAT, GGT, GLDH were determined by standard technique. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Blood pressure was taken in a sitting position in the morning three times in 1 min intervals after at least 5 min rest. The results of the second and third measurement were averaged and used for the present analysis.

Carotid intima-media thickness

The common carotid artery was recorded on both sides between the carotid bulb origin and a point 10 mm proximal using high-resolution B-mode ultrasound (General Electric Vivid 3

Expert with a 7,5-MHz linear-array transducer). The intima-media thickness (IMT) was assessed off-line as the average of at least 5 consecutive measurements in 1-mm steps on both sides of the distance from the media-adventitia interface to the intima-lumen interface on the far wall in a region free of plaques (only one plaque in one participant was detected) by a trained investigator according to the standardized protocol of the Study of Health in Pomerania (SHIP) [START_REF] Lüdemann | Methoden zur Qualitätssicherung im medizinischen Untersuchungsbereich epidemiologischer Feldstudien: Die "Study of Health in Pomerania" (SHIP)[END_REF][START_REF] Völzke | Cohort Profile: The Study of Health in Pomerania[END_REF].

Statistical analyses

Baseline clinical and lifestyle characteristics are expressed as means ± 1 standard deviation or as categories (percentage, number). Comparisons between groups were performed twotailed using ANOVA for continuous variables, and χ 2 -test for categorical variables. The association between IMT and daily alcohol consumption was determined by multiple linear regression analyses adjusted for potential confounders and in participants with lower and higher alcohol consumption as determined by univariate tests. P-values below 0.05 were considered statistically significant. All statistical analyses were performed using STATA version 10 (StataCorp, College Station, TX).

RESULTS

Baseline characteristics

Analyses were performed separately in men and women because of the sex-specific differences in alcohol metabolism and consumption. Baseline characteristics are given in Table 1. Mean age did not differ between men and women. On average, both genders were characterized by a relatively favorable profile of cardiovascular risk factors, particularly regarding lipids, fasting and postprandial glucose and blood pressure. Notably, though in the reference range, in men almost all laboratory values were significantly higher, in the case of HDL-cholesterol lower, as compared to women. Mean serum levels of liver enzymes as markers of possible alcohol abuse did not show major abnormalities. Gamma-GT was above the upper limit of normal in no woman and eight men, in one man above two times the upper limit of normal (> 2SD). In only two cases this was accompanied by slightly elevated ALAT (data not shown).

Mean BMI of women was in the range of normal, while that of men was within the range of overweight. Yet, both sexes surpassed the defined cutoffs for an elevated waist circumference of 94 cm for men and 80 cm for women. In both gender, the prevalence of smokers was low and levels of physical activity high. Men reported a higher intake of energy particularly as meat and meat products and a lower consumption of fruit and vegetables compared to women. This was even more evident if the intake was calculated per 1000 kcal of energy intake (Table 1).

Alcohol intake and drinking patterns

The participants reported a light to moderate alcohol intake (Figure 1). The mean alcohol consumption was significantly higher in men compared to women. One woman reported to drink no alcohol at all. In 45% of women and 42% of men, the mean daily alcohol intake was above the sex-specific upper tolerable limits (women: 10 g/day, men 20 g/day) (Table 2) (Deutsche Gesellschaft für Ernährung, 2000; [START_REF] Kushi | American Cancer Society Guidelines on Nutrition and Physical Activity for cancer prevention: Reducing the Risk of Cancer With Healthy Food Choices and Physical Activity[END_REF]. The frequency of alcohol consumption was higher in men compared to women (Figure 2). Two-thirds of men (67%), but barely half of the women (47%) consumed at least two or three drinks per week. Among men, 11% reported to consume one or two drinks per day, in women the rate was 7%.

Alcohol intake predominantly comprised alcoholic beverages. The preference differed somewhat between men and women (men/women: wine 55%/68%; beer 34%/15%; spirits including liquor and mixed alcoholic beverages 9%/12%; sparkling wine 2%/5%). However, most participants did not exclusively consume one type of alcoholic beverage. Other sources of alcohol played a minor role (9% in men, 6% in women) (Table 1).

Regarding the variables shown in Table 1, men with a mean alcohol intake of >20 g/d versus ≤ 20 g/d were characterized by a significantly higher energy intake, higher age, higher level of HDL-cholesterol and a lower postprandial glucose (data not shown). Women with a mean alcohol consumption of >10 g/d differed from those with an intake ≤ 10 g/d in a higher energy intake, a lower consumption of fruit and vegetables per 1000 kcal energy, and a higher waist circumference, but not in the remainder of variables shown in Table 1 (data not shown).

Alcohol intake and IMT

In men, a significant positive correlation between daily alcohol intake and IMT was observed (Figure 3). The slope of the correlation was even stronger below an alcohol intake of 60g/d (data not shown). In women a positive trend was also notable, which, however, was much weaker and not significant (Figure 3). The mean IMT was significantly higher in men with an alcohol intake above the recommended upper limit of 20 g/d compared to ≤20 g/d (p<0.001),

but not in women with an alcohol intake >10 g/d versus ≤10g/d (Table 2). By confounder analysis the association between alcohol intake and IMT was not influenced by the type of beverages consumed (data not shown).

In men, a stepwise linear regression model revealed a significant increase of 0.0253 mm IMT per 21.4 g alcohol per day after adjustment for age, HDL-cholesterol and LDL-cholesterol (Table 3). This association remained significant after further adjustment for systolic blood pressure and smoking status (model 2) and additionally for waist circumference and fasting glucose (model 3). Further adjustment for dietary factors such as intake of total energy, meat and meat products, and fruit and vegetables, and physical activity (model 4), or lipoprotein(a)

as a genetic marker for cardiovascular risk (model 5) did not change the result. Yet, in women no significant association was detected in any of the models (data not shown).

DISCUSSION

The STRATEGY study showed a positive association between alcohol intake and carotid IMT in healthy men aged 30 to 70 years. This relationship was independent of nutrition, physical activity, anthropometry, and conventional cardiovascular risk factors. In women, the trend was positive too, but much weaker and not significant. The more favorable risk profile and dietary pattern in women may have mitigated the possible harmful effects of alcohol consumption on IMT.

In men the positive correlation between alcohol consumption and IMT held for any level of alcohol intake. The correlation was even stronger for an alcohol intake below 60 g/d. Most participants reported a light to moderate alcohol intake regularly on several occasions a week. The amount was beyond the upper limit of what is assumed to be tolerable in nearly half of the men and women. Liver enzymes did not show any major abnormalities indicating that the observed positive association between alcohol intake and IMT was caused by alcohol abuse or a binge drinking behavior as reported in several epidemiological studies [START_REF] Kauhanen | Pattern of alcohol drinking and progression of atherosclerosis[END_REF][START_REF] Bagnardi | Does drinking pattern modify the effect of alcohol on the risk of coronary heart disease? Evidence from a metaanalysis[END_REF][START_REF] Roerecke | Irregular heavy drinking occasions and risk of ischemic heart disease: a systematic review and meta-analysis[END_REF].

Most participants of the STRATEGY study consumed various types of beverages. However recent evidence suggested that the protective effect of alcohol is attributable primarily to the alcohol content rather than to other component. Also wine drinking is associated with a higher socioeconomic status including a healthier lifestyle [START_REF] Klatsky | Diet, alcohol, and health: a story of connections, confounders, and cofactors[END_REF][START_REF] Mortensen | Better psychological functioning and higher social status may largely explain the apparent health benefits of wine: a study of wine and beer drinking in young Danish adults[END_REF].

The findings of the present study appear to be in striking contrast to several studies that reported an inverse association between alcohol intake and cardiovascular disease [START_REF] Arriola | Alcohol intake and the risk of coronary heart disease in the Spanish EPIC cohort study[END_REF][START_REF] Castelnuovo | Alcohol dosing and total mortality in men and women: an updated meta-analysis of 34 prospective studies[END_REF][START_REF] Fagrell | The effects of light to moderate drinking on cardiovascular diseases[END_REF][START_REF] Thun | Alcohol consumption and mortality among middle-aged and elderly U.S. adults[END_REF][START_REF] Corrao | Alcohol and coronary heart disease: a meta-analysis[END_REF]. A moderate alcohol consumption correlated with a decreasing risk of cardiovascular events and stroke, whereas a harmful effect became evident only at excessive levels [START_REF] Corrao | Alcohol and coronary heart disease: a meta-analysis[END_REF]Fragell et al., 1999;[START_REF] Roerecke | Irregular heavy drinking occasions and risk of ischemic heart disease: a systematic review and meta-analysis[END_REF]. However, several studies suggest that the benefit of alcohol drinking is largely confined to individuals at increased risk for cardiovascular disease, to older subjects or those with a poor nutrition status [START_REF] Fuchs | Alcohol consumption and mortality among women[END_REF][START_REF] Merchant | Interrelation of saturated fat, trans fat, alcohol intake, and subclinical atherosclerosis[END_REF][START_REF] Bos | Alcohol consumption and risk of cardiovascular disease among hypertensive women[END_REF][START_REF] Zyriax | Nutrition is a powerful independent risk factor for coronary heart disease in women -The CORA Study: a population-based case-control study[END_REF]. In contrast, studies including younger adults suggested that increasing alcohol intake is associated with coronary calcification and vascular damage [START_REF] Pletcher | Alcohol consumption, binge drinking and early coronary calcification: findings from the Coronary Artery Risk Development in Young Adults (CARDIA) Study[END_REF][START_REF] Van Trijp | Alcohol consumption and augmentation index in healthy young men: the ARYA study[END_REF].

The few studies evaluating the effect of alcohol consumption on IMT have provided conflicting results. Data of the Atherosclerosis Risk in Communities Study (ARIC) and the Three-City-Study found no relationship between alcohol consumption and IMT [START_REF] Demirovic | Alcohol consumption and ultrasonographically assessed carotid artery wall thickness and distensibility. The Atherosclerosis Risk in Communities (ARIC) Study Investigators[END_REF][START_REF] Zureik | Alcohol consumption and carotid artery structure in older French adults: the Three-City Study[END_REF]. Data from SHIP showed an inverse correlation of alcohol consumption with IMT in men, whereas in women no correlation was seen [START_REF] Schminke | Association between alcohol consumption and subclinical carotid atherosclerosis. the Study of Health in Pomerania[END_REF]. In the Cardiovascular Health Study (CHS) of adults aged ≥65 years and older only a moderate alcohol intake was inversely related to IMT, whereas a higher amount showed a positive correlation [START_REF] Mukamal | Alcohol consumption and carotid atherosclerosis in older adults: The Cardiovascular Health Study[END_REF]. Similarly, in the Bruneck Study, including men and women aged 40-79 years a J-shaped association between alcohol intake and progression of IMT was detected [START_REF] Kiechl | Alcohol consumption and atherosclerosis: what is the relation? Prospective results from the Bruneck Study[END_REF].

The findings of the STRATEGY study are in line with recent data of the Cardiovascular Risk in Young Finns Study which revealed a positive association between alcohol intake and IMT in young adults aged 24-39 years [START_REF] Juonala | Alcohol consumption is directly associated with carotid intima-media tickness in Finnish young adults. The Cardiovascular Risk in Young Finns Study[END_REF]. The authors suggested a proatherogenetic effect of alcohol consumption. Both populations were characterized by comparatively healthy individuals, due to young age in the Finnish study and by selecting subjects aged 30-70 years without known cardiovascular risk factors and medication in the STRATEGY study. They followed a prudent lifestyle pattern. Also, in a sample of healthy middle-aged French men and women a significant and positive association between alcohol consumption and IMT was found [START_REF] Ferrières | Carotid intimamedia thickness and coronary heart disease risk factors in a low-risk population[END_REF].

The protective effect of alcohol may only be detectable in individuals at higher cardiovascular risk, if alcohol alleviates the effect of risk factors. Evidence for this concept is provided by a population-based, cross-sectional study that suggests that moderate alcohol intake may attenuate the relation between saturated fat and increased IMT [START_REF] Merchant | Interrelation of saturated fat, trans fat, alcohol intake, and subclinical atherosclerosis[END_REF].

Presumably, selecting a population following a prudent dietary pattern and lifestyle, the beneficial effects of moderate alcohol intake on subclinical atherosclerosis may not be relevant. Unfortunately, only few studies have taken dietary habits into account.

However, there are some more potential explanations for the controversial results. Beneficial effects of alcohol consumption are assumed to be mediated by increased HDL-cholesterol and insulin sensitivity, improvement in endothelial function, myocyte resistance to ischemic injury, reduced platelet aggregation or inflammation [START_REF] Rimm | Moderate alcohol intake and lower risk of coronary heart disease: meta-analysis of effects on lipids and haemostatic factors[END_REF][START_REF] Teragawa | Effect of alcohol consumption on endothelial function in men with coronary artery disease[END_REF][START_REF] Imhof | Effect of alcohol consumption on systemic markers of inflammation[END_REF][START_REF] Zhou | Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt[END_REF]. Thus, some effects of alcohol are mediated through mechanisms that are not reflected by IMT and may compete with deleterious effects on the vessel wall. Hence, the protective effect of alcohol on cardiovascular events may differ in older subjects at higher risk from those pathways leading to an increase in IMT in younger or healthy adults. Also, some studies observed that alcohol intake was positively and independently correlated with common carotid arterial diameter, indicating that in early stage of atherosclerosis alcohol may compensate for arterial wall thickening and plaque formation and reducing shear stress [START_REF] Crouse | Arterial enlargement in the atherosclerosis risk in communities (ARIC) cohort. In vivo quantification of carotid arterial enlargement[END_REF][START_REF] Bonithon-Kopp | Factors of carotid arterial enlargement in a population aged 59 to 71 years: the EVA study[END_REF]. In the end, the increase in IMT through alcohol may not reflect atherogenesis, but stabilization, thus may be beneficial as well. This, however, would impact on the interpretation of IMT as a surrogate of atherosclerosis and vascular risk.

Study limitations

The STRATEGY study has several limitations. First, persons who reported morbidities or medication for cardiovascular risk factors were excluded. Accordingly, the findings are confined to explicitly healthy volunteers, characterized by a favorable lifestyle and a low risk profile. Second, our data are based on self-reported food-frequency including portion size for alcohol intake. Underreporting might have occurred particularly in heavier drinking groups, though not very likely, and thereby the ability to evaluate excessive drinking was limited.

Third, because of the sample size, the study may have been underpowered to detect an association between mean daily alcohol intake and IMT in women.

Conclusion

The STRATEGY study shows that alcohol consumption is associated with an increase in IMT in healthy men. In the light of evidence from several studies reporting a protective effect of alcohol on cardiovascular events, higher IMT values may not necessarily reflect early atherosclerosis and vascular risk, at least in healthy subjects with light to moderate alcohol consumption, following a protective nutrition and lifestyle. This, however, raises the fundamental question about the prognostic value of IMT measurements in healthy individuals.

  Stress Atherosclerosis and ECG Study (STRATEGY Study) is a cross-sectional study in a healthy population. Men and women aged 30 to 70 years were recruited in Hamburg through a health insurance via a flyer randomly included in regular mailings between September 2006 and March 2007. On telephone contact, participants were invited for a visit fasting in the morning and one week later in the afternoon, if the following exclusion criteria were not met: acute or chronic diseases or any medication except for hormone replacement therapy or oral contraceptives and thyroxin for stably substituted hypothyroidism. Up to at least 25 participants per age decade and gender were consecutively included. Of 215 eligible subjects, two were excluded from this analysis because of missing data with regard to alcohol consumption or IMT, respectively. The final study population comprised 106 men and 107 women (men/women: 30 to 39 years, n= 26/26; 40 to 49 years, n= 26/29; 50 to 59 years, n= 27/27; 60 to 70 years, n=27/25). The study protocol was approved by the ethical committee of Hamburg and conducted according to the principles of the 'Declaration of Helsinki'.

Figure 1 :

 1 Figure 1: Daily alcohol intake in men and women according to categories of alcohol

Figure 2 :

 2 Figure 2: Frequency of alcohol consumption in men and women.

Figure 3 :

 3 Figure 3: Relation between alcohol intake (g/Day) and IMT (mm) in men and women.

Table 1

 1 Clinical and lifestyle characteristics of the study population as mean ± 1 standard deviation

		Men	Women	Significance
	Variables
		n = 107	n = 107	p-value
	Clinical characteristics
	Age (years)	49.6 ± 11.3	49.3 ± 10.6	n.s.
	BMI (kg/m 2 )	26.2 ± 3.2	23.9 ± 3.3	<0.001
	Waist circumference (cm)	95.9 ± 10.5	81.6 ± 9.4	<0.001
	Fasting glucose (mg/dl)	92.3 ± 9.5	87.5 ± 7.8	< 0.01
	2h glucose (75 g OGTT)	83.6 ± 22.9	83.9 ± 17.6	n.s.
	(mg/dl)
	HDL-cholesterol (mg/dl)	63.2 ± 15.8	80.4 ± 17.6	<0.001
	LDL-cholesterol (mg/dl)	124.1 ± 31.0	110.4 ± 32.2 < 0.01
	Triglycerides (mg/dl)	116.6 ± 66.6	85.0 ± 36.6	<0.001
	Lipoprotein(a) (mg/dl)	25.7 ± 34.5	32.5 ± 37.9	n.s.
	ALAT (IU/l)	28.8 ± 12.0	20.0 ± 9.1	<0.001
	ASAT (IU/l)	26.7 ± 5.9	24.1 ± 6.1	< 0.01
	GGT (IU/l)	31.7 ± 23.5	16.7 ± 9.6	<0.001
	GLDH (IU/l)	3.6 ± 3.5	2.5 ± 1.8	<0.001
	Systolic blood pressure	126.6 ± 15.1	119.9 ± 18.8 < 0.01
	(mm Hg)
	Diastolic blood pressure	78.8 ± 11.7	73.1 ± 9.3	< 0.01
	(mm Hg)
	To be continued next page

Table 3

 3 Multiple linear regression analysis of daily alcohol intake and IMT in men (n = 106) Model 1 adjusted for age, HDL-cholesterol, and LDL-cholesterol Model 2 adjusted for model 1 + systolic blood pressure and smoking status Model 3 adjusted for model 2 + fasting glucose and waist circumference Model 4 adjusted for model 3 + daily intake of energy, meat and meat products, fruit and vegetables, and physical activity Model 5 adjusted for model 4 + lipoprotein(a)

	Explanatory variables	Model 1	Model 2	Model 3	Model 4	Model 5
		β-		β-		β-		β-		β-
		esti-	p	esti-	p	esti-	p	esti-	p	esti-	p
		mate		mate		mate		mate		mate
	Age (years)	0.00 38	< 0.001	0.00 36	< 0.001	0.00 33	< 0.001	0.00 34	< 0.001	0.00 40	< 0.001
	HDL-cholesterol (mg/dl)	-0.00 37	0.65	-0.00 12	0.89	0.00 07	0.94	-0.00 12	0.92	-0.00 17	0.88
	LDL-cholesterol (mg/dl)	-0.00 15	0.84	-0.00 00	0.99	0.00 04	0.96	0.00 24	0.82	-0.00 22	0.83
	Systolic blood pressure (mm Hg)			0.01 12	0.18	0.01 00	0.26	0.00 66	0.54	0.00 03	0.98
	Smoking status			0.00 83	0.77	0.01 13	0.69	0.00 94	0.81	0.01 36	0.72
	Fasting glucose (mg/dl)					0.00 09	0.36	0.00 03	0.79	-0.00 02	0.89
	Waist circumference (cm)					-0.00 01	0.88	0.00 12	0.40	0.00 10	0.48
	Energy intake (kcal/day)							-0.00 21	0.87	-0.00 95	0.47
	Meat and meat products (g/day)							0.00 52	0.67	-0.00 43	0.74
	Fruit and vegetables (g/day)							0.00 32	0.78	0.00 51	0.65
	Physical activity (h/week)							0.00 75	0.48	0.00 21	0.85
	Lipoprotein(a) (mg/dl)									-0.00 11	0.92
	Alcohol consumption (g/day]	0.02 53	<0.05	0.02 47	<0.05	0.02 42	<0.05	0.02 51	<0.05	0.02 74	<0.05

Acknowledgements

We are indebted to all participants of the study for their collaboration. We also thank Ms. E. Kohlsdorf and W. Bernigau from the German Institute of Nutrition for their expert biomathematical assistance in establishing the data base.

Conflict of interest.

The authors declare no conflict of interest.

Financial support was given by Merck Pharma GmbH, Darmstadt, and Energy-Lab Technologies GmbH, Hamburg

Author Disclosure Statement

Competing financial interests of all authors have been appropriately disclosed according to the policy of the Journal. No competing financial interests exist.


Antonia Ribes
email: .aribes@clinic.ub.es

Judit García-Villoria

Laura Gort

Irene Madrigal

Carme Fons

Cristina Fernández-Sierra

Aleix Navarro-Sastre

Montserrat Mila

Mª Paz Briones

Angeles García-Cazorla

Cristina Fernández

Mª Angeles García- Cazorla

Jaume Campistol

Dra Antonia Ribes

X-inactivation of HSD17B10 revealed by cDNA analysis in two females with 17β-hydroxysteroid dehydrogenase 10 (HSD10) deficiency

Keywords: HSD10 deficiency, HADH2, HSD17B10

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

17β-hydroxysteroid dehydrogenase 10 (HSD10) is a mitochondrial enzyme involved in the degradation pathway of isoleucine and branched-chain fatty acids [START_REF] Zschocke | Progressive infantile neurodegeneration caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: a novel inborn error of branched-chain fatty acid and isoleucine metabolism[END_REF]. This enzyme has also been found to be involved in the metabolism of sex steroid hormones, neuroactive steroids and in the detoxification of cytotoxic aldehydes [START_REF] Yang | Multiple functions of type 10 17β-hydroxysteroid dehydrogenase[END_REF][START_REF] Murakami | Cytoprotective role of mitochondrial amyloid β peptide-binding alcohol dehydrogenase against a cytotoxic aldehyde[END_REF]. HSD10 deficiency (OMIM 300256) is an X-linked defect caused by mutations in the HSD17B10 gene. Clinically, the great majority of male patients show normal early development followed by progressive loss of mental and motor skills [START_REF] Zschocke | Progressive infantile neurodegeneration caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: a novel inborn error of branched-chain fatty acid and isoleucine metabolism[END_REF][START_REF] Poll-The | Spastic diplegia and periventricular white matter abnormalities in 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, a defect of isoleucine metabolism: differential diagnosis with hypoxicischemic brain diseases[END_REF][START_REF] Ensenauer | Clinical variability in 3-hydroxy-2methylbutyryl-CoA dehydrogenase deficiency[END_REF][START_REF] Olpin | 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23 year old man[END_REF][START_REF] Sass | 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: impaired catabolism of isoleucine presenting as neurodegenerative disease[END_REF][START_REF] Sutton | 3-hydroxy-2methylbutyryl-CoA dehydrogenase deficiency[END_REF][START_REF] Perez-Cerdá | 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: an X-linked inborn error of isoleucine metabolism that may mimic a mitochondrial disease[END_REF][START_REF] Cazorla | Neuroimage findings in 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency[END_REF][START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF].

However, three patients were identified that presented symptoms in the first days of life [START_REF] Zschocke | Progressive infantile neurodegeneration caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: a novel inborn error of branched-chain fatty acid and isoleucine metabolism[END_REF][START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. It has recently been demonstrated that symptoms of these patients are unrelated to accumulation of metabolites in the isoleucine pathway and that the neurological handicap can be associated with an imbalance in neurosteroid metabolism [START_REF] Yang | Mental retardation linked to mutations in the HSD17B10 gene interfering with neurosteroid and isoleucine metabolism[END_REF] or to defects in general mitochondrial function [START_REF] Rauschenberger | A non-enzymatic function of 17β-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival[END_REF]. In addition, the splice variant c.574C>A of HSD17B10 gene has been associated with a new syndromic form of X-linked mental retardation, choreoathetosis and abnormal behaviour [START_REF] Lenski | The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behaviour[END_REF].

The HSD17B10 gene has been mapped to chromosome Xp11.2 [START_REF] He | A human brain L-3-hydroxyacyl-coenzyme A dehydrogenase is identical to an amyloid beta-peptide-binding protein involved in Alzheimer's disease[END_REF] and has been reported as one of the few genes that escapes X-inactivation [START_REF] Miller | Chromosomal basis of X chromosome inactivation: identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation[END_REF]. To date, 10 female patients with HSD10 deficiency have been described presenting a variety of symptoms, from borderline learning difficulties to psychomotor and speech delay [START_REF] Ensenauer | Clinical variability in 3-hydroxy-2methylbutyryl-CoA dehydrogenase deficiency[END_REF][START_REF] Perez-Cerdá | 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: an X-linked inborn error of isoleucine metabolism that may mimic a mitochondrial disease[END_REF][START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. We previously studied two of these females. One of them was heterozygous for the p.N247S mutation and was severely affected, whereas the other was heterozygous for the p.P210S mutation and presented a slight clinical affectation [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. To elucidate why these two females were so differently affected, we performed HSD17B10 cDNA quantitative analysis in both females and in control fibroblasts, the results of which are reported here.

Materials and Methods

Material

Skin biopsies from patients of two unrelated Spanish families with HSD10 deficiency were obtained. Family 1: a male patient (1IIM) and his carrier sister (1IIF), both with a severe phenotype (Figure 1A). Family 2: a male patient (2IIM), with a severe phenotype and his heterozygous mother (2IF) with a slight clinical affectation (Figure 1A). Both families have been previously described [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. Eight cell lines (four males and four females) from our cell bank were used as controls.

All the samples were obtained according to the declaration of Helsinki and informed consent was signed by all the patients or their parents.

Molecular studies

cDNAs were obtained from cultured fibroblasts, were amplified by PCR and sequenced using standard protocols and oligonucleotides designed in-house (sequences available upon request). All HSD17B10 cDNAs were quantified by the StepOnePlus™ real-time PCR System using the Comparative Ct (ΔΔCt) method from StepOne software v2.0 (Applied Biosystems, Foster City, CA, USA). The Primer Express 3.0 software (Applied Biosystems) was used to design two sets of primers and probes to differentiate wild-type (Wt) and mutant (Mut) alleles corresponding to mutations p.N247S (c.740A>G) and p.P210S (c.628C>T) of the HSD17B10 gene. We used two different endogenous controls: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, PN4310884E) and cyclophilin A (PPIA, PN4310883E) (Applied Biosystems). As additional control, a mixed pool of four healthy male cDNAs was used in each analysis. Gene nucleotide numbering was done according to sequence RefSeq NM_004493 with +1 as A of the ATG start codon. The ATG codon represents +1 for the amino acid numbering according to the HSD10 protein sequence NP_004484.

X-inactivation studies

The androgen-receptor locus (AR) methylation assay was performed in genomic DNA of female carriers, as previously described [START_REF] Allen | Methylation of HpaII and HhaI sites near polymorphic CAG repeat in the human androgen-receptor gene correlates with X-chromosome inactivation[END_REF]. If the AR locus was uninformative, skewing was assessed at FMR1 locus [START_REF] Hećimović | Expand Long PCR for fragile X mutation detection[END_REF]. Briefly, genomic DNA (300ng) was digested with 5U of HpaII (both for the AR and FMR1 assays) in a total volume of 20µL. For each sample, an undigested control was prepared. We define the pattern of X chromosome inactivation as skewed when the inactivation percentage was over 80%.

Statistical methods

Statistical studies for the analyses of Relative Quantification (RQ) in male and female controls were performed using the non parametric two-related sample test, Wilcoxon test, with the SPSS ® software (version 14.0 for Windows ® ).

Results and discussion

HSD17B10 has been reported as one of the few genes that escapes X-inactivation [START_REF] Miller | Chromosomal basis of X chromosome inactivation: identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation[END_REF], which predicts that female carriers would not be affected. However, 10 females with HSD10 deficiency have been described so far, presenting different degrees of clinical affectation, which is in agreement with an X-linked inheritance with different degrees of X-inactivation [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF].

To elucidate if HSD17B10 cDNA doses differed between males and females, we performed RQ of wild-type (Wt) HSD17B10 cDNA alleles in four female and four male controls (Figure 2). Results of the Wilcoxon statistical test did not show any significant difference between the doses in both sexes considering the two endogenous controls (p-value=0.07). Therefore, these results are in favour of an X-linked disease that does not escape X-inactivation.

We previously studied two unrelated females with different degrees of clinical affectation [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. The female of Family 1 (1IIF), like her brother (1IIM), presented a severe phenotype with psychomotor and speech delay, and a clear deficiency of HSD10 activity in fibroblasts [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. When we sequenced her HSD17B10 cDNA it seemed that only the mutant allele was identified (Figure 1A). Results for the RQ of her HSD17B10 cDNA (Figure 3A) showed that amplification levels of the mutant (Mut) probe were much higher than those of the Wt probe and very similar to those of her brother (1IIM), independently of the endogenous control used (Figure 3A). To rule out a Turner Syndrome chromosome analysis was performed, which resulted in a normal karyotype (46,XX). Skewed X-inactivation was confirmed by methylation studies. Patient 1IIF was homozygous for AR locus, consequently this study was uninformative, but FMR1 locus showed a skewed X-inactivation pattern (80/20). These results are in agreement with an unfavourable X-inactivation effect of HSD17B10 gene in the analysed tissue. In addition, as the girl was severely affected, it could be expected a similar unfavourable X-inactivation in other tissues.

The other female (2IF) showed a mild clinical affectation, with learning disabilities and HSD10 activity in fibroblasts within the control range [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF]. HSD17B10 cDNA sequencing showed the presence of both mutant and wild-type alleles (Figure 1B). This observation was in agreement with the results of the RQ studies showing similar HSD17B10 cDNA levels of both Wt and Mut probes, while we were only able to amplify the Mut probe in her severely affected son (2IIM) (Figure 3B). X-inactivation analysis showed a random X-inactivation pattern for AR locus in patient 2IF. These results, suggest the presence of both HSD17B10 alleles in this female, which is in line with normal X-inactivation and the expression of both alleles in different cells (functional mosaicism). In addition, the normal enzymatic activity found in this female [START_REF] García-Villoria | Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA deshydrogenase (MHBD) deficiency: difficulties in the diagnosis[END_REF] might be due to lack of sensitivity of the enzymatic technique, or maybe there was enough dose of wild type HSD17B10 mRNA to produce enough HSD10 protein to obtain normal activity.

However, we did observe that the amplification responses were different for each probe when they were corrected by the two different endogenous controls (Figure 3). This could be explained by the low specificity of the probes and by the variability of the endogenous controls. However, in spite of it, the interpretation of the results did not change.

To summarise, here we present the results of HSD17B10 cDNA analysis in two female carriers compared with affected males and controls. The hypothesis that HSD17B10 is inactivated in one of the X-chromosome is supported by the results in controls, which showed that doses of HSD17B10 cDNA were the same in both sexes (Figure 2). RQ cDNA results for one of the females (1IIF), together with the enzymatic studies and the severe clinical presentation were in agreement with an unfavourable X-inactivation effect. In addition, RQ cDNA results for the other female (2IF) seem to reflect the presence of a mosaicism in the studied tissue, which could explain the normal enzymatic activity and her mild phenotype. Although, we cannot exclude that differences in disease severity between both female carriers is at least partly due to differences in the effect of the mutations, as the male patient with the p.N247S mutation died at age two months whilst the patient with p.P210S mutation is alive at four years of age.

In conclusion, our results suggest that HSD17B10 gene does not escape X-inactivation as previously reported [START_REF] Miller | Chromosomal basis of X chromosome inactivation: identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation[END_REF]. Heterozygous females showed the classical biochemical and clinical variability of X-linked diseases due to random X-chromosome inactivation and the severity of the phenotype will depend on the total dose of mutant mRNA in different tissues as well as on the severity of the mutation.

Titles and legends to figures

Figure 1 .

 1 Figure 1. Pedigree (1A) and cDNA sequence (1B) of Family 1 and Family 2.

Figure 2 .

 2 Figure 2. RQ of HSD17B10 Wt probes named as p.N247N and p.P210P, with two

Figure 3 .

 3 Figure 3. RQ of HSD17B10 Wt and Mut probes with two distinct endogenous controls

Acknowledgments

We thank Carlota Ogg and Patricia Alcalá for their excellent technical assistance. This group is funded by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII and CENIT 06/Nanopharm. Cristina Fernández is a recipient of a grant (FIS, CA 06-0128). This work has been performed in the context of the PhD program of the Department of Biochemistry and Molecular Biology of the University of Barcelona.