Sophia J Makowski

Michael Hörmannsdorfer

Dr Wolfgang Schnick
email: wolfgang.schnick@uni-muenchen.de

⋅ 2 H2O -Crystal Structure of a New Alkali Derivative of the Multidentate Ligand Triazine Tricarboxylate

Keywords: s-Triazine, Potassium, Carboxylate ligand, Crystal structure s-Triazine, Potassium, Carboxylate ligands, Crystal structure

Potassium-1,3,5-triazine-2,4,6-tricarboxylate dihydrate K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O was obtained by saponification of the respective ethyl ester in aqueous solution under mild conditions and subsequent crystallization at 4 °C. The crystal structure of the molecular salt was elucidated by single-crystal X-ray diffraction (P1, a = 6.9663(14), b = 17.485(3), c = 17.560(3) pm, α = 119.73(3), β = 91.96(3), γ = 93.84(3)°, V = 1847.6(6) • 10 6 pm 3 , Z = 6, T = 200 K). Perpendicular to [100] the triazine tricarboxylate and potassium ions are arranged in layers alternating with layers of crystal water molecules. Two thirds of the triazine tricarboxylate units form hexagonal channels being filled with the remaining triazine tricarboxylate molecules. K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O was additionally investigated by means of FTIR spectroscopy, TG and DTA measurements.

Introduction

Research in the field of carbon nitrides is mainly focused on the synthesis of two-and three-dimensional networks which can serve as functional materials for a wide field of applications such as catalysis or optoelectronic uses [1][2][3]. A common approach towards multidimensional networks is the pyrolysis of s-triazine (C 3 N 3 ) or s-heptazine (C 6 N 7 ) based precursors to induce condensation and thereby generating covalently bound products. A variety of precursors with different functional groups has been utilized in this regard (cf. Scheme 1, 1a-d and 2a-d) [4][5][6][7][8].

In general, molecules containing residues that are bound to the s-triazine or s-heptazine core via C-C bonds (cf. Scheme 1, 1e-f and 2e-f) are adverse in this regard. This is due to the strength of the C-C bonds impeding a successful condensation. Furthermore, the synthesis of carbon nitrides necessitates an alternating sequence of carbon and nitrogen both in the precursor and in the resulting polymeric network. Even though such compounds are no suitable precursors for the synthesis of covalently bound true carbon nitrides by condensation reactions they can be regarded as promising linkers in coordination polymers. Metal organic frameworks (MOFs) have been extensively studied in the last years due to their potential applications, e. g. as materials for ion exchange, separation, catalysis or gas storage [9,10]. While numerous MOFs are known in which a hydrocarbon cycle functionalized with several carboxylate groups, e. g. trimesic acid 3 (cf. Scheme 2) [11][12][13], serves as the linker between inorganic centers the analog s-triazine or s-heptazine compounds 1f and 2f have not been employed for this purpose so far.

In known s-triazine and s-heptazine based linkers such as s-triazine-2,4,6-tris(4-pyridyl) 4 [14,15], s-triazine-2,4,6-triyltribenzoic acid 5 [16][17][18][19][20] or s-heptazine-2,5,8-triyl-tribenzoic acid 6 [20,21] (cf. Scheme 2) the s-triazine or s-heptazine cores, respectively, only provide the threefold symmetry but do not contribute to metal coordination. In contrast in 1f and 2f both the carboxylate groups and the nitrogen atoms of the ring can be regarded as coordination positions.

The s-heptazine compound 2f has not been mentioned in the literature so far whereas the synthesis of 1f by a multistep reaction via the respective ethyl ester and potassium salt has already been described over 50 years ago [22]. However, no structural characterization of the product or any intermediate has been performed and since then only very little research regarding triazine tricarbonic acid or its salts has been carried out until 2002 when Dunbar et al. reported the crystal structure and magnetic properties of K{Fe (1,3,5-

triazine-2,4,6-tricarboxylate)(H 2 O) 2 } ⋅ 2 H 2 O [23].
To the best of our knowledge no crystal structure of any other salt of triazine tricarbonic acid has been described so far. However, regarding the potential application of triazine tricarboxylate as a multidentate ligand a deeper understanding of its coordination behavior towards various metal ions is of great interest. In this contribution we report on synthesis and crystal structure of potassium triazine tricarboxylate dihydrate as a further example of a structurally characterized triazine tricarboxylate and thereby providing further insight into the coordination properties of this linker.

Results and Discussion

Crystal Structure

Potassium triazine tricarboxylate crystallizes as a dihydrate in the triclinic space group P1 with six formula units in the unit cell. Crystallographic data and details of the structure refinement are summarized in Table 1. The crystal structure comprises triazine tricarboxylate [C 3 N 3 (COO) 3 ] 3-and potassium ions and crystal water molecules (cf. Figure 1).

The triazine tricarboxylate ions are nearly planar with only the carboxylate groups being slightly rotated out of plane so that the anions exhibit molecular symmetry C 3 . The substituents cause an increase of the adjacent angles (N1-C1-N3 = 124.5°) while the remaining angles of the s-triazine core are accordingly reduced (C1-N1-C2 = 115.5°). In accordance with expectation the bond distances of both the C-N bonds within the ring (133-134 pm) and the C-O bonds of the carboxylate groups (124-125 pm) correspond to conjugated double bonds while the C-C bond lengths of 153-154 pm resemble those of single bonds (cf. Table 2).

The triazine tricarboxylate ions, the potassium atoms and water molecules are arranged in an AA'BB' type stacking pattern perpendicular to [100] (cf. Figure 2) with the A and A' layers consisting of the triazine tricarboxylate and potassium ions and the crystal water molecules being located in the intermediate B and B' layers. Within the A and A' layers the triazine tricarboxylate ions are not coplanar but are slightly tilted out of the bc-plane. Perpendicular to [100] the triazine tricarboxylate units form two different kinds of columns. One third of the columns consist solely of molecules B (cf. Figure 1) with the molecules of two adjacent layers A and A' being rotated by 60° within the plane. In the remaining columns molecules A and C alternate and are rotated by 30°. These columns form hexagonal channels around the B columns (cf. Figure 3).

The interlayer distance of 347 pm between two triazine tricarboxylate layers is in the expected range for molecular striazine derivatives [1,24,25]. Between the layers the triazine tricarboxylate units are interconnected by a network of medium strong [26] hydrogen bonds (cf. Table 3) via the carboxylate groups. All crystal water molecules are part of the hydrogen bonding network although due to the strong disorder of one oxygen atom (O24) no hydrogen bond can be clearly assigned to the linked hydrogen atom H11.

The potassium ions are coordinated in the form of irregular polyhedrons by four to six oxygen atoms of water molecules and carboxylate groups and by one nitrogen atom of the triazine rings. The triazine tricarboxylate units serve as tridentate ligands and coordinate the K + atoms via the O-N-O entities (cf. Figure 1) with the K + atom being slightly shifted towards one of the oxygen atoms. The coordination sphere is completed by the oxygen atoms of further triazine tricarboxylate units and water molecules. The K-O distances (273-297 pm) are in accordance with the sum of the ionic radii [27] while the K-N distances (278-282 pm) are relatively short compared to the potassium salts of other s-triazine or s-heptazine compounds [28][29][30]. In K{Fe(1,3,5-triazine-2,4,6-tricarboxylate)(H 2 O) 2 } ⋅ 2H 2 O the Fe atoms are coordinated by an O-N-O unit in a similar way as in the title compound with a short Fe-N distance of 212 pm while the potassium ions are coordinated solely by oxygen atoms and the K-O distances are comparable in both compounds. Hence in both triazine tricarboxylate salts a strong contribution of the s-triazine core to metal coordination is observed so that the triazine tricarboxylate unit can be regarded as an interesting linker for coordination networks potentially allowing other linkage motifs than those observed in MOFs which contain trimesic acid as the organic linker.

Vibrational Spectroscopy

Potassium triazine tricarboxylate dihydrate was analyzed by FTIR spectroscopy. All signals in the spectrum can be attributed to vibrations of the triazine tricarboxylate entities and water molecules, respectively (cf. Figure 4). The bending and stretching vibrations of the s-triazine ring lead to a characteristic absorption band at 740 cm -1 and a group of signals at 1300-1660 cm -1 , respectively. In the range between 1300 and 1660 cm -1 the stretching vibrations of the carboxylate groups can be observed as well. The broad band at 3000-3600 cm -1 can be attributed to the OH stretching vibrations of the crystal water molecules and in accordance with the crystal structure the red-shift of the ν(OH) signals corresponds to medium strong hydrogen bonds [26].

Thermal Behavior

TG and DTA curves of potassium triazine tricarboxylate dihydrate are displayed in Figure 5. In the temperature range between 65 and 150 °C the crystal water molecules are released (mass loss observed: 10.9 %, calculated: 9.9 %) and the so obtained dehydrated compound is thermally stable up to 325 °C. The decomposition of the sample starts with a mass loss of 17 % accompanied by a sharp endothermic signal at 325-390 °C, probably due to a partial decarboxylation, and proceeds with a slow and steady mass loss at higher temperatures.

Conclusions

We have reported on the crystal structure and thermal behavior of potassium triazine tricarboxylate dihydrate. Triazine tricarboxylate represents a multidentate ligand which can be regarded as the s-triazine analog of trimesic acid and hence as a promising linker for metal organic frameworks but only one triazine tricarboxylate salt has been structurally characterized so far. This study has expanded the investigations of this class of compounds to a new salt and thereby provides further insight into the coordination properties of triazine tricarboxylate. The presented crystal structure shows that the nitrogen atoms of the s-triazine cycle exhibit a strong interaction with the potassium ions and that the coordination of metal ions via the O-N-O units of the triazine tricarboxylate entities is a recurring binding motif for this ligand. Because of the differing coordination properties compared to trimesic acid triazine tricarboxylate seems to be an interesting linker for coordination networks and with the observed high thermal stability of K 3 [C 3 N 3 (COO) 3 ] an important condition for syntheses of three-dimensional networks at elevated temperatures is fulfilled.

Experimental Section

Syntheses

Potassium triazine tricarboxylate dihydrate was synthesized according to the procedure described in [22,31] by saponification of the respective triethyl ester. To prepare the triethyl ester dry HCl was conducted through ethyl cyanoformate (20.0 g, 202 mmol, Aldrich, 99 %) under argon until the trimerization product started to precipitate. Then the conversion was completed by cooling the reaction to 4 °C over night. The product was filtered, washed with cold water and dried at 75 °C yielding 7.15 g (24.1 mmol, 36 %) triethyl-s-triazine-2,4,6-tricarboxylate as colorless needles. Elemental analysis (wt. %): N 14.22 (calc. 14.13), C 47.20 (calc. 48.47), H 4.88 (calc. 5.08).

Small amounts of the triethyl ester (100 mg, 0.34 mmol) were added to 3.5 ml of a 1M aqueous solution of KOH while cooling with an ice bath. The reaction was stirred until the ester was completely dissolved and then 25 ml ethanol was poured into the solution to precipitate the potassium salt. The product was filtered, washed with ethanol and dried at 75 °C to obtain 103 mg (0.28 mmol, 83 %) potassium-1,3,5-triazine-2,4,6-tricarboxylate dihydrate as colorless needles. Single crystals suitable for single-crystal X-ray diffraction were obtained by repeated recrystallization from aqueous solution at 4 °C. Elemental analysis (wt. %): N 11.60 (calc. 11.56), C 20.45 (calc. 19.83), H 0.64 (calc. 1.11).

X-ray Structure Determination

Single-crystal X-ray diffraction data of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O were collected at 200 K on a Kappa CCD diffractometer using monochromated Mo-K α radiation (λ = 71.073 pm). The diffraction intensities were scaled using the SCALEPACK software package [32] and no additional adsorption correction was applied. The crystal structure was solved by direct methods using the software package SHELXS-97 [START_REF] Sheldrick | SHELXS-97, Program for the Solution of Crystal Structures[END_REF] and refined against F 2 by applying the full-matrix least-squares method (SHELXL-97) [START_REF] Sheldrick | SHELXL-97, Program for the Refinement of Crystal Structures[END_REF]. The hydrogen positions could be determined from difference Fourier syntheses and were refined isotropically using restraints for oxygen-hydrogen distances. All non-hydrogen atoms were refined anisotropically.

CCDC 784049 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

FT-Infrared Spectroscopy

A FTIR spectrum was recorded at ambient conditions between 600 and 4500 cm -1 on a Spektrum BX II FTIR spectrometer (Perkin Elmer) equipped with a DuraSampler diamond ATR device.

Thermal Analysis

Thermoanalytical measurements were performed with a Thermoanalyzer TG-DTA92 (Setaram) under inert atmosphere (He). The sample was heated in an alumina crucible from room temperature to 500 °C with a heating rate of 5 °C min -1 . Scheme 1. Molecular precursors for carbon nitride networks comprising the s-triazine (1) or s-heptazine (2) core. Scheme 2. Multidentate ligands which are commonly employed as linkers in metal organic frameworks.

Figure 1 .

 1 Figure 1. Representation of one formula unit of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O. The three triazine tricarboxylate entities present in the crystal structure are labeled A-C. Thermal ellipsoids (except for H) are drawn at the 50 % probability level.

Figure 2 .

 2 Figure 2. Crystal structure of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O representing the layered structure perpendicular to [100]. Thermal ellipsoids (except for H) are drawn at the 50 % probability level.

Figure 3 .

 3 Figure 3. Crystal structure of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O representing the stacked arrangement of the triazine tricarboxylate entities. Crystal water molecules are omitted for clarity. The labels A-C refer to Figure 1, indicating which triazine tricarboxylate unit lies on top of the respective column. Thermal ellipsoids are drawn at the 50 % probability level.

Figure 4 .

 4 Figure 4. FTIR spectrum of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O.

Figure 5 .

 5 Figure 5. TG (solid) and DTA (dotted) curves of K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O, recorded with a heating rate of 5 °C min -1 .

  final R indices [I > 2σ(I)] R1 = 0.0513, wR2 = 0.1068[a] final R indices (all data) R1 = 0.0868, wR2 = 0.1228[a] [a] w = [σ 2 (F 0 2 ) + (0.0389P) 2 + 1.6956P] -1 , with P = (

Table 1 .

 1 Crystallographic data and details of the structure refinement for K 3 [C 3 N 3 (COO) 3 ] • 2 H 2 O.

	molar mass / g mol -1	363.41
	crystal system	triclinic
	space group	P1 (no. 2)
	T / K	200
	diffractometer	Nonius Kappa-CCD
	radiation, λ / pm	Mo-K α , 71.073
	a / pm	696.63(14)
	b / pm	1748.5(3)
	c / pm	1756.0(3)
	α / deg	119.73(3)
	β / deg	91.96(3)
	γ / deg	93.84(3)
	V / 10 6 pm 3	1847.6(8)
	Z	6
	calculated density / g cm -3	1.960
	crystal size / mm 3	0.70 x 0.07 x 0.05
	absorption coefficient / mm -1	1.150
	diffraction range	3.26º ≤ θ ≤ 27.54º
	index range	-8 ≤ h ≤ 9, -22 ≤ k ≤ 22,
		-22 ≤ l ≤ 22
	parameters / restraints	577 / 16
	total no. of reflections	14291
	no. of independent reflections	8452
	no. of observed reflections	5617
	min./max. residual electron
	density / 10 6 pm 3

Acknowledgments

The authors thank the Deutsche Forschungsgemeinschaft DFG (project SCHN 377/12) and the Fonds der Chemischen Industrie (FCI) for financial support and Dr. Peter Mayer (Department Chemie, LMU München) for the single-crystal data collection.


Joseph A Sergeant

Tobias Banaschewski

Jan Buitelar

David Coghill

Marina Danckaerts

Manfred Döpfner

A Rothenberger

Paramala Santosh

E J S Sonuga-Barke

Hans-Christoph Steinhausen

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

Eunethydis: a statement of the ethical principles governing the relationship between the European group for ADHD guidelines, and its members, with commercial for-profit organisations prepare guidelines for the treatment of ADHDthree such guidelines have so far been published [START_REF] Taylor | Clinical guidelines for hyperkinetic disorder[END_REF][START_REF] Taylor | European clinical guidelines for hyperkinetic disorderfirst upgrade[END_REF][START_REF] Banaschewski | Long-acting medications for the hyperkinetic disorders -A systematic review and European treatment guidelines[END_REF] . This clearly raises potential conflicts of interest with the commercial companies who develop and market treatments for ADHD.

This document sets out the ethical principles governing the relations between the Group, and its members, and industry. This is important because many members of the Group have at one time or another entered into financial relations with pharmaceutical companies for a range of different activities including consultancy, advice, presentations to meetings, as well as funding for trials and other studies. The goal of these collaborations varies across activities but the common element for the Group is that they are motivated by a desire to improve the understanding of ADHD and its treatment and to disseminate best practice in both research and therapy.

We are confident that these relations are conducted with both the highest ethical standards. The principles set out here are provided to ensure that this is both done and seen to be done. The impetus for these guidelines comes from within the Group and is linked to the recognition for the need for an open and transparent basis for Groupindustry relations, especially in the light of the present concern that the pharmaceutical industry may be exerting a growing influence on the actions of researchers and clinicians in the ADHD field.

Transparency and register of potential conflicts of interest:

The notion of transparency will be the core principle upon which this statement is built. A fully comprehensive register of any potential competing interests that arise out of work with commercial for-profit companies will be established and maintained by the chair of the Group. This will include a short detailed description of the activity involved as well as its time-frame. Examples of relevant activities include; advisory board membership, consultancy, presentations at company funded meetings, travel bursaries and conference attendance support. Both investigator initiated trials and pivotal phase 3 and phase 4 marketing trials should be declared.

Activities should be declared whether or not funds are paid directly to the individual or to an account within a university or other organisation. In time, these principles and procedures may be extended to not-for-profit organisations. At present it is not envisaged that these declarations will not include reference to the scale of financial involvement; but this will remain under review. Where confidentiality is required in these matters, register of competing interests should acknowledge the interaction in a general sense and note the company and product involved.

Independence of Group decision making:

Members who have conducted trials on a particular product will be allowed to present data to the Group on those trials during the review process. However, these data will be evaluated by members of the Group that are independent of these involvements. In case of conflicting opinions about the merits of a study and the meaning of the results, members involved with a specific project will be excluded from making the final decision. If the situation arises (and it has not to date), when all members of the group are involved in a trial, then the chair will approach appropriately qualified experts to give their evaluation of the trial data.

The value of independent statistical review of procedures and findings will be explored.

Protection of Group members' interests:

In order to protect the interests of members of the group the following procedures will be established.

(i) A register of attempts at undue influence by companies on the opinions and activities of Group members will be established and occasions of such will be placed on public record.

(ii) An informal system of peer review will be established to ensure that documents written or talks prepared for companies and funded by them conform to these ethical guidelines.

(iii) A record will be made of the outcome of this review.

(iv) Before entering into financial arrangements with industrial interests Group members should make a disclosure that their conduct is regulated by the principles set out in this document.

Ethical conduct of Group members:

In order to ensure that for-profit-related activities are conducted within an ethical framework the following principles are set down.

(i). Company funded drug trials should be undertaken by Group members only when there is an agreement that the full trial data will be made available to the investigators and there is no in principle veto on publication of data.

(ii) Involvement of Group member in ghost written papers prepared by professional medical writers for companies is prohibited. Furthermore members performing trials for companies are expected to be independent in their interpretation, able to review all material before it is published, and responsible for the whole of a paper of which they are an author. They should avoid inducements that could lead to bias in reporting.

(iii) When presenting data on company-funded trials the Group member must retain full editorial control and ensure that they operate in a completely unbiased and scientifically balanced way.

(iv) In relation to data reviewed by the Group for published guidelines, to ensure consistency and to protect their interests, a spirit of collective responsibility will be adopted in which Group members present the agreed guidelines position. However, this position needs to be reconciled with the need to maintain the individual academic freedom of Group members and to respond to developments in the field. Where these amendments to the published guideline position are made they will be reported to the Group.

(v) Group members conducting trials that are commercially funded (either investigator initiated or company sponsored) should avoid entering into personal financial relations with the trial sponsor (at least until after publication of the main outcomes).

1 Eunethydis A STATEMENT OF THE ETHICAL PRINCIPLES GOVERNING THE RELATIONSHIP BETWEEN THE EUROPEAN GROUP FOR ADHD GUIDELINES, AND ITS MEMBERS, WITH COMMERCIAL FOR PROFIT ORGANISATIONS

	Saturday, 06 October 2007, Cagliari
	Prof. Dr. J. A. Sergeant (Chairman)
	Prof. Dr. T. Banaschewski
	Prof. Dr, J. Buitelaar
	Dr. D. Coghill

Prof. dr. M. Danckaerts Prof. Dr. M. Döphner Prof. dr. A. Rothenberger Dr. P. Santosh Prof. E.J.B Sonuga-Barke Prof. Dr. H.-Ch. Steinhausen Prof, E. Taylor Prof. Dr. A. Zuddas Preamble: The members of the Eunethydis ADHD Guidelines Group (referred to as Group below) are clinicians and researchers dedicated to the understanding of ADHD/Hyperkinetic Disorder and its effective treatment. Their role has been to


Adrien Kettaneh

Laurence Fardet
email: laurence.fardet@sls.aphp.fr

Nathalie Mario

Aurelia Retbi

Namik Taright

Kiet Tiev

Ingrid Reinhard

Bertrand Guidet

Jean Cabane

Á L Fardet

Á K Tiev

Á J Cabane

Á N Taright

Á N Mario

Á A Retbi

Á I Reinhard

Á B Guidet

The 2003 heat wave in France: hydratation status changes in older inpatients

Keywords:

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

Introduction

During the first weeks of August 2003, a heat wave of uncommon duration and intensity occurred in Europe. With reference to 2000-2002, an excess mortality of 55% was reported in France from August 1st to 20th and 11,731 of the 14,729 excess deaths involved subjects aged 75 years and over, a majority being women [START_REF] Fouillet | Excess mortality related to the August 2003 heat wave in France[END_REF]. About one-third of deaths in excess occurred in Ile-de-France, the region surrounding Paris [START_REF] Gremy | Consequences sanitaires de la canicule d'aout 2003 en Ile-de-France[END_REF]. From 4 to 18 August 2003, our hospital recorded a 66% increase in hospitalization through the emergency department and a 30.1% increase in the number of electrolyte determinations compared to the corresponding period in 2002 [START_REF] Mario | Effect of August 2003 heat wave in France on a hospital biochemistry laboratory activity in Paris[END_REF].

Following the heat wave, the French government introduced a 3 level ''National heat wave response plan'' to monitor and face periods with extreme temperatures. The level 1 was activated every year since 2004, from June 1 to August 31. It consists in a daily monitoring of temperatures and data from an information network involving the National Meteorological Office, the National Health Monitoring Institute, and the National Agency for the Improvement of Working Conditions. All citizens and particularly those at high risk such as elderly, infants' parents, manual workers and sportsmen are advised through an extensive media campaign to protect against heat and drink regularly still water at least 1.5 l per day. Whenever a heat wave is forecast, the level 2, an ''alert and action'' protocol, mobilizes local and national public services in order to prevent public health consequences. The level 3 involves exceptional measures with maximal mobilization as soon as the heat wave began. Since 2003, level 3 has been activated during the July 2006 heat wave.

Little is known about the impact of behavioral changes related to the French 2003 heat wave experience, on hydration status of elderly citizens. We examined the changes in hydration profiles and inhospital mortality in elderly patients admitted in a tertiary center in Paris through the emergency department, after the 2003 heat wave, by comparison.

Patients and methods

Study population

All patients aged 70 years and over, admitted in our hospital after an evaluation in the emergency department from 1 January, 2000 through 31 December, 2006, were eligible for this study. These patients were identified from an administrative data file, providing information about age, gender, date and hour of admission, vital status at discharge and International Classification of Diseases version 10 (ICD-10) [START_REF]ICD-10 : international statistical classification of diseases and related health problems[END_REF] codes for hospital diagnoses. A matching procedure searched the biochemistry laboratory file for the first result within 24 h after admission of five blood tests (sodium, potassium, glucose, urea nitrogen and creatinine).

The administrative and the biochemistry data files have been declared to the Commission Nationale Informatique et Liberte ´. After the matching procedure performed on these files, all records were anonymised.

The study population finally included all patients with a match for the five tests and did not include other patients for whom at least one of these tests was not available within 24 h after admission.

Hydration status measurements

Plasma tonicity, defined as the effective osmolality, equals the sum of the concentrations of the solutes which determine the transcellular distribution of water. Plasma tonicity (mOsm/l) was estimated by calculation as glucose (mMol/l) plus twice the sum of sodium (mMol/l) and potassium (mMol/l) [START_REF] Stookey | High prevalence of plasma hypertonicity among community-dwelling older adults: results from NHANES III[END_REF]. Normal range was defined as 275-300 mOsm/ l. We hypothesized that a tonicity value below 275 mOsm/l reflects an excess of body water, either absolute or relative to total body sodium, and that a tonicity value over 300 mOsm/ l reflects absolute body water deficit.

BUNC ratio was calculated as the ratio between blood urea nitrogen (BUN) (mMol/l) and creatinine (C) (mMol/l). We hypothesized that a BUNC ratio\=100 reflects normal systemic perfusion whereas a BUNC ratio [100 reflects reduced systemic perfusion [START_REF] Aronson | Elevated blood urea nitrogen level as a predictor of mortality in patients admitted for decompensated heart failure[END_REF].

Temperature measurements

Daily minimum temperatures measured in the meteorological station Paris-14 e `me Parc Montsouris, were obtained from the website of the European Climate Assessment & Dataset (ECA&D) project [START_REF]Climate Assessment & Dataset (ECA&D) project[END_REF][START_REF] Klein Tank | Daily dataset of 20th-century surface air temperature and precipitation series for the European climate assessment[END_REF].

Comorbidity variable

The Charlson index (CI) takes into account 19 comorbid conditions, each with an associated weight ranging from 1 to 6 [START_REF] Charlson | Validation of a combined comorbidity index[END_REF][START_REF] Charlson | A new method of classifying prognostic comorbidity in longitudinal studies: development and validation[END_REF]. The relative risk of death from an increase of one in the comorbidity score has been estimated approximately equal to that from an additional decade of age [START_REF] Charlson | Validation of a combined comorbidity index[END_REF][START_REF] Charlson | A new method of classifying prognostic comorbidity in longitudinal studies: development and validation[END_REF]. Charlson comorbidity scores based on three digits ICD-10 administrative data for hospital diagnoses were calculated with the STATA Ò Charlson procedure adapted by V. Stagg from a SAS Ò procedure [START_REF] Quan | Coding algorithms for defining comorbidities in ICD-9-CM and ICD-10 administrative data[END_REF].

Statistical analysis

Three distinct time periods were compared: the 3 years prior to heat wave year (2000 to 2002) used as the reference period, the heat wave year (2003) and the three next years (2004)(2005)(2006), in which the ''National heat wave response plan'' was operational.

Study and excluded patients characteristics were compared, in each time period separately, with the Student t test (unequal variances in independent samples) for continuous variables and the Chi-square test for categorical variables. Patients characteristics were compared between groups defined by tonicity thresholds (3 groups: \275, 275-300, and [300 mOsm/l) and BUNC ratio threshold (2 groups: \=100 and [100) with the Student t test (unequal variances in independent samples) or a one way analysis of variance, respectively, for continuous variables, and the Chi-square test for categorical variables. Bartlett test checked equality of variances among groups in one way analyses of variance. If this test was significant, p values comparing groups were alternately estimated by the Kruskall-Wallis non parametric test.

Logistic models estimated adjusted risks for each hydration disorder index separately (tonicity \ 275, tonicity [ 300 and BUNC ratio [ 100) as odds-ratios and 95% confidence intervals in the periods 2003 and 2004-2006 with reference to 2000-2002. For high and low tonicity indexes the reference was the group of patients in the range 275-300 mOsm/L.

In each time period separately, hazard ratios with 95% confidence intervals for inhospital mortality associated with any index of hydration disorder were estimated simultaneously in a Cox proportional hazard model and compared between time periods. The time variable was defined as days from admission to death or discharge, whichever came first. The proportional-hazard assumption was checked by analyzing Schoenfeld residuals and Kaplan-Meier survival graphs.

To take into account the correlations between variables measured repeatedly in patients admitted several times, confidence intervals of odds-ratios and hazard ratios were based on the robust estimator of variance with the subject as clustering unit. All models were adjusted for age, gender, Charlson comorbidity index, and mean of the lowest temperature during the 3 days prior to admission. The choice of combining temperatures over the 3 days prior to admission was empirical in an intend to fit the recommendations of the 2004 National heat wave response plan which recommends Meteo France to alert INVS when minimal or maximal temperatures reach the alert threshold over 3 consecutive days. Additional analyses conducted with the mean of minimal, maximal and mean daily temperatures, over 3 days showed similar results (results not shown).

Analyses were conducted in all patients then stratified for patients admitted in June-August and September-May.

All tests were two-sided with a significance level of 5%. All analyses were performed with STATA Ò Statistical Software: Release 8.2 (Stata Corporation, College Station, TX).

Results

Study population

Table 1 shows characteristics of the 23,022 study patients and excluded 3,490 patients for whom we could not establish hydration status within 24 h after admission. In all periods, study patients had higher mean age, higher mean stay duration, higher Charlson comorbidity scores, and a higher proportion was admitted in intensive care unit, than non participants. Proportion of men was lower in excluded patients admitted in 2003 than in study patients admitted in 2003 and study or excluded patients admitted before or after 2003. In 2003 a higher proportion of excluded than study patients had been admitted in autumn.

Characteristics of patients associated with hydration status

Table 2 shows characteristics of patients associated with hydration status on admission. Proportion of men was lower in patients with tonicity \275 mOsm/l or BUNC ratio [100 than in others. Compared to others, patients with any hydration disorder had higher mean stay duration, a higher mean value for Charlson comorbidity index, a higher proportion with stay in intensive care unit, and a higher inhospital mortality rate. A higher proportion of patients with tonicity \ 275 mOsm/l were admitted during hot periods whereas tendency was inverse for patients with BUNC ratio [ 100 or tonicity [ 300 mOsm/l. In hospital mortality rate was similar in 2003 and in 2000-2002 and further decreased in 2004-2006. Figure 2 shows the same features limited to the subgroup of patients admitted between June 1 and August 31, the period during which level 1 of the ''National heatwave response plan'' has been activated every year since 2004. In this subgroup tendency was similar than in the whole study population except for a marked peak for inhospital mortality rate and a higher proportion of patients with tonicity [ 300 mOsm/l or \275 mOsm/l during the 2003 summer compared to other periods.

Hydration status profile over periods

Hydration status over periods adjusted for covariates

Inhospital mortality associated with hydration disorders

Table 4 shows inhospital mortality in 2000-2002, 2003 and 2004-2006 according to hydration status. Mortality risk associated with plasma tonicity\275 or[300 mOsm/l did not vary significantly between these periods, whereas mortality risk associated with BUNC ratio higher than 100, was lower in 2003 (HR 1.27; 95% CI: 1.01-1.60) than in other periods.

Discussion

In this study we have identified significant shifts in intracellular hydration profiles of patients through and after the 2003 heat wave. These shifts observed in an elderly population are consistent with the impact from the incitation to drink regularly still water through level 1 of the National Heatwave Plan since 2004. Furthermore these results suggest that the effect of incitation have been maintained beyond the June to August periods of level 1 activation. The changes in plasma tonicity indexes, after taking into account potential confounding effects, suggest that since 2004 mean body water content had increased resulting in a lower relative risk for intracellular dehydration and a higher risk for intracellular water inflation.

Our results also show a higher risk for inhospital mortality with the 3 indexes of hydration disorders in patients aged 70 years and over, with a wide spectrum of diseases, in the same range as previously reported with indexes based on plasma sodium concentration [START_REF] Klein | Lower serum sodium is associated with increased short-term mortality in hospitalized patients with worsening heart failure: results from the Outcomes of a Prospective Trial of Intravenous Milrinone for 2003 French heat wave and hydration status of the elderly 523 Exacerbations of Chronic Heart Failure (OPTIME-CHF) study[END_REF][START_REF] Tierney | The prognosis of hyponatremia at hospital admission[END_REF][START_REF] Brunsvig | Hyponatremi. En retrospektiv studie med henblikk pa forekomst, arsak og mortalitet[END_REF] and BUN In patients admitted between June and August, as only the risk for tonicity [300 mOsm/l has increased in 2003 with reference to 2000-2002, intracellular dehydration may have been, among hydration disorders, the main contributor to the excess in mortality observed during the heat wave.

During the 2003 heat wave, records of medical causes of death suggested a 20-fold increase for causes directly related to the heat wave (heatstroke, hyperthermia and dehydration), and a major impact of circulatory, respiratory and nervous systems diseases as well as ill-defined morbid conditions [START_REF] Fouillet | Excess mortality related to the August 2003 heat wave in France[END_REF]. However, accuracy of these declarative data about mortality causes may be questioned and the harvesting hypothesis suggests that death may have favored people that were at any rate doomed to die soon, during the following winter [START_REF] Valleron | Epidemiologie et canicules : analyses de la vague de chaleur 2003 en France[END_REF]. A cohort study carried out in Ilede-France in 31603 dependent subjects C60 years of age, has identified dependency as a risk factor associated with mortality during the 2003 heat wave [START_REF] Belmin | Level of dependency: a simple marker associated with mortality during the 2003 heat wave among French dependent elderly people living in the community or in institutions[END_REF]. Conversely a retrospective observational study conducted in the nursing homes of the Assistance Publique-Ho ˆpitaux de Paris has reported higher mortality rates in the less frail patients suggesting beneficial effect of medical care focused on more fragile patients [START_REF] Holstein | Were less disabled patients the most affected by 2003 heat wave in nursing homes in Paris, France?[END_REF]. A questionnaire-based study identified risk factors for death among 345 patients admitted for heatstroke, to 80 intensive care units in France during the 2003 heatwave [START_REF] Misset | Mortality of patients with heatstroke admitted to intensive care units during the 2003 heat wave in France: a national multiple-center riskfactor study[END_REF]. Risk factors included a higher temperature, pre-existing cardiac disease, diuretic use, and serum creatinine over 120 lmol/l. Davido et al. [START_REF] Davido | Risk factors for heat related death during the August 2003 heat wave in Paris, France, in patients evaluated at the emergency department of the Hopital Europeen Georges Pompidou[END_REF] have identified risk factors for heat related death among the 841 patients evaluated in the emergency department of a teaching hospital in Paris during the 2003 heat wave. Risk factors included a greater degree of dependent living, more severe clinical condition an admission, higher values of blood glucose, troponin and white blood cell count, lower values of serum protein and prothrombin levels, pre-existing ischaemic cardiomyopathy, pneumonia as associated infection, and previous psychotropic treatment. Diuretics, angiotensin converting enzyme inhibitors and psychotropic treatments may contribute to hydration disorders and mortality during heat waves. A study from the French Network of Pharmacovigilance Database has reported no significant excess in records for serious adverse drug reactions during the 2003 summer with reference to 2002 [START_REF] Michenot | Adverse drug reactions in patients older than 70 years during the heat wave occurred in France in summer 2003: a study from the French PharmacoVigilance Database[END_REF]. Indeed common side effects of widely prescribed drugs are seldomly declared and may then be scarce in this database.

During one heat wave in July 2006, the whole National Heatwave Plan has been activated effectively and INSEE, the national polling organization, reported ''only'' 2000 additional deaths whereas 6400 had been expected [START_REF] Fouillet | Estimation de la surmortalite ´observe ´e et attendue au cours de la vague de chaleur du mois de Juillet 2006[END_REF].

The specific effect of hydration shifts on inhospital mortality cannot be precisely evaluated from our data. However, overall inhospital mortality has significantly decreased in 2004-2006, with reference to 2000-2002, as well as the relative risk for intracellular dehydration. Moreover, inhospital mortality risks associated with the intracellular dehydration index were higher than the risk associated with intracellular water inflation, in all study periods. As in 2004-2006, with reference to 2000-2002, the proportion of patients with intracellular water inflation has significantly increased and the proportion of patients with intracellular dehydratation has significantly decreased, it seems reasonable to hypothesize that the shift in intracellular hydration disorders was beneficial on mortality.

Our study has several potential limitations. Independently of extracellular water content, the BUNC ratio may be influenced by several morbid processes unrelated to extracellular water content, such as cardiovascular failure, digestive bleeding, and infection which are also potential risks factors for mortality. Finding a lower risk for BUNC [ 100, and a lower corresponding mortality rate in 2003 compared to 2000-2002 was unexpected and confirms low specificity of BUNC as a marker for extracellular dehydration. The Charlson index was not originally designed for the prediction of hydration disorders risks. However, it is a valid and reliable method for assessing comorbidity in a variety of clinical research situations [START_REF] De Groot | How to measure comorbidity. A critical review of available methods[END_REF]. It predicted significant increase in the risks for the 3 hydration disorders and mortality in all the multivariate analyses we have conducted (data not shown). Data about hydration status were not available in about 15% of our eligible population. Patients who were not included in our analyses were younger and with a lower Charlson comorbidity index, thus they may represent a group at lower risk for hydration disorders. Excluding these patients may have resulted in overestimating the magnitude of risks for hydration disorders and mortality in all study periods. However, the proportion of excluded patients was similar in each period and analyses were adjusted for age and Charlson index. Therefore, it seems reasonable to expect minimal effect of selection bias on estimates. The proportion of male patients was similar among periods in study patients, and was similar in study and excluded patients except for the 2003 subgroup (35.9 vs. 29.3%). However, we did not find significant difference among periods for the odds-ratios between gender and hydration status, and no significant difference among periods in hazard ratios for gender related survival. Therefore, it seems unlikely that the gender difference in excluded patients in the 2003 subgroup may impact substantially our results. Referral patterns into this tertiary centre could have varied over time resulting in a lack of a defined denominator population. Also, many diseases are seasonal which would create biases when using all admitted patients as the denominator. To limit the effect of potential biases in the denominator population we restrained the population study to patients admitted through the emergency department, most of whom live in the neighbourhood, adjusted analyses for the Charlson index and atmospheric conditions and performed and performed subgroup analyses for periods with distinct climatic conditions.

Since the 2003 heat wave, significant shifts in prevalence of intracellular hydration disorders indicate behavioral changes with positive impact on hydration status in elderly patients admitted to tertiary a center. These behavioral changes instigated by the deep psychological impact of the 2003 heat wave are in accordance with the repercussions expected from a prevention policy activated yearly between June and August.

Figure 1

 1 Figure 1 shows unadjusted prevalence of hydration disorders and inhospital mortality rates in the periods 2000-2002, 2003, and 2004-2006. With reference to 2000-2002, the proportion of patients with plasma tonicity \275 mOsm/l has increased in 2003, and further increased in 2004-2006, whereas the proportion of patients with plasma tonicity [300 mOsm/l has increased in 2003, and further decreased in 2004-2006 to a level lower than in 2000-2002. The proportion of patients with BUNC ratio [100 has decreased in 2003 and further decreased in 2004-2006.In hospital mortality rate was similar in 2003 and in 2000-2002 and further decreased in 2004-2006. Figure2shows the same features limited to the subgroup of patients admitted between June 1 and August 31, the period during which level 1 of the ''National heatwave response plan'' has been activated every year since 2004. In this subgroup tendency was similar than in the whole study population except for a marked peak for inhospital mortality rate and a higher proportion of patients with tonicity [ 300 mOsm/l or \275 mOsm/l during the 2003 summer compared to other periods.

Fig. 1 Fig. 2

 12 Fig. 1 Prevalence of hydration disorders on admission, and inhospital mortality. P values are for global comparison between the 3 periods

Table 3

 3

	shows odds-ratios of hydration disorders in 2003
	and 2004-2006 with reference to 2000-2002, adjusted for

Table 1

 1 Characteristics of patients

	N	2000-2002			2003			2004-2006
		Study	Patients	P value Study	Patients	P value Study	Patients	P value
		patients	excluded		patients	excluded		patients	excluded
		9,484	1,409		3,115	458		10,423	1,623
	Men (%)	35.3	33.4	0.15	35.9	29.3	0.006 35.8	34.3	0.24
	Age (years)	83.1 ± 7.3 82.0 ± 7.3	\0.0001 83.1 ± 7.4 82.2 ± 7.4	0.009 83.3 ± 7.3 82.5 ± 7.4	\0.0001
	Stay duration (d)*	13.5 ± 15.2 11.1 ± 11.3 \0.0001 12.2 ± 14.7 10.7 ± 13.9	0.04	11.0 ± 13.0 9.0 ± 11.4 \0.0001
	Death during stay (%) 10.9	11.6	0.97	10.8	8.7	0.19	9.0	7.5	0.04
	Mean day lowest T°( 9.0 ± 4.9	8.9 ± 4.9	0.41	9.4 ± 6.8	9.0 ± 6.0	0.26	9.1 ± 5.8	8.7 ± 5.7	0.01
	°C)**
	Season of stay (%)
	Winter	27.8	26.7	0.48	27.3	16.8	\0.0001 26.6	30.3	0.009
	Spring	24.5	26.3		23.4	17.0		24.9	24.7
	Summer	22.8	22.9		25.9	20.5		23.2	20.8
	Automn	24.9	24.1		23.4	45.6		25.4	24.2
	Charlson comorbidity	1.24 ± 1.5 0.99 ± 1.4 \0.0001 1.26 ± 1.6 1.04 ± 1.6	0.009	1.36 ± 1.6 1.02 ± 1.5 \0.0001
	index
	Index = 0	37.0	49.2	\0.0001 38.0	52.6	\0.0001 35.7	49.1	\0.0001
	Index = 1	34.2	27.4		33.0	24.7		31.7	26.6
	Index C2	28.9	23.4		29.1	22.7		32.6	24.3
	Intensive care stay	7.1	4.1	\0.0001 6.4	3.9	0.04	7.0	2.7	\0.0001
	(%)
	Results are given as mean value ± standard deviation or %
	* In survivors at discharge

** Mean of lowest temperature in the 3 days prior to admission, recorded at the weather station Paris-Montsouris concentration

[START_REF] Aronson | Elevated blood urea nitrogen level as a predictor of mortality in patients admitted for decompensated heart failure[END_REF][START_REF] Hartz | BUN as a risk factor for mortality after coronary artery bypass grafting[END_REF][START_REF] Kirtane | Serum blood urea nitrogen as an independent marker of subsequent mortality among patients with acute coronary syndromes and normal to mildly reduced glomerular filtration rates[END_REF]

mainly in hospital patients with heart failure or coronary disease.

Table 2

 2 Patients characteristics according to plasma tonicity and blood urea nitrogen/creatinine ratio on admission

	N	Tonicity (mOsm/l)			BUNC*** ratio
		\275	275-300	[300	P value	\=100	[100	P value
		1,945	19,208	1,869		15,926	7,096
	Men (%)	27.5	36.0	39.4	\0.0001	39.1	27.6	\0.0001
	Age (years)	83.4 ± 7.1	83.1 ± 7.3	84.3 ± 7.5	\0.0001	82.7 ± 7.2	84.4 ± 7.3	\0.0001
	Stay duration (d)*	13.1 ± 14.3	12.0 ± 14.2	13.6 ± 14.9	\0.0001	11.8 ± 14.0	13.1 ± 14.6	\0.0001
	Death during stay (%)	12.2	8.7	21.1	\0.0001	7.8	14.7	\0.0001
	Mean day lowest T°(°C)**	10.2 ± 5.8	9.0 ± 5.6	9.0 ± 6.0	\0.0001	9.2 ± 5.6	9.0 ± 5.6	0.01
	Season distribution (%)
	Winter	21.4	27.6	29.0	\0.0001	26.4	28.9	0.001
	Spring	26.1	24.5	23.7		24.7	24.1
	Summer	31.0	22.7	22.8		23.9	22.3
	Automn	21.5	25.3	24.6		25.0	24.7
	Period distribution (%)
	2000-2002	36.4	41.5	42.6	\0.0001	38.5	47.3	\0.0001
	2003	12.8	13.2	17.4		13.5	13.5
	2004-2006	50.8	45.2	40.0		48.0	39.2
	Charlson comorbidity index	1.50 ± 1.8	1.26 ± 1.6	1.47 ± 1.5	\0.0001	1.29 ± 1.6	1.32 ± 1.6	0.11
	Index = 0	36.5	37.7	24.6	\0.0001	36.9	35.7	0.20
	Index = 1	28.0	32.8	38.7		32.6	33.5
	Index C2	35.5	29.5	36.7		30.5	30.8
	Intensive care stay (%)	8.8	6.2	12.0	\0.0001	6.8	7.3	0.14

Table 3

 3 Multiple logistic regression. Adjusted odds-ratios for hydration disorders before and after the 2003 heat wave All models are adjusted for age, gender, mean day lowest temperature during the 3 days prior to admission. and Charlson comorbidity index Confidence intervals were based on the robust estimator with the subject as cluster * Reference is patients in the tonicity range 275-300 mOsm/l ** BUNC Blood urea nitrogen/creatinine

		All patients	Patients admitted between	Patients admitted between
			June and August	September and May
	Tonicity \ 275 mOsm/l*
	2000-2002	1 (Ref)	1 (Ref)	1 (Ref)
	2003	1.08 (0.92-1.27)	1.08 (0.81-1.45)	0.94 (0.77-1.14)
	2004-2006	1.26 (1.11-1.41)	1.23 (1.00-1.52)	1.24 (1.09-1.42)
	Tonicity [ 300 mOsm/l*
	2000-2002	1 (Ref)	1 (Ref)	1 (Ref)
	2003	1.28 (1.11-1.47)	1.43 (1.06-1.92)	1.13 (0.96-1.33)
	2004-2006	0.85 (0.76-0.95)	0.83 (0.65-1.05)	0.84 (0.74-0.95)
	BUNC** ratio [ 100
	2000-2002	1 (Ref)	1 (Ref)	1 (Ref)
	2003	0.81 (0.74-0.88)	0.72 (0.59-0.89)	0.82 (0.74-0.91)
	2004-2006	0.65 (0.61-0.70)	0.54 (0.47-0.62)	0.69 (0.64-0.74)
	Results are given as odds-ratio (95% confidence interval)

Table 4

 4 Cox proportional hazard model.

				Independent hazard ratios
	for in-hospital mortality according to patient characteristics, and
	hydration status
		2000-2002	2003	2004-2006	P value
	Plasma tonicity (mOsm/l)
	275-300 1 (ref)	1 (ref)	1 (ref)
	\275	1.40	1.06	1.26	0.50
		(1.12-1.74)	(0.70-1.61)	(1.03-1.53)
	[300	1.96	1.76	1.95	0.80
		(1.64-2.34)	(1.31-2.35)	(1.60-2.38)
	BUNC* ratio
	B100	1 (ref)	1 (ref)	1 (ref)
	[100	1.64	1.27	1.77	0.04
		(1.45-1.86)	(1.01-1.60)	(1.55-2.02)
	Results are given as hazard ratios (95% confidence interval)
	All models are adjusted for age, gender, mean day lowest temperature
	during the 3 days prior to admission and Charlson comorbidity index.
	All hydration status indexes are adjusted on each other
	Confidence intervals were based on the robust estimator with the
	subject as cluster
	* BUNC Blood urea nitrogen/creatinine
	Time variable in days (median [range])/total events: in 2000-2002: 10
	[1-481]/1021, in 2003: 8 [1-332]/335, in 2004-2006: 7[1-337]/938

A. Kettaneh et al.

French heat wave and hydration status of the elderly 519

A. Kettaneh et al.