Piotr Golinski

Agnieszka Waskiewicz

Halina Wisniewska

Irena Kiecana

Elzbieta Mielniczuk

Karolina Gromadzka

Marian Kostecki

Jan Bocianowski

Ewa Rymaniak

Reaction of winter wheat (Triticum aestivum L.) cultivars to infection with Fusarium spp. -mycotoxin contamination in grain and chaff

Keywords: Fusarium culmorum, F. graminearum, F. avenaceum, Fusarium head blight, wheat, zearalenone, deoxynivalenol, nivalenol, moniliformin, HPLC

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Introduction

Fusarium species are common necrotrophic pathogens of small grain cereals in most European countries. Three species F. culmorum (W.G. Smith) Sacc., F. avenaceum (Corda ex Fries) Sacc., and F. graminearum Schwabe, have been considered as the most important in Poland and other central European countries, while several other species as less important [START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain in Europe[END_REF]. F. culmorum and F. graminearum have been shown to be frequent and most aggressive to cereals in countries of moderate and colder climates. FHB severity depends on several genetic, agronomic and climatic factors. The disease can result in Fusarium-damaged kernels (FDK), which are smaller, shriveled and discoloured (Kiecana et al. 2004. FHB can also cause economical losses because of reduced kernel weight and size, low grain quality, and accumulation of several mycotoxins (mostly deoxynivalenol -DON and zearalenone -ZEA) in infected kernels, chaff and rachises [START_REF] Jones | Quality parameters in small grains from Minnesota affected by Fusarium head blight[END_REF]. In some regions disease epidemics have been observed, with high yield losses particularly in wheat and other cereals. Observations of disease occurrence showed a lower susceptibility of selected cultivars and breeding lines to most fungal pathogens [START_REF] Mielniczuk | Susceptibility of oat genotypes to Fusarium crookwellense Burgess, Nelson and Toussoun infection and mycotoxin accumulation in kernels[END_REF][START_REF] Wisniewska | Scab response and deoxynivalenol accumulation in spring wheat kernels of different geographical origins following inoculation with Fusarium culmorum[END_REF]. Profiles of mycotoxins produced by the above mentioned two species (F. graminearum and F. culmorum) were found to be similar both under laboratory and field conditions, with DON, nivalenol (NIV) and ZEA being the most important [START_REF] Quarta | Assessment of trichothecene chemotypes of Fusarium culmorum occuring in Europe[END_REF]. The mycotoxin content in infected grains is also genotype-dependent [START_REF] Chelkowski | Effects of Fusarium culmorum head blight on mycotoxin accumulation and yield traits in barley doubled haploids[END_REF][START_REF] Wisniewska | Scab response and deoxynivalenol accumulation in spring wheat kernels of different geographical origins following inoculation with Fusarium culmorum[END_REF]).

The major Fusarium toxin found in the climatic conditions of Poland is zearalenone, detected in cereals during plant growth and vegetation, as a secondary metabolite of fungi of genus Fusarium [START_REF] Golinski | Fusarium head blight of common Polish winter wheat cultivars -comparison of effects Fusarium avenaceum and F. culmorum on yield components[END_REF]. The amounts of ZEA accumulated in cereals depend on many factors such as temperature, duration of Fusarium infection, substrate and strain of fungal species [START_REF] Bennett | Mycotoxins[END_REF]Klich 2003, Nuryono et al. 2005). This toxin is recognized as a phytoestrogen with reported cytotoxic and genotoxic activity. Toxic properties of ZEA are related to the metabolite binding to estrogen receptors and disrupting the endocrine regulation of the reproductive functions in females [START_REF] Zinedine | Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: An oestrogenic mycotoxin[END_REF]).

An important toxin of the group of trichothecenes is deoxynivalenol (DON), being the product of metabolism of such fungi as F. graminearum and F. culmorum. DON was detected in such cereals as wheat, oat, barley, rye, rice, corn, as well as cereal-based products, e.g. flour, bread or muesli. Accumulation of DON and its derivatives, including NIV, in spring wheat accessions was on average 3.1 µg g -1 (0.7-9.0 µg g -1 ) and it was lower than in winter wheat accessions, with an average of 6.7 µg g -1 (1.6-38.5 µg g -1 ) [START_REF] Goral | Characteristic of triticale, wheat and rye varieties on their resistance and tolerance to head infection by Fusarium spp[END_REF].

DON, similarly to other trichothecenes, has a significant effect on biochemical processes, blocking e.g. protein biosynthesis or elongation of the polypeptide chain. It is assumed that DON, being a highly toxic compound, does not exhibit carcinogenic properties. The most frequently observed DON toxicosis symptoms in animals include vomiting and body weight loss with successive numerous physiological changes in internal tissues [START_REF] Wisniewska | Components of resistance against Fusarium culmorum in spring wheat[END_REF][START_REF] Wisniewska | Scab response and deoxynivalenol accumulation in spring wheat kernels of different geographical origins following inoculation with Fusarium culmorum[END_REF]. Pigs were found to be the most susceptible animals to DON, despite the reported potential detoxication by the intestinal microflora. Intake of feed containing DON may also have a significant effect on reproduction of animals, causing in most cases fatal toxicoses.

Fusarium avenaceum is reported as a very potent producer of moniliformin (MON), a metabolite in the form of sodium or potassium salt of 3-hydroxycyclobut-3-ene-1,2-dione, is toxic to plant and animal species [START_REF] Kiecana | Scab response and moniliformin accumulation in kernels of oat genotypem inoculated with Fusarium avenaceum in Poland[END_REF][START_REF] Jestoi | Presence and concentrations of the Fusarium-related mycotoxins beauvericin, eniatins and moniliformin in Finnish grain samples[END_REF]). MON also is suspected to cause the Keshan disease, a human myocardial impairment reported in rural areas of China and South Africa (Pineda-Valdes and Bullerman 2000). Inhibition of pyruvate dehydrogenese, i.e. the blockage of entry of pyruvate into the Krebs's cycle, and a decrease in mitochondrial respiration are probably responsible for the acute toxicity of the compound [START_REF] Reams | Development of an L6 myoblast in vitro model of moniliformin toxicosis[END_REF].

The aim of this study was to compare susceptibility of winter wheat (Triticum aestivum L.) cultivars to FHB and accumulation of mycotoxins in kernels and chaff, in different climate conditions at two locations in Poland.

Materials and methods

Wheat samples

Material for the study comprised 10 winter wheat cultivars most common in East Europe, differing in their reaction to Fusarium pathogens (6 quality cultivars -group A: cv. Tonacja, Zyta, Sukces, Finezja, Ludwig, Rywalka; 3 bread cultivars -group B: cv. Mewa, Nutka, Soraja; 1 forage cultivar -group C: cv. Symfonia) (

Inoculation procedure

At mid-anthesis (zadoks scale 65) 30 wheat heads of each replication were inoculated individually (by brushing) with the conidial suspension (100 000 spores ml -1 ) isolates of three Fusarium species: F. graminearum (KF 2870), F. culmorum (KF 846) and F. avenaceum (NN 1337), with plot size 1m × 1m. During two days after inoculation micro-irrigation was used.

Non-inoculated plots of the same genotypes were used as a control. Two weeks after inoculation, disease progress was visually evaluated as the disease score of head (Fi) on a scale of 0-5, where 0 means no infection and 5 denotes the highest infection of the head.

Inoculated and control heads were harvested and threshed manually, then kernels were separated into two fractions: Fusarium-damaged kernels (FDK) and in healthy looking kernels (HLK). The percentage of Fusarium-damaged kernels (%FDK) was calculated as a percentage by weight.

Chemicals and reagents

Mycotoxin standards (ZEA, DON, NIV, MON) were supplied by Sigma-Aldrich (Steinheim, Germany). Acetonitrile and methanol (HPLC grade) were also purchased from Sigma-Aldrich (Steinheim, Germany). Potassium chloride, di-potassium phosphate, n-hexane and t-butyl-ammonium hydroxide were purchased from POCh (Gliwice, Poland) and water (HPLC grade) was from our own Millipore water purification system.

Mycotoxin analysis

Mycotoxins (ZEA, DON, NIV and MON) were analyzed as well in the two fractions of wheat kernels described above -FDK and HLK, as in the fraction of chaff.

Deoxynivalenol, nivalenol and moniliformin analysis

Finely ground material was extracted with acetonitrile-methanol-water (16:3:1, v/v/v) using 5 ml of solvent per 1 g of sample. Extracts were defatted with n-hexane (3×50 ml) and then concentrated. Half of the extract was used for MON analysis, while the remaining part was used for DON and NIV determination. The extract of MON was purified on a Florisil column according to the method described by [START_REF] Kostecki | The effects of cereal substrate and temperature on production of beauvericin, moniliformin and fusaproliferin by Fusarium subglutinans ITEM -1434[END_REF]. To analyze the trichothecenes (DON, NIV), extracts were purified by filtration on a column according to the method described by [START_REF] Tomczak | Deoxynivalenol, nivalenol and moniliformin in wheat samples with head blight (scab) symptoms in Poland (1998-2000)[END_REF]. DON, NIV and MON were quantified by HPLC using a Waters 501 apparatus (Waters Division of Millipore, Milford, MA, USA) with a C-18 Nova Pak column (3.9 × 300 mm) and a Waters 486 UV detector (λ max = 224 nm for DON and NIV, while λ max = 229 nm for MON).

DON and NIV were eluted from the column with a 25% water solution of methanol (flow rate 0.7 ml min -1 ) with retention times of 11.72 and 7.46 min, respectively. Regarding MON, the acetonitrile:water solvent (15:85, v/v) buffered with 10 ml of 0.1 M K 2 HPO 4 in 40% t-butylammonium hydroxide in 1 L of solvent (Sharman et al. 1991) was used as a mobile phase (flow rate 0.6 ml min. -1 ). Retention time of MON was 11.5 min. Detection limits for DON, NIV and MON were 0.01 µg g -1 . Positive results (on the basis of retention time) were confirmed by HPLC analysis and by comparison with the relevant calibration curve (correlation coefficients for NIV, DON and MON were 0.9994, 0.9997 and 0.9990, respectively). Recoveries for NIV, DON and MON: 75, 87 and 90%, respectively, were estimated in triplicates by extracting mycotoxins from blank samples spiked with 0.01-0.10 µg g -1 of the compounds. The relative standard deviation (R.S.D.) value was below 5% for DON and NIV, and below 7% for MON.

Zearalenone analysis

ZEA content was determined by HPLC with a fluorescence detector according to the method described by [START_REF] Visconti | Determination of zearalenone in corn by means of immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detector[END_REF]. The chromatographic system was a Waters 2695 HPLC, with a Waters 2475 Multi λ Fluorescence Detector and a Waters 2996 Array Detector. The excitation and emission wavelengths were 274 and 440 nm, respectively. The reverse-phase column was a C-18 Nova Pak column (3.9 × 150 mm), while the mobile phase was acetonitrile-water-methanol (46:46:8, v/v/v), at a flow rate of 0.5 ml min -1 . Quantification of ZEA was performed by measuring the peak areas at the ZEA retention time according to the relevant calibration curve (correlation coefficient r=0.9998). The limit of zearalenone detection was 0.003 µg g -1 . The recovery of the compound was measured in triplicates by extracting ZEA from blank samples spiked with 0.005-0.10 µg g -1 of compound. The results of experiments confirmed literatures data and contained in the range from 97 to 99%. The relative standard deviation (R.S.D.) value was below 1%. In order to confirm the presence of ZEA the Photodiode Array Detector was used.

Statistical analysis

The two-way analysis of variance was carried out to determine the effects of locations, cultivars and locations × cultivars interactions on the variability of disease score of head (Fi), the percentage of Fusarium-damaged kernels (%FDK), ZEA and DON concentrations in FDK as well as in HLK fractions and in chaff (ZEA in FDK, ZEA in HLK, ZEA in chaff, DON in FDK, DON in HLK, DON in chaff), HLK kernels per spike, FDK kernels per spike, HLK weight per spike, FDK weight per spike and thousand kernels weight (TKW).

The least significant differences (LSD) for each trait were calculated, and on these basis homogeneous groups for the analyzed traits were determined. The relationship between ZEA and DON for different traits (mycotoxin concentration in FDK and HLK fraction and in chaff) as well as Fi, HLK kernels per spike, FDK kernels per spike, HLK weight per spike, FDK weight per spike and TKW were estimated on the basis of correlation coefficients.

Results and discussion

Susceptibility of tested wheat cultivars to the Fusarium spp. and mycotoxin biosynthesis depends on pathogen virulence and weather conditions.

Analysis of variance indicated that the main effects of locations (Poznan and Lublin area) were significant for all the traits in the study (Tables 234), except for FDK kernels per spike for F. culmorum (Table 2), FDK weight per spike for F. graminearum (Table 3) and moniliformin concentration in the HLK fraction (MON in HLK) (Table 4); the main effects of cultivars were significant for all the traits, except for TKW for F. culmorum (Table 2). The locations × cultivars (L×C) interactions were not significant only for number of as well HLK and FDK kernels per spike, FDK kernels per spike and TKW for F. culmorum (Table 2), and for MON in FDK number of HLK and FDK kernels per spike and HLK weight per spike for F. avenaceum (Table 4).

Very high variation was found in concentrations of mycotoxins (ZEA, DON, MON) in fractions (HLK and FDK ) and in chaff for individual cultivars in both locations (Tables 56789). Moreover, in the most cases significantly higher concentrations of toxins were found in wheat of Lublin area origin than area of Poznan (p<0.05) (Tables 56789), what confirms earlier findings [START_REF] Golinski | Scab response of selected winter wheat cultivars after inoculation with Fusarium avenaceum (Fr.) sacc[END_REF][START_REF] Golinski | Fusarium head blight of common Polish winter wheat cultivars -comparison of effects Fusarium avenaceum and F. culmorum on yield components[END_REF].

The highest Fusarium infection rate as well as mycotoxin biosynthesis level were observed in the Lublin location with percentage of the FDK fraction in the range from 8.1 to 81.6. In this region the ZEA concentration (µg g -1 ) after inoculation with F. culmorum and F. graminearum ranged from 0.02 to 0.48 and 0.32-1.04, respectively (Tables 5 and6). In the Poznan area the toxin concentrations were considerably lower, ranging from 0.01 to 0.10 and from 0.03 to 0.13 for both isolates, respectively (Tables 5 and6).

The differences in climate conditions are in our opinion the reason of differences in ZEA concentrations in both locations (Tables 5 and6). Inoculation in Cerekwica was performed May 30 and June 5 for all Fusarium pathogens isolates with few days of average temperature increase with peaks of the highest temperature May 31, June 6 and 10. At the same time rainfalls level was lower than it was before inoculation. In contrast, inoculation in Lublin area was few days later (June 10) than in Cerekwica which is the reason because of differences in climate and vegetation (cereals flowering). After inoculation -in Lublin areathere were a few days with lower temperature and higher air relative humidity than previously and during inoculation (weather reports for Lublin and Cerekwica). Such a situation explains that lower temperature in Lublin area during disease development induced ZEA contamination of kernels. Such observation was also reported by others [START_REF] Klix | Effects of cultivars, agronomic practices, geographic location, and meteorological conditions on the composition of selected Fusarium species on wheat heads[END_REF].

NIV was reported only in trace amounts in the FDK fraction of two cultivars after inoculation with F. culmorum in the Lublin region. The concentration of DON was significantly higher when compared to ZEA and NIV levels. In the Poznan region contamination with DON did not exceed 80.8 µg g -1 in FDK fraction (Tables 7 and8). It was also noticed that the infection was higher after inoculation with F. culmorum both in the Poznan and Lublin area, where the DON concentration in FDK fraction was in the range from 21.6 up to 114.6 µg g -1 (Table 7).

It is worth noting that in several samples especially in Lublin area, but also in Cerekwica, the level of DON was higher in kernels of FDK fraction than in chaff (Table 8).

We assume that dry summer in both locations was responsible for the fungus penetration to inner parts of kernels (where humidity and water content is higher), followed by the disease development and higher yield of the mycotoxins biosynthesis and/or their transport with nutrients into the kernels centre, what effected in higher the metabolites concentration level than it was in outer parts -in chaff temperature had to be higher (sun operation) in a contrary to low moisture. To some extend this concept is similar not only to the results indicating penetration of DON into kernels of cereals with FHB symptoms and pathogen development but also in asparagus spears without fusariosis symptoms but more often MON and fumonisins positive than diseased ones. This phenomenon was elucidated as follows: mycotoxins accumulated in overgrown with pathogenic fungus and/or fungi crowns are transported with nutrients to developing spears. In fact the mycotoxins presence is definitely related to abundance of pathogen(s) in crown and not in situ of chemical analysis (spears) (Waskiewicz at el. 2010). It is necessary to point out the differences in the structure of kernels and spears, differences resulting in only limited penetration of mycotoxins in hard tissue.

Moreover, a significant positive correlation for F. culmorum were observed between FDK weight per spike and FDK kernels per spike in both locations as well as between HLK weight per spike and TKW in Lublin (Table 9). The negative significant correlation was observed only between HLK weight per spike and Fi (Table 9). As mentioned above metabolite penetration into the kernels might be limited when humidity is high, mycotoxins accumulate -in such situation -mostly on the kernels surface. The literature and our earlier findings [START_REF] Young | Appearance of fungus, ergosterol and Fusarium mycotoxins in the husk, axial stem, and stalk after inoculation of field corn[END_REF]Miller 1985, Perkowski et al. 1990) indicate that usually the outer parts chaff, husk are higher in the mycotoxin contamination levels, which is an interesting finding with practical implications. During kernel threshing, hulling, dry and wet cleaning (washing), grinding and milling significantly reduce the concentration levels of the mycotoxins not only in kernels but also in such final products as flour, groats and flakes. In turn, for F. graminearum a significant relationship was observed between FDK weight and kernels per spike and HLK weight and kernels per spike in both locations as well as between DON in HLK and ZEA in HLK, HLK weight per spike and TKW in Poznan (Table 10). In Lublin, HLK kernels per spike was correlated with FDK kernels per spike and FDK kernels per spike (Table 10). [START_REF] Edwards | Fusarium mycotoxin content of UK organic and conventional wheat[END_REF] observed that the relationship between DON and ZEA concentrations was not stable between years. The ZEA distribution was a similar pattern to DON which is to be expected as they are produced by the same Fusarium species.

In studies conducted in Austria, kernel samples of durum wheat predominantly infected by F. graminearum and with a lower incidence of F. culmorum contained high amounts of DON (up to 8.2 µg g -1 ) together with low levels of ZEA (up to 0.33 µg g -1 ) [START_REF] Adler | Occurrence of moniliformin, deoxynivalenol, and zearalenone in durum wheat (Triticum durum Desf.)[END_REF], while in other studies DON was found in all tested wheat samples, ranging from 0.2 to 4.1 µg g -1 [START_REF] Berthiller | Occurrence of deoxynivalenol and 3-β-D-glucoside in wheat and maize[END_REF]). In Germany DON, together with ZEA and NIV, was found in 87% of wheat samples at very high levels, mean being 3.96 µg g -1 and the highest 43.4 µg g -1 (Lepschy von [START_REF] Lepschy Von Gleissenthal | A survey on the occurrence of Fusarium mycotoxins in Bavarian cereals from the 1987 harvest[END_REF]). The occurrence of DON (up to 1.18 µg g - 1 ) was reported in Finland [START_REF] Hietaniemi | Contents of Fusarium toxins in Finnish and imported grains and feeds[END_REF] and in the Netherlands (up to 0.5 µg g -1 ) together with ZEA (De [START_REF] Nijis | Fungal infection and presence of deoxynivalenol and zearalenone in cereals grown in The Netherlands[END_REF]. In France, [START_REF] Bakan | Toxigenic potential of Fusarium culmorum strains isolated from French wheat[END_REF] analyzed strains of F. culmorum isolated from wheat grain, which produced ZEA (1700 µg g -1 ), DON (up to 51.9 µg g -1 ) and NIV (up to 12.0 µg g -1 ), whereas in Italy isolates of F. culmorum produced DON up to 1700 µg g -1 [START_REF] Quarta | Assessment of trichothecene chemotypes of Fusarium culmorum occuring in Europe[END_REF]. In Poland high amounts of DON (2.30- 15.32 µg g -1 ) related with a high incidence of F. culmorum and F. graminearum were observed, whereas DON concentration was in most samples higher than those of NIV (0.01-0.74 µg g -1 ) [START_REF] Chelkowski | Identification of toxigenic Fusarium species in wheat ears using PCR assay and their mycotoxins in kernels[END_REF]. Analysis of mycotoxins of six winter wheat genotypes inoculated with F. graminearum revealed DON content ranging from 0.89 up to 1.53 µg g -1 and a very low level of NIV [START_REF] Ittu | Deoxynivalenol accumulation and other scab symptoms in six Romanian wheat genotypes inoculated with Fusarium graminearum[END_REF]. In other studies the highest contamination with DON of inoculated wheat was observed in Polish cultivars, at 38.5 and 35.7 µg g -1 for cv.

Olimpia and Henika, respectively [START_REF] Wisniewska | Scab response and deoxynivalenol accumulation in spring wheat kernels of different geographical origins following inoculation with Fusarium culmorum[END_REF]).

In Cerekwica the levels of MON accumulation (µg g -1 ) in HLK were between not detectable level (ND) and 0.13, while in the FDK fraction from 0.14 and 1.73, and in chaff at levels between ND and 1.07 (Table 11). F. avenaceum infection rate ranged between 7 and 35% in the samples where the toxin was detected. In the Lublin region the MON content (µg g -1 ) in the HLK fraction ranged between ND and 0.11, in FDK in the range of ND-2.51, while in chaff between 0.03 (cv. Rywalka) and 0.48 (cv. Sukces) (Table 11). In this area the infection rate with F. avenaceum, in the analyzed cultivars varied, from 3 to 41%. For F. avenaceum we observed positive correlation between number and weight of HLK and FDK kernels per spike, FDK kernels and weight per spike, MON in FDK and MON in HLK in both locations (Table 12). Additionally, MON in chaff was correlated with MON in HLK and MON in FDK as well as HLK weight per spike with TKW in Lublin (Table 12).

The achieved results indicate that MON content depends on the susceptibility of particular genotype to infection with F. avenaceum, what is confirmed as well by studies in Norway, where moniliformin was found in 76% tested wheat samples (with the highest toxin concentration 0.95 µg g -1 ) [START_REF] Uhlig | Moniliformin in Norwegian grain[END_REF] as studies conducted in Finland by [START_REF] Jestoi | Presence and concentrations of the Fusarium-related mycotoxins beauvericin, eniatins and moniliformin in Finnish grain samples[END_REF], where significant correlation between F. avenaceum contamination level and the level of MON (0.03-0.10 µg g -1 ) was observed. In Poland, wheat samples with F. avenaceum inoculum contained the toxin in the range from 0.01 to 0.35 µg g -1 [START_REF] Chelkowski | Identification of toxigenic Fusarium species in wheat ears using PCR assay and their mycotoxins in kernels[END_REF] and 1.72 µg g -1 [START_REF] Tomczak | Deoxynivalenol, nivalenol and moniliformin in wheat samples with head blight (scab) symptoms in Poland (1998-2000)[END_REF].

In cereals four types of plant resistance mechanisms to fusariosis have been suggested: resistance of the host plant to infection with pathogenic fungi (mechanism I); resistance of the host to pathogen spread in the tissue (II); ability of the plant to degrade mycotoxins (III) and tolerance of the host tissue to a high concentration of toxic metabolites [START_REF] Kiecana | Scab response and moniliformin accumulation in kernels of oat genotypem inoculated with Fusarium avenaceum in Poland[END_REF].

In the district of Lublin, with suggestion to the other parts of Poland two cultivars Sukces and Tonacja are recommended for general purpose kernels while cv. Symfonia as a feedcomponent. In our present studies cultivars Ludwig and Finezja were of the lowest

  to F. avenaceum inoculation and MON accumulation (Mewa, Ludwig and Finezja in Lublin; Rywalka, Ludwig and Finezja in Poznan) while cv. Sukces exhibited the lowest susceptibility in both locations. After inoculation with F. culmorum the lowest susceptibility and the mycotoxin formation were observed for cv. Nutka, Zyta and Rywalka for both localities (Finezja and Soraja in Lublin; Rywalka in Poznan), while the highest for cv. Symfonia and Sukces (Soraja in Poznan and Mewa and Symfonia in Lublin). Cultivars Tonacja and Nutka (Finezja and Ludwig in Lublin and Symfonia and Zyta in Poznan) were the lowest susceptibility to F. graminearum inoculation and with the lowest toxic metabolite biosynthesis while cv. Mewa (Soraja in Lublin and Mewa in Poznan) of the highest. Recorded results indicates on winter wheat cultivars with low susceptibility to infection under different climate conditions, therefore experiments described in this paper should be continued to select winter wheat cultivars of the lowest susceptibility to FHB, mycotoxin formation and the most suitable for different climate and cultivation conditions in Poland.

F

F

F

Table

  ). The experiment was performed in 2008, in a randomized complete block design in triplicate, at two different locations of Poland: Cerekwica (82 m a.s.l.), Central West Poland -30 km northwest of Poznan, and Sitaniec (210 m a.s.l.), South East Poland -90 km southeast of Lublin. Weather conditions for both locations received from Institute of Meteorology and Water Management.

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

Table 1 .

 1 Characteristics of the cultivars used in the experiment (date from Research Centre for Cultivar Testing)

	Cultivar	Technological value/group	Frost resistance	Lodging resistance Soil acidity tolerance Protein and gluten contents
	Finezja	quality/A	medium	medium	medium	high
	Ludwig	quality/A	low/medium	low	medium	high
	Mewa	bread/B	high	low	medium	high
	Nutka	bread/B	low	high	medium	low/medium
	Rywalka	quality/A	medium	low	medium	high
	Soraja	bread/B	medium	medium	medium	medium
	Sukces	quality/A	low	high	high	high
	Symfonia	forage/C	high	medium	medium	medium
	Tonacja	quality/A	high	high	medium	high
	Zyta	quality/A	low	medium	medium	high

Table 2 .

 2 Mean squares from analysis of variance for F. culmorum

	Mean squares

Table 3 .

 3 Mean squares from analysis of variance for F. graminearum

	Mean squares

Table 4 .

 4 Mean squares from analysis of variance for F. avenaceum

	Mean squares

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Table 6 .

 6 The average values for ZEA in FDK, ZEA in HLK and ZEA in chaff (µg g -1 ) for F. graminearum

					Food Additives and Contaminants		Page 22 of 27
	Cultivar	Lublin	ZEA in HLK Poznan	Means	Lublin	ZEA in FDK Poznan	Means	Lublin	ZEA in chaff Poznan	Means
	Finezja 0.091	0.0000	0.046cd	0.315	0.032	0.173c	14.925	0.130	7.527b
	Ludwig 0.119	0.0001	0.060c	0.598	0.003	0.300c	22.455	0.020	11.238a
	Mewa	0.115	0.0002	0.057c	1.037	0.025	0.531b	10.994	0.040	5.517b
	Nutka	0.189	0.0000	0.094b	0.563	0.026	0.295c	4.015	0.044	2.029d
	Rywalka 0.040	0.0022	0.021d	0.468	0.007	0.237c	4.152	0.014	2.083d
	Soraja	0.912	0.0003	0.456a	4.617	0.065	2.341a	10.990	0.037	5.514b
	Sukces 0.068	0.0101	0.039cd	0.442	0.032	0.237c	10.609	0.084	5.347bc
	Symfonia 0.205	0.0009	0.103b	0.387	0.126	0.257c	14.321	0.173	7.247b
	Tonacja 0.130	0.0004	0.065c	0.687	0.018	0.352bc	5.591	0.002	2.797cd
	Zyta	0.068	0.0000	0.034cd	0.401	0.043	0.222c	12.240	0.172	6.206b
	Means 0.194A 0.0014B		0.951A	0.038B		11.029A	0.072B
	LSD 0.05 C: 0.026, L: 0.011, C×L: 0.036	C: 0.194, L: 0.087, C×L: 0.274	C: 2.658, L: 1.189, C×L: 3.759
	Means with the same letter are not significantly different

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Table 7 .

 7 The average values for DON in FDK, DON in HLK and DON in chaff (µg g -1 ) for F. culmorum

	Cultivar	Lublin	DON in HLK Poznan	Means	Lublin	DON in FDK Poznan	Means	Lublin	DON in chaff Poznan	Means
	Finezja	0.47	0.54		0.505cd	84.8	43.5	64.14abc	67.5	41.4	54.45b
	Ludwig	1.36	0.21	0.785bcd 114.6	41.3		77.94ab	9.4	33.0	21.20d
	Mewa	0.91	1.11	1.010abc	68.7	44.1		56.38bc	7.1	62.2	34.68cd
	Nutka	1.37	1.26		1.315ab	101.1	54.2		77.66ab	13.3	37.2	25.25d
	Rywalka	1.22	1.92		1.570a	90.3	21.6		55.96bc	43.9	21.7	32.79cd
	Soraja	0.85	0.38		0.615cd	88.4	80.8		84.62a	14.6	97.6	56.09b
	Sukces	2.63	0.29		1.460a	78.6	28.3		53.45c	115.9	45.6	80.75a
	Symfonia 2.10	0.55		1.325ab	60.0	51.8		55.92bc	14.2	71.2	42.73bc
	Tonacja	0.27	0.44		0.355d	84.0	29.1		56.53bc	33.2	37.6	35.44cd
	Zyta	1.15	0.90	1.026abc	82.0	55.5	68.77abc	22.8	25.4	24.07d
	Means	1.23A	0.76B			85.2A	45.0B			34.2B	47.3A
	LSD 0.05	C: 0.59, L: 0.26, C×L: 0.83	C: 22.17, L: 9.91, C×L: 31.35 C: 17.01, L: 7.61, C×L: 24.05
	Means with the same letter are not significantly different

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Table 8 .

 8 The average values for DON in FDK, DON in HLK and DON in chaff (µg g -1 ) for F. graminearum

	Cultivar	DON in HLK Lublin Poznan Means	Lublin	DON in FDK Poznan	Means	Lublin	DON in chaff Poznan	Means
	Finezja 0.21	0.69	0.45bcde	71.2	36.3	53.76ab	12.5	41.9	27.19cd
	Ludwig 0.24	0.13	0.19e	43.5	18.2	30.86e	45.2	13.3	29.27cd
	Mewa 0.08	0.95	0.52bcde	58.7	42.6	50.62abc	60.0	102.3	81.16a
	Nutka 0.22	1.27	0.75b	54.2	27.1	40.62bcde	19.7	35.7	27.70cd
	Rywalka 0.30	0.22	0.26de	77.4	21.4	49.40abcd	35.9	19.0	27.44cd
	Soraja 0.32	0.31	0.32cde	62.7	56.6	59.65a	68.9	16.9	42.90b
	Sukces 0.15	2.97	1.56a	52.6	29.1	40.84bcde	31.8	55.2	43.50b
	Symfonia 0.76	0.29	0.53bcd	41.8	33.6	37.67cde	18.4	22.5	20.45d
	Tonacja 0.77	0.44	0.61bc	60.2	14.2	37.23de	60.5	9.7	35.12bc
	Zyta	0.51	0.13	0.32cde	71.2	16.4	43.84bcde	48.6	8.8	28.72cd
	Means 0.36B 0.74A		59.3A	29.5B		40.1A	32.5B
	LSD 0.05 C: 0.33, L: 0.15, C×L: 0.47	C: 14.81, L: 6.62, C×L: 20.94	C: 11.06, L: 4.95, C×L: 15.65
	Means with the same letter are not significantly different

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Table 9 .

 9 The correlation matrix for the traits studied in Poznan (above diagonal) and Lublin (below diagonal) for F. culmorum

	Page 25 of 27
		Fi	ZEA in HLK	ZEA in FDK	DON in HLK	DON in FDK	HLK kernels per spike	FDK kernels per spike	HLK weight per spike	FDK weight per spike	TKW
	Fi	1.00	0.30	-0.26	0.22	0.05	-0.25	0.04	-0.66*	-0.16	-0.38
	ZEA in HLK 0.34	1.00	0.45	-0.16	0.05	-0.24	0.15	-0.39	-0.09	-0.21
	ZEA in FDK -0.38 -0.18	1.00	-0.24	0.43	-0.38	0.25	-0.27	0.12	-0.11
	DON in HLK -0.16 0.46	0.27	1.00	-0.11	-0.24	-0.03	-0.09	-0.02	0.11
	DON in FDK 0.09 -0.26	0.20	0.10	1.00	-0.18	0.24	-0.09	0.10	0.13
	HLK kernels per spike	-0.42 -0.11	0.15	-0.20	0.14	1.00	-0.10	0.54	-0.14	-0.33
	FDK kernels per spike	0.49	0.42	-0.17	0.35	0.32	-0.16	1.00	-0.12	0.84**	-0.02
	HLK weight per spike	-0.05 0.29	-0.12	0.05	-0.03	0.58	0.19	1.00	0.16	0.55
	FDK weight per spike	0.23	0.29	-0.11	0.34	0.54	0.09	0.77**	0.14	1.00	0.29
	TKW	0.09	0.37	-0.29	0.30	0.05	-0.03	0.26	0.63*	0.37	1.00
	* significant at 0.05 level, ** significant at 0.01 level

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Table 10 .

 10 The correlation matrix for the traits studied in Poznan (above diagonal) and Lublin (below diagonal) for F. graminearum significant at 0.05 level, ** significant at 0.01 level, *** significant at 0.001 level

		Fi	ZEA in HLK	ZEA in FDK	DON in HLK	DON in FDK	HLK kernels per spike	FDK kernels per spike	HLK weight per spike	FDK weight per spike	TKW
	Fi	1.00 -0.23	0.20	-0.14	0.56	-0.42	0.34	-0.61	0.12	-0.45
	ZEA in HLK 0.40	1.00	-0.02	0.72*	-0.02	-0.12	0.15	-0.09	0.28	-0.01
	ZEA in FDK -0.01 0.20	1.00	-0.09	0.47	-0.29	0.19	-0.28	0.33	-0.09
	DON in HLK 0.16	0.39	0.30	1.00	0.09	-0.44	0.43	-0.33	0.47	-0.19
	DON in FDK 0.09 -0.38	0.04	0.14	1.00	-0.52	0.50	-0.58	0.42	-0.39
	HLK kernels per spike	-0.31 -0.28	-0.06	0.11	-0.12	1.00	-0.59	0.81**	-0.54	0.39
	FDK kernels per spike	0.20	0.03	0.27	-0.10	0.21	-0.72*	1.00	-0.51	0.82**	-0.33
	HLK weight per spike	-0.20 -0.16	0.09	0.20	-0.16	0.93***	-0.62	1.00	-0.24	0.71*
	FDK weight per spike	0.19 -0.06	0.32	-0.11	0.25	-0.63*	0.93***	-0.53	1.00	0.02
	TKW	0.16	0.09	0.48	0.16	0.00	-0.04	0.25	0.27	0.45	1.00

*

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Acknowledgements

Part of this study was supported by PMSHE Project no: NN 3103019 34


Julie Vaysse

Stéphane Balayssac

Véronique Gilard

Denis Desoubzdanne

Myriam Malet-Martino

Robert Martino
email: martino@chimie.ups-tlse.fr

Paul Sabatier

Spcmib Lab

Myriam ; Université

Spcmib Martino

Denis Desoubdzanne

Analysis of adulterated herbal medicines and dietary supplements marketed for weight loss by DOSY 1H NMR

Keywords: slimming products, herbal medicines, dietary supplements, adulteration, synthetic drugs, 1 H NMR, 2D and 3D DOSY 1 H NMR, sibutramine, phenolphthalein

Introduction

According

to WHO (http://www.who.int/mediacentre/factsheets/fs311/en/), approximately 1.6 billion adults were overweight and at least 400 million were obese in 2005.

WHO further projects that by 2015, approximately 2.3 billion adults will be overweight and more than 700 million will be obese. Overweight and obesity are associated with premature mortality, chronic morbidity, diabetes, heart disease, osteoarthritis and cancer. Given (i) the medical and social impact of being overweight or obese, (ii) the few drugs marketed for these indications, and (iii) the safety concerns of these medications, more and more patients turn to weight-loss natural herbal medicines (HM) or dietary supplements (DS). Indeed, in contrast to conventional pharmaceuticals, these products are regarded by many as being harmless because of their natural origin for HM or of their safe composition (vitamins, minerals, herbs or other botanicals, amino acids, and substances such as enzymes, organ tissues, glandular, and metabolites) for DS.

The WHO's definition indicates that medicines containing plant material combined with chemically defined active substances, including chemically defined, isolated constituents of plants, are not considered to be HM (http://www.who.int/medicines/areas/traditional/ definitions/en/index.html). Moreover, FDA regulations require that a complete list of ingredients and the net contents of the product appear on DS labels (http://www.fda.gov/ food/DietarySupplements/default.htm). Despite these clear definition and regulation and as it is very easy for the consumer to purchase these formulations over the Internet, common problems affecting the safety of these preparations include fraudulent adulteration with synthetic actives used in conventional pharmaceutical drugs which can lead to severe sideeffects.

Reported methods for screening illegal adulterations in HM or DS are mainly HPLC-DAD, GC-MS and LC/MS/MS [START_REF] Liu | HPLC and GC-MS screening of Chinese proprietary medicine for undeclared therapeutic substances[END_REF][START_REF] Lau | Analysis of adulterants in a traditional herbal medicinal product using liquid chromatography-mass spectrometry-mass spectrometry[END_REF][START_REF] Bogusz | Application of LC-ESI-MS-MS for detection of synthetic adulterants in herbal remedies[END_REF] [START_REF] Chen | Determination of synthetic drugs used to adulterate botanical dietary supplements using QTRAP LC-MS/MS[END_REF]. Recently, [START_REF] Zhu | Modified local straight-line screening to detect synthetic drugs in adulterated herbal medicines[END_REF] developed an algorithm, termed local straight-line screening (LSLS), to resolve complex IR spectra of possibly adulterated HM.

The analytical methods reported concerning more specifically the adulteration of herbal formulations marketed for weight loss are chromatographic methods coupled with various detectors [START_REF] Tseng | GC/MS analysis on anorectics adulterated in traditional Chinese medicines[END_REF][START_REF] Jung | Anorectic sibutramine detected in a Chinese herbal drug for weight loss[END_REF][START_REF] Yuen | Adulteration of overthe-counter slimming products with pharmaceutical analogue-an emerging threat[END_REF][START_REF] Zou | Detection of sibutramine, its two metabolites and one analogue in a herbal product for weight loss by liquid chromatography triple quadrupole mass spectrometry and time-of-flight mass spectrometry[END_REF][START_REF] Date | Rapid determination of medical components found in the health food for weight loss by liquid chromatography/tandem mass spectrometry (LC/MS/MS)[END_REF][START_REF] Huang | Simultaneous determination of sibutramine and N-di-desmethylsibutramine in dietary supplements for weight control by HPLC-ESI-MS[END_REF][START_REF] Wang | Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS[END_REF], capillary electrophoresis (CE) [START_REF] Cianchino | Analysis of potential adulteration in herbal medicines and dietary supplements for the weight control by capillary electrophoresis[END_REF]) and IR-LSLS [START_REF] Lu | A new method for testing synthetic drugs adulterated in herbal medicines based on infrared spectroscopy[END_REF]). None of these methods is ideal as HM often contain mixture of herbs and other natural products, and the formulas of those products are very complex. Chromatographic methods do not provide global information as they are mainly targeted screening methods and thus sometimes too selective. Moreover, false-positive results might occur with both chromatographic and IR-LSLS methods because it is in fact an analogue of a known active compound with a very close chemical structure which is detected instead of the target compound.

In this study we show an application of 2D Diffusion Ordered SpectroscopY 1 H Nuclear Magnetic Resonance (2D DOSY 1 H NMR) spectroscopy as an untargeted screening method for the detection and unequivocal identification of adulterants in "herbal" slimming products.

The main advantage of NMR is to provide global information as it allows for considering the drug preparation as a whole. Moreover, the method is non-selective and requires no prior knowledge of the structures of the various components present in the mixture, which is a major advantage for screening of adulteration.

Experimental

Samples

Twenty commercial formulations of HM or DS marketed for weight loss were analyzed. 1. All samples, as received, were stored in the dark at ambient temperature and humidity. They were all analyzed within their expiration dates (when indicated).

H and 2D DOSY H NMR

The 1 H NMR experiments were performed on a Bruker Avance 500 spectrometer equipped with a 5 mm cryoprobe at 298K. Typical acquisition parameters were as follows: Relaxation delay was lengthened to 3 s and 128 scans were recorded for the quantitative analysis of sibutramine, phenolphthalein, synephrine, methylsynephrine, caffeine and vitaberine. The concentrations of adulterant compounds were measured by comparing the expanded areas of their respective NMR signals with that of the internal standard for quantification TSP. The areas were determined by manual integration using TopSpin software. Each data is the mean of at least five integrations. In order to check that the NMR conditions used allowed an accurate quantitation of adulterants, a longer interval between pulses (10 s) was used in some experiments. The same concentration values were obtained, thus demonstrating that a pulse interval of 3 s for a flip angle of 35° is sufficient to record the spectra under conditions of full T 1 relaxation. Sibutramine was assayed from the singlet at 7.47 ppm, phenolphthalein from the two doublets at 7.17 and 6.84 ppm, synephrine from the 6 two doublets at 7.23 and 6.83 ppm, methylsynephrine from the two doublets at 7.20 and 6.83 ppm, caffeine from the singlet at 7.77 ppm in formulation 12 and from the two singlets at 3.93 and 3.50 ppm in formulation 19, and vitaberine from the triplet at 4.04 ppm. The limit of quantification of the NMR assay in the recording conditions used in this study is 5 µM.

Stimulated echo bipolar gradient pulse experiments were used for 2D DOSY 1 H NMR with a pulse field gradient length between 1.4 and 1.8 ms, a delay of 3 ms after each gradient and a diffusion delay between 50 and 100 ms. Sequence parameters were optimized in order to have the intensity of the main signals of the spectrum in the aromatic region strongly decreased (at least divided by 50) at 95% of the full gradient strength. The longitudinal eddy current delay was 20 ms, spoiler gradients were 1 ms long with a field strength of -7.92 and -6.09 G.cm -1 and other acquisition parameters were the same as those reported above for 1D

1 H NMR except for the relaxation delay fixed to 2 s. Forty experiments were recorded with gradient intensity linearly sampled from 5 to 95%. The gradient system had been calibrated to 46.25 G.cm -1 at maximum intensity.

All data were processed using the Gifa 5.2 software with the inverse Laplace transform method using the Maximum Entropy (MaxEnt) algorithm. The processing parameters were 2048 points along the Laplace spectrum diffusion axis and 20000 MaxEnt iterations. The inverse Laplace transform was computed only on the columns presenting a signal 32 times greater than the noise level of the experiment. DOSY spectra are presented with chemical shifts on the horizontal axis and diffusion coefficients expressed in µm 2 .s -1 on the vertical axis.

The 3D DQF-COSY iDOSY experiment was recorded on formulation 7. In the diffusion dimension, 28 gradient steps were used with 50 ms for the diffusion delay, 1.9 ms for the gradient pulse length and 3 ms for the recovery delay. and9.25 G.cm -1 strength, were applied for 1 ms. The relaxation delay was 1.3 s and the number of scans was 8 (half-day experiment). The spectrum was processed using the NPK software [START_REF] Tramesel | Modeling of NMR processing, toward efficient unattended processing of NMR experiments[END_REF]

MS-MS analysis

An Applied System QTRAP triple-quadrupole mass spectrometer, equipped with a Turbo Ion Spray (TIS) interface and controlled by the Agilent Analyst software (version 1.4),

was used for analysis. The mass spectrometer was operated in positive ionization mode.

Nitrogen served both as auxiliary, collision gas and nebulizer gas. The operating conditions for TIS interface were as follows: (i) in MS mode: mass range 100-600 u (1 s), step size 0.1 u; Q1 TIS MS spectra were recorded in profile mode, IS 5000 V, DP 40 V; (ii) in MS-MS mode: precursor mass 280 u; mass range 30-300 u; step size 0.1 u; MS-MS spectra were recorded in profile mode, IS 5000 V, DP 40 V, CE 20 V. All samples were dissolved in MeOH and analyzed after direct introduction.

Preparation of samples for NMR analysis

The tablets were powdered (or capsule emptied) and dissolved in 5 mL of CD For the quantitative analysis of sibutramine, phenolphthalein, synephrine, methylsynephrine, vitaberine and caffeine found in some formulations, the tablet was powdered (or capsule emptied), and 10% of the powder was dissolved in 10 mL of methanol under magnetic stirring during 15 min and then sonicated for 10 min. An aliquot of 1 mL was evaporated to dryness and the residue dissolved in 1 mL of MeOH-d 4 . TSP was added at a final concentration of 0.2 mM before the NMR analysis. The experiments were done in triplicate.

Results

Conventional 1 H NMR and 2D DOSY 1 H NMR analyses

All 4.

Even if sensitivity is not an issue with respect to adulteration, we determined that in the experimental conditions used in this study (1h of recording), 2D DOSY 1 H NMR allows an accurate determination of diffusion coefficients for compounds at a concentration of 50 µM.

The limit of detection corresponds to 10 µM with only CH 2 and CH 3 signals being detected.

3D DOSY-COSY 1 H NMR analysis

Quantitative analysis

The contents of sibutramine and phenolphthalein in the herbal drugs analyzed were measured by NMR (Table 5). Eight formulations (2, 3, 4, 8, 10, 11, 13, 14) contain sibutramine alone at doses ranging from 4.4 to 30.5 mg/capsule. Five formulations (1, 5, 7, 9, 15) contain sibutramine (from 5.0 to 19.6 mg/capsule or tablet) and phenolphthalein (from 4.4 to 66.1 mg/capsule). Formulation 12 contains 19.5 ± 0.5 and 10.0 ± 0.5 mg/capsule of synephrine and caffeine, respectively. Formulation 19 contains 38.3 ± 0.9, 184.9 ± 5.6 and 75.4 ± 1.6 mg/capsule of methylsynephrine, caffeine and vitaberine, respectively.

Mass spectrometry

The aim of mass spectrometry experiments was to cross-validate NMR data. MS/MS mass spectrometry with ESI ionization was carried out in order to confirm the presence of sibutramine and to investigate the presence of its previously described mono-or didesmethylated analogs [START_REF] Wang | Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS[END_REF]. Molecular

Discussion

To date, identification of synthetic drugs adulterating HM to inhibit appetite and achieve weight loss using NMR and more specifically 2D DOSY 1 H NMR has not been reported. The objective of this study was to apply this method to analyze the content of 20 different HM or DS.

Chromatographic methods are preferred by most analysts for mixture analysis as they are very common, easy to handle, quantitative, sensitive, relatively cheap and do not require highly trained personnel. Nevertheless, NMR has also several advantages; it is non-selective, quantitative, highly powerful for structural elucidation and it requires no prior knowledge of the structures of the various components present in a mixture. 2D DOSY 1 H NMR allows the virtual separation of several components of a mixture in a single run based on the difference in their translational self-diffusion coefficients (Figure 1). Moreover, DOSY experiments do not need complicated setup procedures or separation steps and the method can be used routinely. This technique has been used successfully for analyzing several kinds of counterfeit drugs [START_REF] Trefi | Analysis of illegally manufactured formulations of tadalafil (Cialis®) by 1 H NMR, 2D DOSY 1 H NMR and Raman spectroscopy[END_REF][START_REF] Nyadong | Combining two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy, imaging desorption electrospray ionization mass spectrometry and direct analysis in real time mass spectrometry for the integral investigation of counterfeit pharmaceuticals[END_REF]. Table 6 shows a comparison on information obtained with various analytical methods on slimming herbal drug composition and highlights the interest of DOSY NMR for a global analysis of these formulations.

Among the 20 formulations analyzed, 2 were strictly herbal (formulations 17 and 18), and 4 had a composition corresponding to declared ingredients on the packaging or the leaflet ). The two metabolites of sibutramine, N-mono-and N-di-desmethylsibutramine, previously described by some authors [START_REF] Blachut | Identification of N-desmethylsibutramine as a new ingredient in Chinese herbal dietary supplements[END_REF][START_REF] Lai | Isolation and identification of a sibutramine analogue in a healthy food for weight loss[END_REF][START_REF] Yuen | Adulteration of overthe-counter slimming products with pharmaceutical analogue-an emerging threat[END_REF][START_REF] Huang | Simultaneous determination of sibutramine and N-di-desmethylsibutramine in dietary supplements for weight control by HPLC-ESI-MS[END_REF] Other actives found may also cause serious adverse effects. Phenolphthalein is a laxative that was withdrawn as a medicine several years ago after concerns about carcinogenicity (No authors listed 1999). Moreover, interaction profiles are unknown for the combination sibutramine/phenolphthalein. After the ban on ephedrines in 2004, manufacturers changed to using the weaker stimulant synephrine that has purported thermogenic and lipolytic properties due to its α-adrenergic agonist property [START_REF] Haaz | Citrus aurantium and synephrine alkaloids in the treatment of overweight and obesity: an update[END_REF]. The safety of synephrine is 13 controversial but it could pose cardiovascular health risks [START_REF] Haaz | Citrus aurantium and synephrine alkaloids in the treatment of overweight and obesity: an update[END_REF], especially in combination with caffeine [START_REF] Haller | Hemodynamic effects of ephedra-free weight-loss supplements in humans[END_REF]. Formulation 12, claimed as purely herbal, contains both compounds and formulation 19 contains methylsynephrine (parahydroxyephedrine) in combination with a high dose of caffeine, a potentially hazardous combination.

Conclusion

This study presents a new application of 2D DOSY 1 H NMR for the analysis of HM and DS, which are undeniable complex mixtures. Among the twenty formulations marketed for weight loss studied thirteen were adulterated with sibutramine alone or with a combination of sibutramine and phenolphthalein. Adulteration of "natural herbal medicines" with undeclared synthetic drugs is a common and dangerous phenomenon and all the analytical methods that ensure the quality and safety of these products have thus to be developed. In this context, 2D

DOSY 1 H NMR spectroscopy is a powerful method for providing a multivariate fingerprint of a very complex mixture especially in situations where the identity of the components is not known beforehand as it permits to consider the drug preparation as a whole. The technique should be now considered as a useful and complementary tool among a standard 2D NMR analytical package. Moreover, its evolution towards 3D DOSY-COSY experiments noticeably increases its interest as structural data are easily obtained. .36 d (3.5;g1),4.10 d (8.5;f3),4.00 t (8.5;f4),g5,f6,g6,g3),3.61 AB system (12.5;f1),3.48 dd (3.5,10.0; g2), 3.38 t (9.5; g4) a Spectral data were measured from authentic standards in CD 3 CN:D 2 O (80:20). b s: singlet; d: doublet; dd: doublet of doublet; t: triplet; td: doublet of triplet; q: quadruplet; quin: quintuplet; sext: sextuplet; sept: septuplet; m: multiplet; app: apparent. c The chemical shifts were measured in the spectrum of formulation 19. d The structure of sinapine (choline ester of sinapic acid) was determined with 1D and 2D 1 H and 13 C NMR experiments on formulation 14 and confirmed by MS ([M + ] 310). e g: glucose; f: fructose.

  with Fourier transform in magnitude mode in the COSY dimension and with the inverse Laplace Transform method (MaxEnt algorithm) in the DOSY dimension. The processing parameters were 192 points along the diffusion axis and 20000 MaxEnt iterations. The algorithm was computed only on the columns with a signal-to-noise ratio above 32. The corresponding 2D COSY and DOSY experiments were acquired with the 3D DQF-COSY iDOSY parameters. These experiments were analyzed with the NMRnotebook package (NMRtec 2007).

  3 CN:D 2 O 80:20 v/v (CD 3 CN for formulation 8 and D 2 O for formulation 16) under magnetic stirring and then sonicated for 10 min. The suspension was then centrifuged (5 min, 3000 rpm) and the supernatant (550 µL) analyzed.

  formulations were analyzed with 2D DOSY 1 H NMR. 2D DOSY spectra of formulations 3, 12 and 19 along with their corresponding 1D spectrum are presented in Figure1. All the peaks of a same compound are lined up. The peaks at 3.62 and 1.99 ppm correspond to the signals of residual HOD in D 2 O and CHD 2 CN in acetonitrile-d 3 , respectively. As shown in Figure1, the separation according to diffusion coefficients (D) in the diffusion dimension allows an easier identification of the components of a mixture. Indeed, in formulation 3 (Figure1A), the active pharmaceutical ingredient (API) adulterant is sibutramine (D = 1107 ± 9 µm 2 .s -1 ) citrate (D = 959 µm 2 .s -1 ) and low amounts of natural compounds including glucose, sucrose, and fatty acids (D = 931 ± 29, 1006 ± 29, and 872 ± 29, respectively) are also detected. In formulation 12 (Figure1B), the main signals are those of caffeine (D = 1156 ± 39 µm 2 .s -1 ) and synephrine (D = 734 ± 20 µm 2 .s -1 ), whereas formulation 19 (Figure1C) contains caffeine, methylsynephrine, vitaberine (thiamine-O-isobutyryl disulphide) and coefficients of 1288 ± 93, 851 ± 41, 624 ± 9 and 918 µm 2 .s -1 , respectively.All the ingredients detected in the herbal drugs studied are reported in Table2, which shows the comparison between the indications provided by the manufacturer and the compounds found in each formulation. Several unexpected actives, sibutramine (formulations 1-5, 7-11, 13-15), phenolphthalein (formulations 1, 5, 7, 9, 15) synephrine and caffeine (formulation 12), were found. The unambiguous identification of the components in the herbal formulations analyzed was achieved by comparison of 1D 1 H NMR data of standards, addition of authentic standards, and comparison of 1D NMR spectra extracted from the 2D DOSY 1 H NMR spectrum with those of authentic standards (Figure1D).The NMR spectral data of sibutramine measured in CD 3 CN:D 2 O (80:20) are reported in

Figure 2

 2 Figure 2 illustrates the different NMR spectra obtained in the 3D DOSY-COSY 1 H

  [M+H] + ions corresponding to sibutramine, N-mono-desmethylsibutramine and N-di-desmethylsibutramine were searched at m/z 280, 266 and 252, respectively[START_REF] Wang | Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS[END_REF]. Only sibutramine was found. MS/MS was then carried out on the m/z 280 ion giving characteristic fragments at m/z 153, 139, and presence of sibutramine was thus confirmed by MS/MS in thirteen formulations (1-5, 7-11, and 13-15). MS was also used to confirm the presence of methylsynephrine, vitaberine and caffeine ([MH] + ions at m/z 182, 703, and 195 respectively) in formulation 19 and synephrine ([MH] + ion at m/z 168) in formulation 12.

(

  formulations 6, 16, 19, 20). The others were all adulterated. Thirteen formulations (1-5, 7-11, contained sibutramine alone or in combination with phenolphthalein (1, 5, 7, 9, and 15

  Figure 1. 2D DOSY 1 H NMR spectra recorded in CD 3 CN:D 2 O (80/20) of formulations 3 (A), 12 (B) and 19 (C). (D) Comparison of 1D 1 H NMR spectra of caffeine (D1) and vitaberine (D2) extracted from the 2D DOSY 1 H NMR spectrum of formulation 19 with

Figure

  Figure 2. NMR spectra of formulation 7 in CD 3 CN:D 2 O (80/20). (A) 2D DOSY 1 H

Table 3

 3

. The 1 H NMR resonances were assigned by 2D NMR (gCOSY, gHSQC, gHMBC, gNOESY) and 1D selective NOE experiments. The chemical shifts and coupling constants of all the other ingredients are reported in Table

Table 2 .

 2 Comparison of formulation composition with indications provided by the manufacturer.

					Food Additives and Contaminants
	12	Lida	20080924	09/2010	Kunming Dali Industry and Trade Co.	China	Capsule	Green
		Onting
	13	quianweisu slimming	Unknown	03/2010	Menova	China	Capsule	Blue
		herbs a
	14	Meekeyes a	95882108	Unknown	Marlian	China	Capsule	Green and white
	15	Perfect slim a	Unknown	Unknown	Unknown	China	Capsule	Pink
	16	Garslim	22	05/2011	Balsam	Syria	Tablet	Beige
	17	Alattar psyllium	4	06/2009	Alattar Medical Co.	Syria	Capsule	Green
		Alattar
	18	slimming	255161M2	06/2009	Alattar Medical Co.	Syria	Powder	-
		mixture
	19	Hyper drive 3.0 +	110802	10/2013	ALR industries	USA	Capsule	Red
	20	Elan-Sil	9.026.6	01/2001	Vitaline Ltd	UK	Capsule	White

a This formulation has been seized by French Customs.

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		Botanical slimming soft gel	Only natural compounds	Sibutramine Soybean oil	Soybean oil is used to prepare the gel
				Sibutramine
	9	Botanical slimming herbs	Only natural compounds	Phenolphtaleine Very low amounts of natural compounds
				including glucose, sucrose, fatty acids
	10	2 Day Diet Japan Lingzhi slimming formula	Only natural compounds	Sibutramine Very low amounts of natural compounds including glucose, fatty acids
				Sibutramine
	11 Lipro diet pills 12 Lida F o r Only natural compounds (Chinese herbs) Only natural compounds (Yunnan herbs) 13 Onting quianweisu slimming herbs No indication b P e	Stearate Very low amounts of natural sugars including glucose Caffeine Synephrine Low amount of natural compounds Sibutramine Low amounts of natural compounds including glucose, fructose, fatty acids	Identical to formulation 4
	14	Meekeyes	No indication b e r	Sibutramine Sucrose Fatty acid
			R e	High amount of sinapine and very low amounts of other natural compounds Sibutramine Phenolphtaleine
	15 16	Perfect slim Garslim	No indication b Fruit extracts with 50% hydroxycitric acid and citric acid v i e w Very low amounts of natural compounds Vitamin C a including glucose, fructose, fatty acids Hydroxycitric acid Citric acid	Identical to formulation 1
				O n l
				y

Table 3 .

 3 NMR characteristics of sibutramine detected in the herbal formulations analyzed. Spectra were recorded in CD 3 CN:D 2 O (80:20). The chemical shift of this carbon was measured in a spectrum recorded in CD 3 CN.Table4. 1 H NMR characteristics of the main signals of ingredients found in herbal formulations (except sibutramine given in Table3).

			Sibutramine
				3
			2		4
		6	5	1	11	14	15	16
		7
		8		10	N	17
		Cl	9	12		13
	Atom	δ 1 H (ppm) a			Multiplicity b	δ 13 C (ppm)
	number					(J in Hz)
	1							49.2
	2 or 4	2.60 (1H syn aromatic ring) 2.49 (1H anti aromatic ring)		m m		32.5
	3	1.98 (1H) 1.78 (1H)			m m		15.5
	4 or 2	2.37 (1H syn aromatic ring) 2.41 (1H anti aromatic ring)		m m		33.7
	5	-				-		140.2
	6,10 7,9	7.47			s		128.7 129.9
	8							132.9
	11	3.67			t (6.3)	71.7
	12 or 13	2.19			broad s	39.6 c
	13 or 12	2.81			broad s	47.0 c
	14	1.53 (1H) 1.39 (1H)			m m		33.5
	15	1.78			m		25.4
	16 or 17	1.01			d (6.5)	22.0
	17 or 16	1.04			d (6.5)	21.2
	a The value given is the chemical shift of the centre of the signal.
	b s: singlet; d: doublet; t: triplet; m: multiplet.

c

Table 5 .

 5 Amounts of adulterant sibutramine and phenolphthalein found in claimed herbal medicines analyzed in this study.

		Sibutramine	Phenolphtalein
	Formulation number	mg/capsule or tablet mean±SD	mg/capsule or tablet mean±SD
		(n = 3)	(n = 3)
	1	13.1 ± 0.3	47.0 ± 3.0
	2	4.4 ± 0.3	-
	3	23.3 ± 0.1	-
	4	15.6 ± 0.5	-
	5	19.6 ± 0.5	66.1 ± 1.4
	7	5.0 ± 0.1	36.2 ± 0.5
	8	12.7 ± 0.2	-
	9	12.0 ± 0.2	4.4 ± 0.2
	10	10.6 ± 0.3	-
	11	30.5 ± 0.1	-
	13	15.1 ± 0.3	-
	14	14.0 ± 0.7	-
	15	13.7 ± 0.6	40.8 ± 1.3

Table 6 .

 6 Adulterants found in herbal products or dietary supplements marketed as slimming products reported in literature

	References	Analytical methods for detection	Number of formulations analysed	Active Pharmaceutical Ingredients detected	Other compounds detected
				Mazindol
				Clobenzorex
	Tseng et al. 2000	GC-MS	4	Diazepam
				Phentermine
				Caffeine
	Jung et al. 2006	GC-MS HPLC-DAD	1	Sibutramine
	Blachut et al. 2007	GC-MS	1 (Lida)	N-desmethylsibutramine
	Sein Anand and
	Chodorowski	GC-MS	1 (Meizitanc)	Sibutramine
	2007
				Fenfluramine
	Bogusz et al. 2006 LC-ESI-MS/MS	1	Phentermine
				Caffeine
				Sibutramine
	Zou et al. 2007	LC-MS/MS LC-TOF-MS	1	N-desmethylsibutramine N-didesmethylsibutramine
				Homosibutramine
				Phenolphthalein
				Sibutramine
	Date et al. 2008	LC-ESI-MS/MS	10	Mazindol Sennosides A and B
				Bisacodyl
				Hydrochlorothiazide
	Huang et al. 2008	LC-ESI-MS	15	Sibutramine N-didesmethylsibutramine
				Sibutramine
	Wang et al. 2008	LC-ESI-MS	6	Phenolphthalein
				N-desmethylsibutramine
	Chen et al. 2009	QTRAP LC-MS/MS	18	Fenfluramine Sibutramine Phenolphthalein
				Ephedrine
	Cianchino et al. 2008	CE	4	Norephedrine Furosemide
				Caffeine
					Carnitine
					Vitamin C
					Dehydroascorbic acid
	This study	1 H NMR and 2D DOSY 1 H NMR	20	Sibutramine Phenophthalein Synephrine Methylsynephrine Vitaberin Caffeine	Vitamins B1, B3, B5, B6, E Glucose Fructose Sucrose Stearate Linoleate
					Citrate
					Hydroxycitrate
					Tartrate
					Sinapine

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Acknowledgements

The authors wish to thank Marie-José Parent and Dr Saleh Trefi for helpful discussion on herbal drug adulteration and Catherine Claparols for mass spectrometry experiments.

Tartrate was not detected. The amounts of vitamins B2, B7, B9 and B12 are too low to be detected.

a Vitamin C is slightly unstable in the extraction conditions used. It was thus detected as a mixture of ascorbic and dehydroascorbic acids. b This formulation was provided by French Customs with no packaging. c Incorrect chemical nomenclature as propionylcarnitine is the propionic ester of carnitine.


MD Elodie Sellier

BSc Geraldine Surman

MD Kate Himmelmann

MD Guro Andersen

MD Allan Colver

MD Ingeborg Krägeloh-Mann

MD Javier De-La-Cruz

MD Christine Cans
email: ccans@chu-grenoble.fr

Trends in prevalence of cerebral palsy in children born with a birthweight of 2,500 g or over in Europe from 1980 to 1998

Keywords: Cerebral palsy, Children with birthweight ≥ 2500 g, Europe, Prevalence, Statistical interaction

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

INTRODUCTION

Cerebral palsy (CP) is the most common cause of significant motor impairment in children. Approximately 2 children in every 1000 born alive suffer from CP. Although the risk of having CP is greatly increased for those born very preterm or with very low birthweight, [START_REF] Himpens | Prevalence, type, distribution, and severity of cerebral palsy in relation to gestational age: a meta-analytic review[END_REF] children with birthweight ≥ 2500 g, what we shall call normal birthweight (NBW) for the purpose of this paper still account for most children affected. More than half of the children born during the 1970s and 1980s with CP and recorded on a European database, weighed more than 2500 g at birth;[2] the situation is similar in the USA [START_REF] Grether | The California Cerebral Palsy Project[END_REF], Australia [START_REF] Watson | Report of the Western Australian cerebral palsy register[END_REF] or China. [START_REF] Liu | Prevalence of cerebral palsy in China[END_REF] Less research has been carried out on the epidemiology of CP in children of NBW than in those of very low birthweight (< 1500 g). As the number and severity of impairments increase with increasing gestational age [START_REF] Himmelmann | Gross and fine motor function and accompanying impairments in cerebral palsy[END_REF][START_REF] Hemming | The influence of gestational age on severity of impairment in spastic cerebral palsy[END_REF] and birthweight, [START_REF] Surman | Children with cerebral palsy: severity and trends over time[END_REF] the type of brain lesion among term or NBW children tends to exhibit a different pattern to that of preterm or low birthweight births. [START_REF] Hagberg | The origins of cerebral palsy[END_REF][START_REF] Krageloh-Mann | The role of magnetic resonance imaging in elucidating the pathogenesis of cerebral palsy: a systematic review[END_REF][START_REF] Ashwal | Practice parameter: diagnostic assessment of the child with cerebral palsy: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society[END_REF] Time trends for CP prevalence in NBW children vary across studies. [START_REF] Cans | Epidemiology of cerebral palsy[END_REF][START_REF] Paneth | The descriptive epidemiology of cerebral palsy[END_REF] Most of them reported stable prevalence rates from the 1970s to the 1990s. [START_REF] Watson | Report of the Western Australian cerebral palsy register[END_REF][START_REF] Drummond | Analysis by gestational age of cerebral palsy in singleton births in north-east England 1970-94[END_REF][START_REF] Topp | Cerebral palsy births in eastern Denmark, 1987--90: implications for neonatal care[END_REF][START_REF] Dolk | Trends in the prevalence of cerebral palsy in Northern Ireland, 1981-1997[END_REF][START_REF] Wu | Cerebral palsy in a term population: risk factors and neuroimaging findings[END_REF] However, a small increase was reported in USA between 1975 and 1991 [START_REF] Winter | Trends in the prevalence of cerebral palsy in a population-based study[END_REF] while in Sweden the monitoring from the 1950s to the 1990s showed an increase in prevalence from the beginning of the 1970s to the mid 1980s followed by a decrease. [START_REF] Himmelmann | The changing panorama of cerebral palsy in Sweden. IX. Prevalence and origin in the birth-year period 1995-1998[END_REF] Also, in Iceland a decrease in prevalence for term babies was observed in 1997-2003 in comparison with 1990-1996. [START_REF] Sigurdardottir | Trends in prevalence and characteristics of cerebral palsy among Icelandic children born 1990 to 2003[END_REF] In 1998, a network of population-based CP registers, Surveillance of Cerebral Palsy in Europe (SCPE), [START_REF]Surveillance of cerebral palsy in Europe: a collaboration of cerebral palsy surveys and registers. Surveillance of Cerebral Palsy in Europe (SCPE)[END_REF] was established. It is the largest international collaboration of CP registers in the world. Anonymous data, now covering 19 registers, are held in the common database. Data are recorded and coded according to agreed definitions. [START_REF] Cans | Recommendations from the SCPE collaborative group for defining and classifying cerebral palsy[END_REF] This paper aims to establish whether the prevalence of CP in NBW children altered in Europe between 1980 and 1998 using multicentre analysis methods. Reporting of secular trends for main neurologic subtypes and severity of impairment were also made possible as a result of the large database available.

MATERIAL AND METHODS

Definition and classification

All registers contributing to the network are population-based, covering either a region or a whole country. Data were collated from SCPE common database as described previously.[2, 21] CP is defined as a group of permanent, but not unchanging, disorders of

Study population

Children with CP born from 1980 to 1998, whose mothers lived in an area covered by a contributing register at birth, were eligible for this study. We selected children with birthweight ≥ 2500 g. Post-neonatal CP cases were excluded. Annual population data for live births, stratified by birthweight were provided by each register except four, for which these data were not available by birthweight (SCPE_C02 in Toulouse, France; SCPE_C07 in Dublin, Ireland; SCPE_C14 in Arnhem, Netherlands and SCPE_C21 in Lisbon, Portugal).

Data from these four registers were consequently excluded from analysis. One register, SCPE_C10 in Tübingen, Germany, recorded only bilateral spastic CP cases and its data were included only when analyzing the bilateral spastic subgroup. The SCPE_C01 register in Grenoble, France, has specific migration patterns and cases were defined as living in the area at the time of registration.

Characteristics of the children

Classification of CP subtypes followed SCPE classification. [START_REF] Cans | Recommendations from the SCPE collaborative group for defining and classifying cerebral palsy[END_REF], [START_REF] Platt | Surveillance of cerebral palsy in europe: reference and training manual[END_REF] Severe intellectual impairment was defined as IQ below 50; epilepsy as a history of two unprovoked seizures before confirmation of CP diagnosis, excluding febrile or neonatal seizures; severe visual impairment as visual acuity of < 6/60 (Snellen scale) or 0.1 (Decimal scale) in the better eye following correction and severe hearing impairment as a loss of > 70 dB in the better ear before correction. Severity of CP was assigned to three categories. [START_REF] Beckung | Probability of walking in children with cerebral palsy in Europe[END_REF] Severe CP was defined as children with IQ < 50 and unable to walk, even with assistive devices.

Moderate CP was defined as children with IQ < 50 and able to walk; or children with IQ ≥ 50 and unable to walk without assistive devices. Mild CP was defined as children with IQ ≥ 50 and able to walk without assistive devices. To analyze trends in prevalence, we combined moderate and severe CP into a moderate-to-severe group. A Z-score for birthweight by gestational age was derived for each child. For Swedish children, Marsal fetal growth standard curves were used; [START_REF] Marsal | Intrauterine growth curves based on ultrasonically estimated foetal weights[END_REF] Gardosi fetal growth standard curves were used for children of all other registers. [START_REF] Gardosi | An adjustable fetal weight standard[END_REF]

Statistical methods

We report descriptive statistics as percentages or as mean with standard deviation (SD). To study changes in characteristics of children with CP over time, we considered 4 periods: 1980-1984; 1985-1989; 1990-1994, and 1995-1998, and we adjusted on register effect to take account of the fact that the number of registers contributing data differed between the periods. Binary variables were analyzed using the Chi-square test for trend adjusting on register, provided that the Chi-square test for non-linearity was not significant. Continuous variables were analyzed using linear regression analyses adjusted for period and register.

Poisson regression was used to investigate trends in prevalence of CP. Given that data were provided by different registers covering varying periods, we followed several steps.

Firstly, we analyzed trends in prevalence within each register with a linear term for individual birth year. We tested non-linearity of the trend using polynomial terms for birth years up to third order. Secondly, we used pooled data to investigate trends in Europe. The initial model contained just individual birth years. Addition of a term for register allowed testing for a register effect. Then, adding an interaction term between register and birth year provided a test for variation in trends between registers. We also tested non-linearity of the trend using polynomial terms for birth years up to third order. Likelihood-ratio Chi squared tests were used to compare nested models. The same modeling strategy was used when analyzing time trends by CP subtype and by severity.

To assess the robustness of our results, we performed cross-validation by sequentially removing each register from the model. We verified that the registers responsible for interaction were also those responsible for the main changes in the prevalence estimates when removed sequentially from analysis.

In order to minimize findings that were of statistical significance but little clinical relevance in such a large dataset, the threshold selected for overall analyses was P<.005. The threshold for analyses of individual registers data was P<.05. We present prevalence rates with Poisson 99% confidence intervals for pooled data and with 95% confidence intervals for individual registers. Statistical analyses were performed using Stata Statistical software (version 10.0, Stata Corp., College Station, TX, USA).

RESULTS

Characteristics of the children with cerebral palsy included in the study

Fifteen SCPE registers provided data on 7507 children with known birthweight from 1980-1998. Of those, 4002 (53%) were born with birthweight ≥ 2500g and were included in the study (Figure 1). The type of CP was spastic in 84.9% of cases (bilateral in 45.7%, unilateral in 39.2%), dyskinetic in 9.3%, and ataxic in 5.8% (Table 1). Among these children 2290 (58.0%) were male, 3520 (91.2%) were ≥ 37 weeks gestational age, and 3772 (97.4%) were singletons. Of 2518 mothers with available data, 1697 (67.4%) were primigravida. At the time of registration, 29.1% of the children were unable to walk without assistive devices, 30.3% had severe intellectual impairment, 10.4% had severe visual impairment, and 1.7% severe hearing impairment. Nine hundred and forty-five children (23.9%) were known to have been admitted to a neonatal care unit. Apgar score at five minutes had been recorded since 1990 in four registers, and was available for 761 children. Among them, 610 (80.2%) had an Apgar score ≥ 7 and 57 (7.5%) scored below 4.

Change over time in characteristics of children with cerebral palsy and birthweight ≥ 2500g (Table 2)

There was no significant change in the proportion of children with CP who were female, or from a multiple birth. The mean Z score for birthweight increased significantly.

Mean maternal age increased from 26.7 years to 28.8 years, P<.001. The proportion of children with severe intellectual impairment decreased from 33.4% in 1980-1984 to 27.9% in 1995-1998, but the trend did not reach significance.

Prevalence of cerebral palsy in children with birthweight ≥ 2500g

The overall prevalence, including 14 registers, was 1.14 per 1000 births (CI, 1.09-1.18) during the study period. Prevalence rates in each register are presented in Table 3. For 12 of them, trends in prevalence were not significant. Two registers from United Kingdom showed significant change in prevalence: an increase was seen in the area covered by Newcastle register, SCPE_C08 (P=.01) whilst a decrease was observed in Oxford register, SCPE_C09 (P<.001).

When analyzing trend in overall prevalence of CP in Europe, there was a significant interaction between register and birth year (P<.001). This interaction indicates that the trend was heterogeneous across registers and therefore does not allow reporting of an overall trend for all registers. After removing the two registers (Newcastle and Oxford) which showed a significant trend in prevalence, the interaction was no longer significant (P=.83). We therefore analyzed trends pooling data from the 12 remaining registers whilst data from Oxford and Newcastle were analyzed separately. The overall prevalence for the 12 registers was 1.10 per 1000 live births (1.05-1.15) from 1980 to 1998; 1.16 per 1000 live births (CI, 0.88-1.48) in 1980 and 0.99 per 1000 live births (CI, 0.80-1.20) in 1998 (Figure 2). The decrease was not significant (P=.14). The prevalence of bilateral spastic CP decreased significantly from 0.58 (CI, 0.41-0.80) in 1980 to 0.33 (CI, 0.22-0.46) in 1998, P<.001. At the same time, the prevalence of unilateral spastic CP increased from 0.37 (CI, 0.23-0.58) to 0.46 (CI, 0.34-0.62), P=.004. There was no significant change in the prevalence of dyskinetic forms over the period, P=.37. The prevalence of moderate-to-severe CP decreased from 0.52 (CI, 0.34-0.75) to 0.42 (CI, 0.30-0.57) but the trend was not significant (P=.009). The prevalence of mild cases was stable (P=.67) with a mean rate of 0.53 per 1000 live births (CI, 0.50-0.56).

In Newcastle, the prevalence increased significantly (Figure 3, P=.01), mainly because of the increase in bilateral spastic type (P=.005) from 0.54 per 1000 (CI, 0.18-1.27) to 0.71 (CI, 0.44-1.08). There were no significant changes in rates of other CP subtypes or in the moderate-to-severe group. In Oxford, the prevalence decreased significantly (Figure 3, P<.001), mainly due to a decrease in unilateral spastic type from 0.37 (CI, 0.18-0.66) to 0.16 (CI, 0.05-0.37), P=.006. In this register, there was a "borderline" decrease in bilateral spastic forms, P=.06 and a decrease in the prevalence of moderate-tosevere forms, P=.02.

During the same period covered by our study, i.e. between 1980 and 1998, there was a significant reduction in neonatal mortality of nearly 50% from 1.7 (CI, 1.4-2.1) to 0.9 (CI, 0.7-1.1) per 1000 NBW live births in the areas covered by registers which contributed data throughout (n=9).

DISCUSSION

The SCPE population-based registers and surveys feed a unique population-based database for Europe using standardized procedures to include and classify children with CP.

In the present study, we found that in 12 out of 14 registers, there was no significant change in prevalence of CP in children with birthweight ≥ 2500 g born between 1980 and 1998. Our findings issuing from population-based data are unlikely to be influenced by a selection bias typically encountered in hospital-based studies. And this result is in agreement with previous studies conducted outside Europe. A recent report from the Western Australia cerebral palsy register [START_REF] Watson | Report of the Western Australian cerebral palsy register[END_REF] showed stable trends in prevalence of CP in NBW children, with a prevalence slightly higher than we observed (1.55 per 1000 live births in 1980-1984 to 1.62 in 1995-1999). A study from California, USA, also reported a stable prevalence of CP in term children of 1.1 per 1000 live births in 1991-2002. [START_REF] Wu | Cerebral palsy in a term population: risk factors and neuroimaging findings[END_REF] Robustness of our results was assessed in analyzing prevalence rates and trends of CP in children born at term (≥ 37 gestational age) in the 11 registers able to provide population data by gestational age. Results were very similar to those observed on NBW children (data not shown). Obviously, data quality of the SCPE common database remains dependent on the quality of data provided by the contributing registers. Some of them have already shown the reliability of their data in previous studies, [START_REF] Topp | Validation of a cerebral palsy register[END_REF][START_REF] Johnson | Can routine information systems be used to monitor serious disability?[END_REF][START_REF] Parkes | Does the Child Health Computing System adequately identify children with cerebral palsy?[END_REF][START_REF] Guillem | How to calculate the completeness of ascertainment of a morbidity register? Example of the Isere childhood handicap register in Grenoble, France[END_REF] and continuous data quality improvement work is being performed at the network level.

The clinical profile of children with CP was generally stable over time. However, there was a non-significant trend toward a decrease in the frequency of associated impairments, i.e. severe intellectual impairment and, in the last period, severe visual impairment. Twenty-four percent of the children were known to have been admitted to a neonatal care unit. This percentage may be an underestimate due to numerous missing data in three registers. Two recent studies in the USA and Australia showed that 30% to 35% of children born at term who developed CP were admitted to a neonatal intensive care unit (NICU). [START_REF] Wu | Cerebral palsy in a term population: risk factors and neuroimaging findings[END_REF][START_REF] Mcintyre | The changing picture of cerebral palsy for those born at term: 16 years of population data[END_REF] Whilst most moderate (1500 g to 2499 g) or very low birthweight children are born in maternity with NICU, this is not observed for NBW children. The significant decrease in neonatal mortality for NBW children reflects advances in obstetric and neonatal care between 1980 and 1998 and it is reassuring that this was not accompanied by a rise in CP prevalence. Probably the impact on morbidity of these improvements in neonatal care is first seen in children born in maternity with NCIU. The significant decrease in CP prevalence seen in very low birthweight [START_REF] Platt | Trends in cerebral palsy among infants of very low birthweight (<1500 g) or born prematurely (<32 weeks) in 16 European centres: a database study[END_REF] children is yet to be observed in NBW children.

Several explanations can be proposed regarding the different trends observed in two UK registers. In Oxford, the decline in prevalence was particularly marked for births after 1996. We can not exclude a true decline in the number of children with CP in this area.

However, this was more likely the result of under-ascertainment due to the unwillingness of clinicians to provide data for the CP register following the enactment of the 1998 Data Protection Act. Both in Scotland and Liverpool registers have ceased collecting data because of these difficulties. Moreover, the register in Northern Ireland was allowed to move to a nonconsent based system because of incompleteness when seeking consent. The explanation for the observed increase in Newcastle may be related to the expansion of its catchment area from 1991 births. If the prevalence for the additional area covered since 1991 was higher than the pre-1991 area, the inclusion of this additional area may have increased the overall prevalence. This explanation is supported by the stability of prevalence over the period 1964-1993 in the initial catchment area. [START_REF] Colver | Increasing rates of cerebral palsy across the severity spectrum in north-east England 1964-1993. The North of England Collaborative Cerebral Palsy Survey[END_REF] We observed a significant decrease in the bilateral spastic group, the most common subtype, of more than 40%. At the same time, there was an increase in unilateral spastic subtype. It is unlikely that the changes in prevalence of unilateral and bilateral CP types resulted from classification bias between the two subtypes. A reliability study performed in 2003 within the SCPE network showed more discrepancies between bilateral spastic and dyskinetic CP than between bilateral and unilateral spastic CP. [START_REF] Gainsborough | Validity and reliability of the guidelines of the surveillance of cerebral palsy in Europe for the classification of cerebral palsy[END_REF] However, we do not believe that the trends reflect a different approach to classification of dyskinetic and bilateral spastic as there was no significant change in the prevalence of dyskinetic CP. In addition, if the increase in unilateral spastic CP was related to improved ascertainment, an overall increase in milder cases could be expected and this was not observed. Few papers have analyzed trends over time by CP subtypes and birthweight and usually it is proportions of subtypes that are compared, not prevalence. 9, [START_REF] Colver | Increasing rates of cerebral palsy across the severity spectrum in north-east England 1964-1993. The North of England Collaborative Cerebral Palsy Survey[END_REF] In our study, we found a non-significant increase in prevalence of dyskinetic forms. A recent study, also based on the SCPE common database, reported a significant increase in the prevalence of dyskinetic CP among NBW children for the period 1976-1984, followed by a plateau. [START_REF] Himmelmann | Dyskinetic cerebral palsy in Europe: trends in prevalence and severity[END_REF] Our analysis covered a later period, 1980-1998, and confirmed the leveling off of prevalence of dyskinetic CP.

The changes observed in spastic subgroups do not have a straightforward interpretation. We agree with recent comment of Pharoah, regarding the fact that trends in incidence, not prevalence are crucial to determining etiology. [START_REF] Pharoah | Dyskinetic cerebral palsy in Europe: trends in prevalence and severity, on behalf of the SCPE Collaboration[END_REF] Nevertheless, few hypothesis might be mentioned. The decrease in prevalence of bilateral spastic CP may be explained by improvements in neonatal care, but there is no obvious reason for the increase in unilateral spastic CP. Neuro-imaging studies [START_REF] Krageloh-Mann | The role of magnetic resonance imaging in elucidating the pathogenesis of cerebral palsy: a systematic review[END_REF][START_REF] Ashwal | Practice parameter: diagnostic assessment of the child with cerebral palsy: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society[END_REF]suggest that slightly different patterns of brain abnormalities are observed in unilateral and bilateral spastic CP. Thus, it might be possible that prevention has affected the two subtypes differently.

In conclusion, this study was the first to analyze European trends in prevalence of CP in children with birthweight ≥ 2500 g. We showed that the prevalence was stable in spite of decreasing neonatal mortality. The reduction in prevalence of bilateral spastic CP, suggests that prevention of the more severe forms may be occurring. Data were available for children born until 1998, thus ongoing monitoring of the trends, including trends in the different subtypes, is necessary.

  movement and/or posture and of motor function, due to a non-progressive interference, lesion, or abnormality of the developing/immature brain. Progressive motor disorders, nerve or muscular diseases and brain metabolic disorders are excluded. The diagnosis of CP is confirmed at around age 5 years, before submission by each register of its data to the SCPE common database. Subtypes of CP are defined as unilateral spastic, bilateral spastic, dyskinetic or ataxic. Hierarchic trees developed by SCPE were used to implement inclusion and exclusion criteria and classify CP subtypes consistently among registers.[2,[START_REF]Surveillance of cerebral palsy in Europe: a collaboration of cerebral palsy surveys and registers. Surveillance of Cerebral Palsy in Europe (SCPE)[END_REF]

Table 1 .

 1 Characteristics of children with cerebral palsy and birthweight ≥ 2500 g, summary from 14 SCPE registers a (n=3948).

	N=3948
	(%)

a Data from Tübingen registering only bilateral spastic cases were excluded

Table 2 .

 2 Changes over time in characteristics of children with cerebral palsy and birthweight ≥ 2500g. From 14 SCPE registers Mother's age was available for 2831 children, multiple birth status for 3872 children, Z score for 3763 singleton children, ability to walk for 3621 children, intellectual impairment level for 3433 children, epilepsy for 3357 children. Percentages are calculated using the total known cases for each characteristic as denominators b Test for linear trend was adjusted for register

	(n=3948).
	Characteristics a	1980-1984	1985-1989	1990-1994	1995-1998	Test for linear trend b
		n=758	n=1252	n=1057	n =881	P value
	Female, n (%)	344 (45.4)	530(42.3)	427 (40.4)	357 (40.5)	.06
	Maternal age over 34 years, n (%)	51 (9.5)	91 (10.5)	111 (15.2)	111 (15.7)	.001
	Multiple birth, n (%)	21 (2.9)	22 (1.8)	30 (2.8)	26 (3.0)	.15
	Z score, mean (SD)	-0.25 (1.22)	-0.15 (1.31)	-0.06 (1.36)	0.08 (1.42)	<.001
	Unable to walk, even with aids, n (%)	203 (28.2)	380 (30.9)	286 (27.8)	185 (28.8)	.77
	Severe intellectual impairment, n (%)	226 (33.4)	303 (29.5)	300 (30.9)	211 (27.9)	.05
	Severe visual impairment, n (%)	78 (10.6)	140 (11.9)	129 (12.3)	65 (7.4)	.29
	Severe hearing impairment, n (%)	8 (1.1)	30 (2.4)	17 (1.6)	12 (1.4)	.56
	Epilepsy, n (%)	214 (37.9)	343 (35.2)	391 (38.6)	291 (36.2)	.98
	Severe-to-moderate CP, n (%)	350 (50.2)	598 (51.5)	474 (47.8)	383 (48.2)	.42

a

Table 3 .

 3 Children with cerebral palsy and birthweight ≥ 2500g by SCPE register: number of cases and prevalence by period.

		Period covered Number of cases Prevalence rate a	Prevalence rate a	Prevalence rate a	Prevalence rate a
				1980-1984	1985-1989	1990-1994	1995-1998
	Grenoble, France (SCPE_C01)	1980-1998	234	0.88	0.99	1.21	0.80
	Edinburgh, UK (SCPE_C03)	1984-1989	301	0.83	0.83	-	-
	Cork, Ireland (SCPE_C04)	1986-1998	89	-	0.96	0.93	0.91
	Belfast, UK (SCPE_C05)	1981-1998	472	1.21	1.04	1.16	1.13
	Gothenburg, Sweden (SCPE_C06)	1980-1998	474	1.32	1.32	1.19	1.16
	Newcastle, UK (SCPE_C08)	1980-1998	501	1.09	1.27	1.42	1.55
	Oxford, UK (SCPE_C09)	1984-1998	543	1.28	1.40	1.00	0.88
	Tübingen, Germany (SCPE_C10) b	1980-1986	54
	Liverpool, UK (SCPE_C11)	1980-1989	340	1.23	1.26	-	-
	Copenhagen, Denmark (SCPE_C12)	1980-1998	738	1.46	1.30	1.25	1.29
	Rome, Italy (SCPE_C13)	1983-1998	40	1.54	1.20	1.24	0.62
	Tonsberg, Norway (SCPE_C15)	1991-1998	98	-	-	1.05	0.89
	Bologna, Italy (SCPE_C16)	1991-1996	25			0.74	0.69
	Galway, Ireland (SCPE_C17)	1990-1998	62			0.83	1.16
	Madrid, Spain (SCPE_C18)	1991-1998	31			0.65	0.72

a Prevalence rates are presented per 1000 live births b The SCPE_C10 register included only children with bilateral spastic cerebral palsy.

ACKNOWLEDGMENTS

We thank all SCPE participants: C Cans, S Rey (RHEOP, Grenoble, FR), J Chalmers (ISDSHS, Edinburgh, UK), V McManus, A Lyons (Lavanagh Centre, Cork, IE), J Parkes, H Dolk (Belfast, UK), P Uvebrant, K Himmelmann (Göteborg University, Göteborg, SW), A Colver (University of Newcastle, Newcastle, UK), J Kurinczuk, G Surman (NPEU, Oxford, UK), I Krägeloh-Mann, V Horber (Tübingen University, Tübingen, DE), MJ Platt (University of Liverpool, Liverpool, UK), P Udall, S Holst-Ravn (NIPH, Copenhagen, DK), MG Torrioli, S Matricardi (Lazio Cerebral Palsy Register, Rome, IT), G Andersen, A Meberg (CPRN, Tonsberg, NO), M Bottos (Bologna, IT), G Gaffney (Galway, IE), J De la Cruz, C Pallas (DIMAS-SAMID, Madrid, SP).

FUNDING

This work was supported by grants from the European Commission [Research DG-QLG5-2001-30133, DGSANCO-2003-3131]. Geraldine Surman was funded by the Policy Research Programme in the Department of Health, England and the views expressed are not necessarily those of the Department.