Stéphanie Lheureux
email: s.lheureux@baclesse.fr

Bernard Lambert

Sophie Krieger

Angelina Legros

Min Do

Ique Vaur

Christophe Denoyelle

Pascaline Berthet

Laurent Poulain

Agnès Hardouin

Corres Ponding

Lheureux Stéphanie

Two novel variants in the 3'UTR of the BRCA1 gene in familial breast and/or ovarian cance r

Keywords: Breast, ovarian, inherited cancer, 3'UTR, BRCA, miRNA

For the majority of breast and/or ovarian cancer patients tested for BRCA1/2 genes, mutation screening of the coding regions remains negative. Micro RNAs wh ich negatively regulate mRNA translation by binding to 3' untranslated region (3'UTR) are imp licated in cancer. Genetic changes in the 3'UTR of several genes were reported to be associated with higher susceptibility to particular tu mor types . The aim of the present study was to analyze the BRCA1 3'UTR in patients tested negative for BRCA1/2 deleterious mutations, in order to find variants implicated in the decrease of BRCA1 expression through modificat ion of miRNA b inding.

Genotyping analyses were perfo rmed on genomic DNA of 70 BRCA negatives index cases, selected among patients with breast or ovarian cancer, less than 50 years old, with a strong family history. The co -occurrence of the identified variants with deleterious BRCA1 mutations was then determined in a control population of 210 patients. A luciferase gene reporter assay was used to investigate the impact of the variants on the BRCA1 gene expression.

Two novel variants, c.*750A>G and c.*1286C>A, were identified in the 3'UTR of BRCA1 gene, in two patients.

The former was found three times in the control population, whereas the latter was absent. The used functional assay did not reveal any effect on the luciferase expression.

This study reveals a weak genomic variability in the 3'UTR of the BRCA1 gene. All together, the results led us to classify the variant c.*750A>G as probably neutral, the variant c.*1286C>A remain ing unclassified.

Introduction

BRCA1 and BRCA2 genes are two inherited breast and/or ovarian cancer susceptibility genes [1;2]. A recent meta-analysis of ten studies estimated that the risk of developing breast cancer by age 70 years was 57% and 49% for BRCA1 and BRCA2 mutation carriers, respectively. The corresponding risks for ovarian cancer were 40% and 18% [START_REF] Chen | Meta-analysis of BRCA1 and BRCA 2 penetrance[END_REF]. As risk-reduction strategies, including chemoprevention and prophylactic surgeries, have been demonstrated to be effective in high-risk individuals [START_REF] Robson | Clinical pract ice. Management of an inherited predisposition to breast cancer[END_REF], genetic testing for BRCA gene mutations has been increasingly utilized in clin ical settings. The identification of mutat ion in these genes is ext remely beneficial for patients pursuing risk reduction strategy and more recently for their cancer t reatment. In fact, a novel class of promising drug, the PARP (poly ADP-ribose polymerase) inhibitors, is particularly effective in patients with BRCA1/2 mutations [START_REF] Hennessy | So matic Mutations in BRCA 1 and BRCA2 Could Expand the Nu mber of Patients That Benefit Fro m Poly (ADP Ribose) Poly merase Inhibitors in Ovarian Cancer[END_REF]. These news drugs, like o laparib, are currently developed in this target BRCA positive population [6;7]. Despite our understanding of the clinical phenotypes most predictive of the presence of BRCA1 and BRCA2 mutations, a lot o f families with both breast and/or ovarian cancers do not have identifiable mutations [START_REF] Ford | Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium[END_REF]. About 80% o f the mutation screening remains negative making the identification of the risk of developing a cancer difficu lt for the concerned families [START_REF] Fackenthal | Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations[END_REF] and depriving the access to clinical trials. Most of the techniques used for genetic screening of BRCA1 and BRCA2 genes are performed by analysing the entire coding region, including the intron-exon junctions, while introns and proximal untranslated regions remain unexplored.

However, d iscovery of micro RNA (miRNA) has pointed out the important role of these untranslated regions in gene regulation. MicroRNAs are a class of small non-coding regulatory RNAs ranging in size fro m 17 to 25 nucleotides. They post-transcriptionnally repress expression of target genes by recognizing comp lementary sites mostly in their 3'untranslated region (3'UTR). Their exp ression deregulation has been implicated in the onset and progression of many diseases, including cancers [START_REF] Nilsen | Mechanisms of micro RNA-mediated gene regulation in animal cells[END_REF].

In spite of the potential importance of the 3'UTR in gene regulat ion, the role of geno mic variat ions in the 3'UTR of the tu mor suppressor BRCA1/2 genes remains unclear. Until now, few studies analysed the 3'UTR of BRCA1 and most of them focused on the role of single nucleotide poly morphis ms (SNP) [START_REF] Kontorovich | Single nucleotide polymorphis ms in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high-risk wo men[END_REF][START_REF] Pongsavee | The BRCA1 3'-UTR: 5711+421T/T_5711+1286T/T genotype is a possible breast and ovarian cancer risk factor[END_REF][START_REF] Nico Loso | Single-nucleotide poly morphis ms inside micro RNA target sites influence tumor susceptibility[END_REF][START_REF] Puget | Screening for germ-line rearrangements and regulatory mutations in BRCA 1 led to the identification of four new delet ions[END_REF]. Fro m Pongsavee et al., one SNP c.* 421T/T (rs 8176318) seems to be linked to a decrease of BRCA1 gene exp ression associated with an increased risk of cancer [START_REF] Pongsavee | The BRCA1 3'-UTR: 5711+421T/T_5711+1286T/T genotype is a possible breast and ovarian cancer risk factor[END_REF]. Authors proposed that the BRCA1 gene expression alteration was the result of the creation of a new miRNA binding site by the SNP, although the link between both events has not been clearly established. However, to our knowledge, no mutation has been already described in the 3'UTR of BRCA1/2 genes. These observations led us to assume that unknown genomic variations in the 3'UTR o f BRCA1/2 genes could be imp licated in the inherited breast or ovarian cancer through their effect on BRCA gene expression. In order to test this hypothesis, we analysed, in a first attempt, the BRCA1 3'UTR fro m patients tested negative for BRCA1 and BRCA2 mutations in highly selected families.

Material and Methods

Patient population

Genotyping analyses were performed on genomic DNA of 70 index cases selected from patients with breast or ovarian cancer diagnosed at an age of less than 50 years. These index cases had a strong history family at least on two generations: 3 breast cancers at first degree, one of which before 40 years, or 1 ovarian cancer associated with a breast cancer before 40 years. They have been tested negative for BRC A1 and BRCA2 mutations.

Screening for BRCA1 and BRCA2 mutations was performed using two techniques, depending on the exons analyzed, i.e.denaturing High Performance Liquid Chro matography (dHPLC) and High Resolution Melt ing (HRM), both follo wed by direct sequencing. The screening methods for BRCA1 and BRCA2 large rearrangements were perfo rmed using a co mbination of the mu ltip lex ligation -dependent probe amplification (MLPA) and quantitative multip lex PCR of short fluorescent fragments (QMPSF ).

All patients have been selected from a database of families undergoing genetic counseling in the Western French Network. The patient gave signed informed consent.

Control population

The control group consisted of 210 unrelated probands with a BRCA1 deleterious mutation. Th is population was chosen as control population to determine the frequency of the variants found in the 3'UTR. Arguing the fact that BRCA1 compound heterozygotes for two deleterious mutations are embryonically lethal [START_REF] Liu | Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimp lantation development[END_REF], this population was used to determine the frequency of the identified variants in 210 BRCA1 wild type alleles.

Moreover, the choice for this specific control population could allow to exclude the identified variant as a deleterious mutation if found in trans status with an other deleterious mutation. These patients were checked in our database between 1997 and 2009 and gave signed informed consent.

No menclature

The DNA sequence numbering is based on the cDNA sequence for BRCA1 (NCBI RefSeq NM_ 007294), following the recommendations of the Hu man Geno me Variation Society (HGVS, translation init iation codon ATG=1).

PCR and sequencing

Patient peripheral blood samp les were collected in EDTA tubes. Genomic DNA was isolated using an automated procedure (EZ1 DNA blood kit on biorobot EZ1 workstation, Qiagen, Hilden, Germany). The 3'UTR of BRCA1 gene (1376 bp) was studied in 4 overlapping frag ments about 500 nucleotides.

Each frag ment was amplified by PCR (poly merase chain reaction) by a Taq Go ld poly merase (Invitrogen Corporation, Madison, Carlsbad, California) with specific fo rward and reverse primers (50µM, Eurogentec, Liege, Belgiu m). These primers were tailed with M13 co mmon sequences (50 µM , Eurogentec) (Table 1).

PCR was performed with a co mmercial kit (Taq Go ld poly merase, Invit rogen Corporation, Madison, Carlsbad, California), fro m 250 ng of DNA. All amplifications were performed using a touch -down PCR program. The thermocycler parameters for PCR were 95°C for 10 minutes for the initial DNA poly merase activation, 26 cycles of 94°C for 20 seconds, 63°C for 20 seconds with a decrease of 0.5°C/cycle, and 72°C for 45 seconds, follo wed by 20 cycles of 94°C for 20 seconds, 50°C fo r 20 seconds and 72°C for 45 seconds, and a final extension at 72°C for 7 minutes. PCR products were separated by electrophoresis on a 1.5 % agarose gel containing ethidiu m bromide and visualized by exposure to ultraviolet light. The amplified products were purified by ExoSap kit (USB Co rp., Cleveland, Oh io, USA), accord ing to the manufacturer's instructions. The purified templates were sequenced in both directions with the M13 forward/reverse primers. A dye terminator cycle sequencing kit ( Big Dye Terminator kit v1.1; Applied Biosystems) was used and templates analysed on an ABI 3130 sequencer (Applied Biosystems, USA). HRM HRM has only been performed fo r the exp loration of the fragment 3 in the control population. Twenty ng of genomic DNA was added to a reaction master mix consisting of 1×LightCycler® 480 High Resolution Melting Master (containing the proprietary ds -DNA saturating binding dye), with 2.5 mM MgCl 2 (Roche Diagnostics, Germany) and 0.5 μM of F3F and F3R (same PCR section). All the samples were perfo rmed under the same conditions in a 96-well p late in the LightCycler® 480 Real-Time PCR System (Roche Diagnostics, Germany).

The PCR program started with an init ial denaturation step of 10 min at 95 °C, continued with 42 cycles of 15 s at 95 °C, 15 s at 65 °C with a decrease of 1°C/cycle and 25 s at 1 min fo llowed by 10 cycles of 15 s at 95°C, 15 s at 55°C and 25 s at 72°C. To induce heteroduplexes formation, samples were heated to 95 °C for 1 min and cooled to 40 °C for 1 min. Samp les were melted fro m 60 °C to 95 °C perfo rming 25 acquisitions per 1 °C. HRM melting curve analysis was performed using the Light-Cycler® 480 Gene Scanning software version 1.2 (Roche Diagnostics, Germany), which allo ws clustering the samples into groups on the basis difference plot obtained analyzing the differences in melting curve shapes. All the samples with a late amplification, as monitored by real-t ime PCR or associated with fluorescence of less than the 60% of the maximu m, were excluded fro m the analysis. According to manufacturer's recommendations, unreliable melt ing profiles could be generated. All samples studied were clustered at the default sensitivity setting (0.3) with a pre -melt slider settings (100% of fluorescence) between 85.9 and 86.4°C and post-melt slider settings (0% fluorescence) between 88. (30s) and an extension step of 72°C (80s). Th is first cycle was followed by 20 cycles of 98°C (30s), 66°C (30s), 72°C (80s) and 1 cycle of 72°C (2min30). After gel purificat ion of PCR products, 3'-A overhangs were added to the blunt-ended 3'UTR frag ment using the standard A-tailing protocol described by Promega. The 3'A -tailed 3'UTR frag ment was cloned into p GEMT easy vectors (Pro mega). Several clones were analyzed by sequencing.

Luciferase reporter gene constructs

Contrary to what it was described above, in order to obtain the 4 possibilities of BRCA1 3'UTR containing the 750A or 750G and 1286C or 1286A, we performed a BRCA1 3'UTR amp lification under experimental conditions favouring the PCR-mediated reco mbination. For such a purpose, we performed the PCR with a shorter extension time (50s instead of 80s) for 35 cycles instead of 20. PCR p roducts were purified, cloned into pGEMT easy vector and sequenced. Among the sequenced clones were retrieved clones with the wild type 3' UTR BRCA1 (* 750A;*1286C) (p GEM T-BRCA1-WT), the (* 750G;* 1286C) (p GEMT-BRCA1-750G), the (*750A;*1286A) (p GEMT -BRCA 1-1286A) and the (*750G;*1286A) (p GEMT -BRCA1-750G/1286A). The generated plasmids were d igested by MluI and HindIII, releasing a BRCA1 frag ment containing the entire 3'UTR region (1376 bp). Each restricted frag ment was inserted between the MluI and HindIII restriction sites of the pMIR-Report TM miRNA Expression reporter vector (Applied Biosystems). In such constructions all the BRCA1 3'UTR are immed iately downstream of the coding sequence of firefly luciferase.

Dual luciferase assay

All transfections were performed using the JetPRIM E TM transfection reagent (Po ly-plus transfection). A breast cancer cell lines (MCF-7) and two ovarian cancer cell lines (S KOV3 and IGROV1) at 50-70% confluence were transiently co-transfected with each pMIR-Report-BRCA1 construction (200ng) and the control reporter plas mid pRL-TK fro m Pro mega (20ng). p RL-TK contains a cDNA encoding Renilla luciferase, the expression of which is under the control of the thymidine kinase promoter. Luciferase assays were performed after 24h using the Dual Luciferase reporter assay kit (Pro mega) and the lumino meter Centro XS LB960 (Berthold Technologies) according to the manufacturer's instructions. Firefly luciferase activity was normalized relative to Ren illa luciferase activity.

After 24h, the luciferase activ ity of each transfected population was compared with a wild type (* 750A;*1286C) BRCA1 3'UTR pMIR construction (pMIR-BRCA 1-WT).

Results

Study population

Functional assay

In order to determine whether the c.*750A>G and/or the c.*1286C>A base substitutions in the BRCA1 3' UTR could alter gene expression, MCF-7 breast cancer cells were transfected with luciferase repo rter plas mids containing either one base substitution (pMIR-BRCA 1-750A, pMIR-BRCA 1-1286C) or both (pMIR-BRCA 1-750A;1286C). The results showed that the presence of c.*750A>G and/or c.* 1286C>A does not alter the luciferase expression (Figure 3). The same res ults were obtained using SKOV3 and IGROV1 ovarian cancer cell lines as recipient cells (data not shown).

Bioanalysis

Analyses of miRNA binding sites on BRCA1 3'UTR were performed using micro RNA.org (http://www.microrna.org/ microrna/home.do), microinspector (http://www.imbb.forth.gr/ microinspector.) and RegRNA (http://regrna.mbc.nctu.edu.tw) softwares. No hu man miRNA binding site has been ide ntified in the zone of interest for the variant c.*750A>G. For the variant c.*1286C>A, the base substitution increased slightly the binding of several miRNAs. The potential miRNAs were d ifferent according to the bioinformatic softwares and the defined thresholds for the free energy change.

Discussion

This is one of the first published studies which identified variants in the 3'UTR of BRCA1. Two novel variants c.*750A>G and c.*1286C>A were identified in the 3'UTR of BRCA1 gene in 2 patients from 70 inde x cases non BRCA1/2 mutation carriers selected for high probability of inherited predisposition. For the doubly heterozygous patient, we showed clearly that the variants c.*750A>G and c.*1286C>T were localized in cis on the same allele.

The variant c.*750A>G was found three times in the control population associated to three different deleterious mutations, thereby probably localized in trans with each mutation. It induced no alteration of luciferase expression in the functional assay and did not create any potential miRNA binding sites. All these results suggest that this variant has probably no impact on the BRCA1 expression.

The lack of association of the variant c.*1286C>A with another deleterious mutation was evocative of a potential deleterious character. This hypothesis was reinforced by the bioinformatics analysis which shows that it increases the binding of several miRNAs on the BRCA1 3'UTR. Conversely, its presence immediately downstream of the coding sequence of firefly luciferase gene, either alo ne or in association with the variant c.*750A>G, did not induce any effect on the expression of this reporter gene in three cell lines. This observation would suggest this variant is not able to modulate BRCA1 exp ression through modified miRNA binding.

However, although the functional testing was carried out on three different cancer cell lines, one cannot completely exclude that the miRNAs identified by bioanalysis could be absent from the tested cell lines thus leading to a false negative result. Such cell specific miRNA expression was already observed by Pongsavee et al. [START_REF] Pongsavee | The BRCA1 3'-UTR: 5711+421T/T_5711+1286T/T genotype is a possible breast and ovarian cancer risk factor[END_REF]. The authors tested the BRCA1 gene expression alteration induced by the SNP c.*1287T/T (rs12516) on three different breast cell lines. A weak inhibitor effect was only observed in MCF-7 cells (p=0.044) [START_REF] Pongsavee | The BRCA1 3'-UTR: 5711+421T/T_5711+1286T/T genotype is a possible breast and ovarian cancer risk factor[END_REF].

Finally, one cannot also rule out the hypothesis that the absence of the variant c.*1286C>A in the control population or its presence in one patient among 70 is the result of chance. Thus, without further studies, it seems difficult to conclude on the potential bio logical effect of the c.*1286C>A variation.

Nevertheless, one has to notice that the sequencing of the 3'UTR of BRCA1 fro m 68 over 70 high selected patients did not reveal any variant in this region. This weak genomic variability, as also observed by Puget et al. [START_REF] Puget | Screening for germ-line rearrangements and regulatory mutations in BRCA 1 led to the identification of four new delet ions[END_REF], suggests that the creation of new miRNA binding sites in this region is not a majo r mechanism to alter the BRCA1 gene expression. Recently, a study revealed that a SNP, asso ciated with an increased risk o f breast cancer, could positively regulate the exp ression of the BRCA1 gene through modified binding of a miRNA in the coding region of this gene [START_REF] Nico Loso | Single-nucleotide poly morphis ms inside micro RNA target sites influence tumor susceptibility[END_REF]. Futhermore, genetic variants in miRNA genes , targeting the 3'UTR of BRCA1, can potentially alter the regulation of this gene although their impact on the genetic susceptibility to breast cancer remains to be determined [START_REF] Shen | Novel genetic variants in micro RNA genes and familial breast cancer[END_REF][START_REF] Gao | The association between two polymorphis ms in pre-miRNAs and breast cancer risk: a metaanalysis[END_REF][START_REF] Catucci | Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in German and Italian familial breast cancer cases[END_REF].

It is a real challenge to identify inherited mutations in such patients with a strong family history, to propose an adaptive follow-up for them and their family. Therefore, in families with a strong history of breast cancer, other studies must be performed on the other regions of BRCA gene which are still unexplored, includ ing the 3'UTR of BRCA2. The increased risk of hereditary breast cancer is not only the result of the alteration of the BRCA genes expression [START_REF] Campeau | Hereditary breast cancer: new genetic developments, new therapeutic avenues[END_REF]. The search for the implication of other genes would beneficiate fro m high throughput methods of analysis [START_REF] Walsh | Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing[END_REF].

In conclusion, the screening of the 3'UTR of BRCA1 in 70 patients with a strong family history of breast or ovarian cancer revealed two novel variants. The variant c.*750A>G alone could be identified as neutral. The variant c.*1286C>A remains unclassified and did not allow us to bring a clear element decision -making for use in routine.

Tables captions

Table 1: Pri mers for the amplificati on of 4 overlapping frag ments from the 3'UTR of the BRCA1 gene The primers were designed in order to have specific sequences of the 3'UTR of the BRCA1 gene (in bold) downstream to M13 sequences (in italic).

Table 2: Genomic variations in the 3'UTR of the BRCA1 gene

The sequencing of the 3'UTR allows the identification of three poly mo rphisms (for co mparison the frequency in the European panel is shown) and 2 novel variants.

Figures captions

  3 and 88.8°C. Significant differences (ie +/-2 of relative signal differences) in fluorescence curves from the horizontal baseline were indicative of mutations. Results were confirmed by sequencing PCR products in the ABI Prism 3130 Genetic Analyser (Applied Biosystems, USA). Determination cis/trans status of the doubly heterozygous c.[*750A>G (+) *1286C>A] variant BRCA1 3'UTR was amp lified using genomic DNA fro m the patient doubly heterozygous for the variants c.*750A>G and c.*1286C>A. Extension time and nu mber of PCR cycles were determined in o rder to limit PCRmed iated recombination [16;17]. In brief, PCR mixture (50µl) contained 100ng of genomic DNA, 200µM dNTP, the fo rward BRF1 5'CTACGCGTCCCCACA GCCA CTA CTGA 3' and reverse BRR1 primers 5'CCCAAGCTTTTGCTACCAA GTTTATTTGCA G3' (300µM each) (the sequences in bold character represent the MluI and HindIII restriction sites for BRF1 and BRR1 respectively) and 2.5U of Taq PHUSION (Finnzy mes). PCR program was undertaken for 1 cycle of 98°C (40s) fo llo wed by an annealing step of 58°C

Three polymorphis ms in the 3 '

 3 UTR o f BRCA1 were identified: c.* 421G>T (rs8176318) with genotype frequencies 0.53, 0.11 and 0.36 for G/ G, T/T and G/T respectively, c.*1287C>T ( rs12516) with genotype frequencies 0.53, 0.11 and 0.36 fo r C/ C, T/T and C/T and c.*1332G>A (rs8176320) found heterozygous in only two patients. The polymorph ism c.*421G>T was always associated with the poly morphis m c.* 1287C>T in our study population. Two novel variants c.*750 A>G and c.*1286C>A were described for th e first time, confirmed on two independent blood tests. Patient A was diagnosed with bilateral breast cancer at 40 and 44 years and presented the variant c.*750A >G (Figure 1A). Pat ient B was diagnosed with breast cancer at 33 years and presented both the c.*750 A>G and the c.*1286C>A variant (Figure 1B). The patients A and B were ho mo zygous for the two polymorphis ms c.*421G/ G and c.*1287C/C. These results are summarized in table 2. Control Population The c.*750A>G variant was screened by HRM. The frag men t 3 of the 3'UTR of BRCA1 was also sequenced in the control population. Three wo men with three d ifferent types of deleterious mutations presented this variant in the 210 patients tested (1.4%). The c.* 1286C>A variant was screened by sequencing analysis. This variant was not found in the control population. Determination cis/trans status of the doubly heterozygous c.[*750A>G (+) *1286C>A] variant The study of 19 clones fro m the patient B showed that the variants c.*750A>G and c.*1286C>A were localized in cis on the same allele (Figure 2).

Fig 1 :

 1 Fig 1: The i dentification of the novel vari ants c.*750A>G and c.*1286C>A by sequencing anal ysis (A) Forward sequencing of patient A frag ment 2 containing the c.*750A >G variant; (B) Reverse sequencing of patient B frag ment 4 containing the c.*1286C>A variant.

Fig 2 :Fig. 3 :

 23 Fig 2: Cis/trans status determination of the doubl y heterozyg ous c.[*750A>G (+) *1286C>A] variant The 3'UTR of the BRCA1 gene was amplified fro m the genomic DNA of the patient doubly heterozygous for the variants c.*750A >G and c.*1286C>A. A mplified 3'UTR was cloned and sequenced. (V): variant; (WT): wild type

  Fig 1

Acknowledg ments

Authors are very grateful to Professeur P. Gauduchon and Dr. M. Tosi for their helpfu l discussions.


Iñigo Navarro-Blasco

Cristina Sola-Larrañaga

Chromium Content of Different Kinds of Spanish Infant Formulae and Estimation of Dietary Intake By Infants Fed on Reconstituted Powder Formulae

Keywords: Metals analysis -AAS Additives/Contaminants: Chromium, Metals -nutritional, Trace elements Food Types: Infant formulae, Water Chromium, Infant formula, Graphite furnace atomic absorption spectrometry (GF-AAS), Drinking water, Daily Intake

That chromium is an essential element for humans is well documented. Trivalent chromium, the main chemical form found in foods, is essential for maintaining normal glucose metabolism. Because of analytical difficulties, several literature reports of chromium content of foods, especially for the lower levels, show large variability and they should be interpreted with caution for a valid interpretation of reliable results. A Zeeman background correction transversely-heated graphite furnace atomic absorption spectrometry was used to determine the chromium content of 104 different infant formulae (cow's milk and soy protein based) marketed in Spain following an acid digestion sample preparation procedure in a closed, pressurized and microwave digestion unit. The mean and range of chromium values, regarding types and main protein-based infant formulae are presented. Additionally, the influence of the type of the type of container used, the impact of industrial process from different manufacturers and the physical state (powder and liquid formulae) on chromium levels is also discussed. In general, the infant formulae contain a higher chromium concentration than that found in human milk (reference range: 0.20 -8.18 µg l -1 ), particularly in the case of hypoallergenic (18.16 ± 7.89 µg l -1 ), lactose-free (11.37 ± 3.07 µg l -1 ), pre-term (11.48 ± 3.15 µg l -1 ) and soya (10.43 ± 4.05 µg l -1 ) formulae. The maximum theoretical estimated intake of infants fed on the studied formulae was lower than the upper limit safety for trivalent chromium of 1 mg kg -1 (14 µg kg -1 b.w. day - 1 ) recommended by the experts of Council for Responsible Nutrition (CRN ULS, 2004), amounting to about 10 %, 15-18 % and 26 % of the standard (adapted and follow-up) and toddler; soya, lactose-free and pre-term; and hypoallergenic formulae, respectively. Therefore, manufacturers are called upon to make continued efforts to routinely monitor chromium levels, mainly for specialised and pre-term formulae, and at the same time, might consider the inclusion of labelling the levels of chromium at least in these complex formulations.

Introduction

That chromium is an essential element for humans is well documented. Trivalent chromium, is the main chemical form found in foods, and is essential for maintaining normal glucose metabolism. Signs and symptoms of deficiency have been shown in infants suffering from kwashiorkor or protein-energy malnutrition, who found improvement in impaired glucose tolerance after chromium supplementation [START_REF] Anderson | Essentiality of chromium in humans[END_REF]).

Nevertheless no literature report have described the response to chromium supplementation in infant fed breast milk or infant formula presently in use. Probably because those infants have not become obviously chromium deficient, being chromium intake likely adequate for pre-or term infants [START_REF] Zlotkin | Special micronutrients concerns in premature infants: Implications for enteral and parenteral feeding. Micronutrients deficiencies in the first month of life[END_REF].

Human milk feeding is the preferred nutritional model for infants during the first four to six months of life. Its composition, including micronutrients, is therefore used as a reference for infant formula manufacturing, due to the fact that infants fed on breast milk do not generally show trace element deficiencies. Upon cautious inspection of the data and references from literature, it is thus possible to suggest a reference chromium range of 0.20 -8.18 µg l -1 for human milk (Table I). Moreover, the chromium concentration does not show significant variation [START_REF] Deelstra | Daily Chromium intake by infants in Belgium[END_REF] or exhibits slight rising tend (not significant) during the lactation period [START_REF] Wappelhorst | Transfer of selected elements from food into human milk[END_REF].

[Insert table I about here]

A reliable assessment of chromium intake is essential to avoid the potential risk of deficiency or, far from it, to notice the principal sings of adverse health and toxic effects for infants. In this respect, the estimation of the theoretical chromium intake by infants exclusively fed on infant formulae and the comparison with the recommended values of Adequate Intake (AI) and Tolerable Upper Intake Level (UL), seems to be advisable (FNB, 2001).

International paediatric organizations found insufficient data on which to recommend a minimum or maximum chromium content of standard infant formulae [START_REF] Raiten | Assessment of nutrient requirements for infant formulas[END_REF].

It was noted that as this micronutrient is ubiquitous in nature a formula based on usual food ingredients does not need any chromium addition. However, the Australia New Zealand Food Authority agreed with no need for the addition or supplementation of chromium in 3 adapted and follow-up formulas but suggests an amount of no less than 0.36 µg 100 KJ -1 , (10.0 µg l -1 ) and no more than 2.0 µg 100 KJ -1 , (56.7 µg l -1 ) for an infant formula product for specific dietary use, as preterm, specialised or soya formulae, listing the chromium sulphate as exclusive additional permitted chemistry form (ANZFA 2002(ANZFA , 2005)).

Furthermore progress in the understanding of chromium nutritional chemistry has been frustratingly slow, hampered by serious analytical difficulties encountered in the accurate measurement of very low chromium concentrations in biological and food samples. Several literature reports of chromium content of foods, especially for the lower levels, show large variability and they should be analysed with caution for a valid interpretation of reliable results [START_REF] Miller-Ihli | Graphite furnace atomic absorption spectrometry for the determination of the chromium content of selected U. S. foods[END_REF].

Today many of analytical problems found have been overcome by the development of closed microwave attack and highly sensitive analytical instruments. Electrothermal atomization atomic absorption spectrometry provided with Zeeman background correction is recognized to be suitable for accurate chromium determination in food digests [START_REF] Tinggi | Determination of manganese and chromium in foods by atomic absorption spectrometry after wet digestion[END_REF]. Regardless chromium analysis at low levels is still considered a challenge to the technical skill of the analyst (Kubadda et al. 2003), in view of the fact that contamination of samples can relatively easily occur, although all kind of precautions were taken, a strict analytical quality assurance programme must be adopted to obtain reliable results [START_REF] Krachler | Concentrations of selected trace elements in human milk and in infant formulas determined by magnetic sector field inductively coupled plasma-mass spectrometry[END_REF].

The aim of the present survey was twofold. First, the quantification of chromium levels in the majority of infant formulae sold commercially in Spain is presented. Additionally, potential sources of exogenous (processing, manufacturing and packaging practices) and endogenous (raw material) origin on chromium levels is also discussed. It might be suggested that elevated chromium levels were the result of the process of manufacture such as different studies have reported about the probability that much of chromium associated with processing foods is exogenous chromium introduced during processing [START_REF] Miller-Ihli | Graphite furnace atomic absorption spectrometry for the determination of the chromium content of selected U. S. foods[END_REF][START_REF] Reilly | Transition metals: chromium, manganese, iron, cobalt, nickel, copper, and molybdenum. Metal Contamination of Food: Its Significance for Food Quality and Human Health[END_REF], 2004). The processing industry uses almost exclusively stainless containing 13 -30 % chromium in its processing equipment material. Thus, contamination in infant formula with chromium should be expected.

And finally, the determination of total chromium is appropriate to evaluate the essential chromium food composition data. In this nutritional context, the obtained information is of paramount importance for the reliable assessment of daily chromium intake of infants fed on studied infant formulae. Therefore, the estimation of theoretical chromium intake from both, infant formulae and additional contribution of tap water to reconstitution of powder formulae; and its comparison with recommended values of Adequate Intake (AI) and Tolerable Upper Intake Level (UL) is advisable, since data on the potential adverse effects of chromium intake are not conclusive (FNB 2001) and, thus caution on fortification practices is requested to manufactures of infant formulae.

Material and Methods

Infant formula and water samples

104 infant formulae samples of different commercial brands, included both powder (n = 97) and ready to use (n = 7) formulae, were acquired from specialised markets and pharmacies in Pamplona (Spain). The infant formulae studied were either milk-(n = 97) or soy-(n = 7) based types. Different types of cow's milk-based formulae were classified in: Preterm formula (n = 7, product specially formulated to satisfy particular needs of infants born prematurely or of low birth weight), Adapted formula (formula which satisfies the nutritional requirements of infants from the first day to 4-6 months of age; Type 1: formula adapted following the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) recommendations (n = 21) [START_REF] Koletzko | Global standard for the composition of infant formula: Recommendations of an ESPGHAN Coordinated International Expert Group[END_REF] and Functional 1: formula adapted with special additives or new ingredients (n = 13), Follow-up formula (formula which constitutes the principal liquid source of nourishment in a progressively diversified diet for infants aged from six months of age; Type 2: formula adapted following the ESPGHAN recommendations (n = 20) and Functional 2: formula adapted with special additives or new ingredients (n = 10), Toddler formula (formula similar to Follow-up but specially designed for infants from 1 to 3 years age old, n = 6), Hypoallergenic formula (specialised formula which are normally based in hydrolysed proteins, n = 10), Lactose-free formula (Formula designed as principal source of food for lactose intolerant infants, n = 10).

Drinking water samples were collected from eighty-eight sampling points, selected to be representatives of the Community of Navarra (Spain), according to a population census provided by the local office of the National Statistical Institute. A strict protocol was established to carry out tap water sampling. Tap water samples were collected in acidwashed low density polyethylene containers. All samples were acidified with sub-boiling

Reagents and solutions

All chemicals used were of highest purity available and all the material were nitric acidwashed (5 %, v/v) for at least 6 days and given them a thorough rinse with ultrapure water.

Standard and sample solutions were prepared with ultrapure deionised water type Milli-Q (resistivity 18 MΩ . cm). Concentrated 65 % nitric acid (suprapur grade, Merck, Darmstadt, Germany) was additionally purified by sub-boiling distillation (Hans Kürner, Rossenheim, Germany). Working standard solution were prepared in subboiling nitric acid to match the acid concentration in digestion solutions from a stock 1000 mg l -1 standard solution supplied by Merck. A solution of magnesium nitrate (0.150 g Mg(NO 3 ) 2

. H 2 O -suprapur Merck-were diluted in 100 ml with ultrapure water) was used as matrix chemical modifier.

The Standard Reference Material 1549 -non-fat milk powder -(National Institute of Standards and Technology, Gaithersburg, USA) was used for validation of the methodology.

Analytical procedures

Special care was devoted to minimize the risk of adventitious contamination when handling. Infant formulae were opened in the clean room under flow laminar bench using talc-free gloves (Rotiprotect® Carl Roth, Karlsruhe, Germany). All steps of sample preparation procedure were carried out in a laboratory equipped for trace element analysis and following a strict handling rules.

A infant formula sample amount of 0.3 -0.5 g accurately weighted (powder formula) or 3 ml of ready to use formula were placed into high pressure teflon digestion vessel and treated with 8 ml of sub-boiling nitric acid in a microwave digestion system (Ethos Plus, Millestone s.r.l., Sorisole, Italy). The optimised microwave digestion programme applied included two steps: 25-170 ºC for 10 min. and 170 ºC for 10 min., both at 1000 W, followed immediately by ventilation at room temperature. Digested samples were diluted to 10 ml in a volumetric flask with ultrapure water and finally, transferred to pre-cleaned polypropylene tubes. Solutions were kept stored frozen at -20 ºC until analysis. All infant formula samples were digested in triplicate. Samples and standards were atomised and all data were taken at 357.9 nm with a slit width of 0.7 nm. Argon of 99.9999 % purity at 250 ml min -1 flow was used as internal gas.

F

Readings on the spectrometer were taken using the peak area mode.

[Insert table II about here]

Injections (10 µl sample and 10 µl matrix modifier) were made in triplicate. Measurements were accomplished by direct calibration using aqueous standards (0-10 µg l -1 ) made up each day by dilution from stock standard solution with enough subboiling nitric acid to a final acid concentration similar to digested samples. The analytic sensitivity expressed as the characteristic mass was calculated to be 4.8 pg.

Quality assurance

A strict analytical quality control programme was employed during the study.

Limit of detection and quantification: Blank reagent values were monitored throughout the survey and subtracted from the measured sample concentration to calculate the final result in infant formulae samples. Detection limit (LOD) in the acid digest solution was calculated according to the definition and criteria established by IUPAC (X b + 3σ b ) as the average of three times the standard deviation of the reagent blank, setting at 0.15 µg l -1 (n = 6) equivalent to 0.51 µg l -1 when expressed in terms of infant formula, calculated on the basis of the analytical sample weight (0.4 g), volume of dilution (10 mL) and the mean percentage of reconstitution (13.5 g of powder formula per 100 mL of water) for powder formulae or analytical sample (3mL) and dilution (10 mL) volume in the case of ready-touse formulae. In the same way,. the limit of quantification (LOQ = X b + 10σ b ) was set at 0.18 µg l -1 (n = 6) that corresponds to 0.62 µg l -1 (expressed in terms of infant formula wet weight). Blank reagents in tap water analysis, consisting of ultrapure deionised water and reagents, were subjected to a similar sample preparation, storage and mixing with buffer solution, setting the LOD at 0.08 µg l -1 .

Accuracy: The accuracy of the analytical chromium method was verified by analyzing the standard reference material SRM 1549 (non-fat milk powder), prior to the analysis of unknown infant formulae samples. 1g. of reference material was digested in the microwave apparatus and diluted up to 10 mL with ultrapure water. The level of concentration determined in the acid solutions (mean ± standard deviation value: 0.240 ± 0.009 µg l -1 ), close to the LOQ, provides the reliability of the analytical method. The found value (mean ± standard deviation at 95 % confidence interval: 2.40 ± 0.06 ng g -1 , n = 12) show acceptable good agreement with SRM chromium certified value (2.6 ± 0.7 ng g -1 ).

Moreover, analytical recoveries of spiked chromium at different amounts (2, 4 and 8 ng) before digestion in SRM 1549 samples were satisfactory (95.5 -104.1 %). Recovery assays were carried out at different weights (0.75 and 1.0 g) of SRM 1549 samples, obtaining similar results. This test is particularly sensitive at low concentration to reveal analytical errors and it could be considered as an evaluation of accuracy, complementary to recovery studies [START_REF] Dabeka | Considerations in the estimation of recovery in inorganic analysis. The use of recovery factors in trace analysis[END_REF].

Precision: The instrumental precision estimated from six consecutive measures of the same infant formula (10.18 ± 0.09 µg l -1 ) was 0.8 %. The relative standard deviation of the method determined in the intra-assay precision (aliquots of samples measured during the same assay session) for the blank reagent (14.0 %), SRM 1549 samples (3.6 %) and internal aqueous control (2.4 %) were comparable with those calculated for the inter-assay precision (aliquots of the same sample measured in different days), 21.6 %, 7.3 % and 4.8 %, respectively.

Quality control: An internal aqueous quality control (1.04 ± 0.02 µg l -1 , range = 0.93 -1.14, n = 36) was run concurrently with blank reagent and SRM 1549 digestion, throughout the course of the analysis and always previously measured to each batch of samples, in order to satisfy the criteria established in the quality program (lower and upper action limits: 0.85 and 1.15 µg l -1 , respectively) and to provide on -going quality control information.

Statistical Analysis

All statistical analyses were run using SPSS v.11.0 for Windows program. The Kolmogorov-Smirnov statistic was used to assess the normality of the data distributions.

Different groups of infant formula samples (classified as types or predominant protein content) were compared through non-parametric Kruskal-Wallis and Mann-Whitney U-test or Wilcoxon Signed Rank test for independent or paired groups, with statistical significance set al p < 0.05). Table III summarizes the chromium concentrations for each of the different types of infant formula studied. The results are expressed as µg l -1 according to the manufacturer's dilution (Adapted: 12.80 -15.9 %, Follow-up: 12.7 -16 %; Toddler: 14.0 -16.0 %, Preterm: 14.7 -15.9, Hypoallergenic: 12.8 -15.2, Lactose-free: 12.6 -14.0 %, Soya: 12.7 -13.5 %) instructions, owing to the fact that usually levels of trace elements in formulae are expressed in literature on a ready-to-use basis.

Results and Discussion

Chromium levels in infant formula

In a first approach, the complete distribution of chromium content in studied infant formula shows a positive skew (skewness value 0.89) with scores clustered to the left at the low values and a positive kurtosis value (0.32) indicating a rather peaked distribution (clustered in the centre). In spite of the result of the test of normality suggests a violation of the assumption of normality, fact quite common in larger samples [START_REF] Pallant | SPSS survival manual : a step by step guide to data analysis using SPSS for Windows[END_REF], the inspection of the normal probability plot (Normal Q-Q plot) and the assessment of normality in the different distributions of distinct types of infant formula allow the use of mean (± standard deviation at 95 % confidence interval) and the range as useful information concerning the statistical description of data. Median is also included in table III as informative value and, at the same time, the most representative parameter in those distributions with short number of samples.

[Insert table III about here]

Globally, considering all 104 formulae together, chromium concentration determined (9.60 ± 1.13 µg l -1 , range 2.06 -46.50) is higher than that found in mature human milk. Chromium values in breast milk reported from other researchers exhibit large differences.

Mainly, older data tended to be high due to the lack of precautions to avoid contamination of the sample and the inadequate analytical quality assurance scheme adopted to obtain reliable results.

In this respect, as might be expected due to analytical difficulties, a wide variability in infant formula chromium content reported in literature is highlighted in table IV. Determined chromium values are in good agreement with the findings of most authors in formulae from different countries. However, values reported in some surveys [START_REF] Biego | Determination of mineral contents in different kinds of milk and estimation of dietary intake in infants[END_REF], MAFF 1999) are considerably higher probably due to the limited number of samples or insufficient control of contamination in sample handling.

[Insert table IV about here]

Undoubtedly, the differences in the chromium contents in different commercial formulae studied are of special relevance. In this respect, the box plots are useful to compare the different types of infant formula (Figure 1) and, simultaneously a non-parametric statistical analysis due to the short number of formulae (n < 12) in six of 9 studied groups, by means of Kruskall-Wallis test was carried out, establishing significant differences (p < 0.001).

Therefore, it is possible to try to establish a relation of dependence based on chromium content in accordance with the type of infant formulae, the complexity of manufacturing process or even better, the predominant protein source used in infant formula manufacture.

[Insert figure 1 about here]

The higher chromium levels provided by hypoallergenic (18.16 ± 7.89 µg l -1 ), lactose-free (11.37 ± 3.07 µg l -1 ), preterm (11.48 ± 3.15 µg l -1 ) and soya (10.43 ± 4.05 µg l -1 ) formulae greatly exceed those values found in adapted (type 1: 6.29 ± 1.21 µg l -1 , functional 1: 7.77 ± 2.22 µg l -1 ) formulae (U-Mann Whitney test; Type 1 -HA: p < 0.001, Type 1 -Preterm: p = 0.004, Type 1 -Lactose-free: p = 0.002, Type 1 -Soya, p = 0.007; Functional 1 -HA: p < 0.001, Functional 1 -Preterm: p = 0.040, Functional 1 -Lactose-free: p = 0.050). Follow-up (type 2: 8.73 ± 2.17 µg l -1 , functional 2: 8.35 ± 1.55 µg l -1 ) and toddler (9.68 ± 6.71 µg l -1 ) formulae contain an intermediate level (U-Mann Whitney test; Type 2 -HA: p = 0.001; Functional 2 -HA: p = 0.001).

Nowadays, manufacturers of infant formulae currently in use do not report any chromium content on the label, probably because no supplemental chromium is added or/and fortification practices is still exercised with caution by manufacturers of infant formulae. Thereby another factor should exists which explain the large variability of chromium observed in infant formulae. In this respect, our findings could clarify the sources or factors with special influence on chromium content in infant formulae.

Firstly, the endogenous chromium content is contributed by raw material (cow's milk or isolated soy protein). Generally, cow's milk contains low and wide variable levels of chromium attributed to a different kind of feed consumed by these animals and to their different chromium content [START_REF] Bratakos | Chromium content of selected Greek foods[END_REF]. In contrast, soy beans been characterized as a good source of chromium though its content is variable owing to soil composition and agricultural practices [START_REF] Jorhem | Levels of lead, cadmium, zinc, copper, nickel, chromium, manganese, and cobalt in foods on the Swedish market[END_REF]. Thereby, higher levels found in soya formulae could point out to the intrinsic chromium naturally present in soy protein.

Following, it is known that chromium in valence state III, has a strong tendency to form coordinate and chelate compounds and interacts with protein molecules in order to make up strong and stable cross-links [START_REF] Bratakos | Chromium content of selected Greek foods[END_REF]. Therefore, it is not surprising that ). Undoubtedly, the intrinsic chromium is likely the most source of this element in the formulae with lower technical complexity of manufacture.

[Insert tables V and VI about here]

On the other hand, migration from equipment and fittings may explain the high chromium contents found in soya, lactose-free and preterm formulae. In addition, the highest levels of chromium in casein and whey hydrolysed formulae are a direct consequence of the aggressive protein hydrolysis treatment which involves the use of chemicals and additives. Moreover, other source of contamination must be considered in these formulae, the addition of several ingredients as calcium and phosphate salts, vitamins, other minerals and trace elements.

Another potential source of chromium contamination to take account in infant formulae is the packaging in metal cans. The pacifying treatment in which chromium compounds are used to increase the lacquer adherence and resistance to oxidation of surface, can contribute significantly to the level of pick-up by canned infant formula [START_REF] Reilly | Transition metals: chromium, manganese, iron, cobalt, nickel, copper, and molybdenum. Metal Contamination of Food: Its Significance for Food Quality and Human Health[END_REF][START_REF] Reilly | Chromium. The Nutritional Trace Elements[END_REF]. Chromium levels for both powder and ready to use formulae are summarised in table VII. It was not possible to establish statistical differences (Wilcoxon's test, p > 0.05) between the pairs of infant formula market in both aggregation form. However, powder formulae showed a tendency to be higher chromium content in ready-to-use formulae.

Furthermore, general chromium levels in canned formula were consistently higher than those commercialised in aluminium bags (results not shown) for each types of infant formulae studied. In conclusion, the chromium concentration of the studied infant formulae come from both origin endogenous and exogenous, although the relatively high levels found in preterm, soya and specialised formulae are most likely the result of leached from contact with stainless steel during manufacturing or packaging rather than fortification practices, given that chromium concentrations is not listed in none infant formula analysed being currently considered as a voluntary element for nutritional information labelling.

F

Daily dietary chromium intake

The daily intake of this element has been estimated using the chromium concentration mean value determined in the different types of formulae according to feeding tables and dose recommended by manufacturers, and taking into consideration the different stages of infancy (0-2 weeks: 600 mL, 3-4 weeks: 900 mL, 2 month: 1050 mL, 3 month: 1100 mL; 4-5 month: 1250 mL, 6 month: 1000 mL, 7-12 months: 960 mL, 2 year: 560 mL, 3 year: 560 mL), under the assumption that most infants observe similar feeding regimens and only formulae. Anyway, it is necessary to underline the fact that most of infants fed on follow-up formulae also receive rusks (complementary feeding) nutrients from four-five months old which may increase the chromium intake from other food sources better than formulae.

The AI was set by the National Research Council based on chromium intake of infants principally fed on human milk, due to no functional criteria of chromium status reflect their dietary intake. These requirements have been established as 0.2 µg day -1 (29 ng kg -1 day -1 ), 5.5 µg day -1 (611 ng kg -1 day -1 ), and 11 µg day -1 (1222 ng kg -1 day -1 ) for infants 0-6 months, 7-12 months and 1-3 years old, respectively (FNB, 2001).

The daily intake values calculated of chromium from the newborn period to the third year of life are show in table VIII. The chromium supplied by different infant formulae is clearly higher, approximately 45 and 75-100 times with standard and specialised (hypoallergenic and lactose-free) formulae respectively, than that provided by human milk. All studied formulae provided amounts of chromium intake higher than recommended AI.

[Insert Unfortunately, the differences in bioavailability of chromium from breast milk and infant formulas are not known [START_REF] Goldhaber | Trace element risk assessment: essentiality vs. toxicity[END_REF], FNB, 2001). However, chromium absorption via the gastrointestinal tract has been inversely related to dietary intake [START_REF] Anderson | Chromium intake, absorption and excretion of subjets consuming self-selected diets[END_REF], depending upon chemical form, oxidation state and other factors (lack or presence of ascorbic acid, carbohydrates, phytate…) [START_REF] Iyengar | Nutritional chemistry of chromium[END_REF]). Chromium absorption is approximately 2 % at daily dietary intakes of 10 µg, reaching 0.5 % when this is increased at least 40 µg. Even though taking into account the percentage of absorption estimated, the studied formulae provide amounts close to the AI.

Preterm infants are a special group of risk that may be susceptible to chromium deficiency.

In this sense, the Canadian Paediatric Society (CPS 1995) has proposed a chromium requirement for low birth weight and premature infants (52-98 ng kg -1 day -1 ) distinct from those for full-term infants. Figure 2 compares the daily chromium intake of infants fed on preterm formulae and human milk (Spanish and Reference range) with this recommended CPS-AI value. These formulae provide a chromium intake slightly greater than standard formulae and greatly exceeds, approximately 55 times, that supplied by both lower level human milk and recommended adequate intake.

[Insert figure 2 about here]

Finally, as mentioned above, several dietary components, different chemical components and trace elements deficiency state could influence chromium absorption in both sense enhancing or decreasing. Therefore, taking into account the markedly amount of chromium present in infant formulae analysed, it seems to be demanded to determine more definitively the safety of infant formulae. It is true that no data or reports have shown categorical adverse effects of chromium III in human nutrition, also it was suggested that tolerable limit for chromium is quite high with an extraordinary wide margin of safety.

Thus, the Council for Responsible Nutrition (CRN) set its Upper Limit Safety (ULS) at 1 mg day -1 (any form of Cr III) for adults [START_REF] Hathcock | Vitamin and Mineral Safety[END_REF]) based on a large number of trials and official reviews (FNB 2004, EVM 2003), that extrapolated to infants according to standard weight tables, reaches a maximum intake of 0.014 mg kg -1 day -1 (0-2 weeks: 55 mg day -1 , 3-4 weeks: 63 mg day -1 , 2 month: 76 mg day -1 , 3 month: 87 mg day -1 ; 4-5 month:

105 mg day -1 , 6 month: 112 mg day -1 , 7-12 months: 126 mg day -1 , 2 year: 182 mg day -1 , 3 year: 224 mg day -1 ).

Luckily, the maximum theoretical estimated intake of infant fed on studied formulae was lower than the ULS (figure 3). Standard (adapted and follow-up formulae) and toddler formulae contribute the lowest chromium intake (about 10 % ULS); soya, lactose-free and [Insert figure 3 about here]

Thereby, taking into account the additional margin of safety established by upper limit, it seems appropriate to estimate the maximum chromium concentration in a hypothetical case of an infant formula to exceed the ULS. Following the recommended feeding of manufacturers, the limit of chromium in infant formula should range from 68 to 91 µg l -1 .

Fortunately, none of infant formula studied reach so great level of chromium concentration. Nevertheless, it should be kept in mind that the level of this element as hexavalent chromium is unknown and, both, increased absorption of this chemistry form and low stomach acidity might represent a particular vulnerability situation for infants. Its quantification could be useful in the risk assessment [START_REF] Soares | Selective determination of chromium (VI) in powder milk infant formulas by electrothermal atomization atomic absorption spectrometry after ion exchange[END_REF].

Adventitious chromium contribution of drinking water

Total chromium concentrations in drinking water are usually less than 2 µg l -1 , although levels as high as 120 µg l -1 have been reported in several countries (WHO 2003). The WHO guidelines (WHO 1996) has considered the level of 50 µg l -1 , as a provisional guideline values, which is considered to be unlikely to give rise to significant risk to health. At this level, tap water appears to be an important source of chromium, contributing substantially to daily intake of infants.

The chromium content found in drinking water samples collected in 88 representative sampling points from the Community of Navarra, was 0.74 ± 0.19 µg l -1 (range 0.18 -4.65 µg l -1 ). This median chromium value was used to calculate, based on feeding tables for infant formula reconstitution, the complementary contribution from tap water to daily chromium intake of infants fed on powder infant formula. The theoretical chromium intake, including minimum and maximum values, is shown in table VIII. Comparatively, the influence of chromium level in drinking water on the final concentration of reconstituted powder infant formulae is limited. Nevertheless, it is representative that the supplied chromium by tap water might find by itself the AI during the first six months of life (approximately 2 -4 times of the considered adequate intake) and at the same time, it match the amount provided by Spanish human milk in the feeding of preterm infants (figure 2).

Conclusions

The findings of this survey demonstrate that chromium levels analysed in infant formula are markedly greater than those found in the reference range from human milk or those level (0.17 µg l -1 ) calculated from the AI value to be necessarily provide by an infant formula to fulfil the recommendation. Therefore, the measurement of total chromium routinely by manufacturers and its inclusion in labelling information list will be of interest, especially in those complex infant formulae designed for special uses. In this respect, viewed from a research standpoint, speciation surveys (Bratter et al. 1998)

S o y a L a c t o s ef r e e H y p o a l l e r g e n i

  approximately pH = 2, frozen and kept at -20 ºC until required for analysis.

  an intermediate intake (15-18 % ULS) and hypoallergenic formulae provided the highest intake (26 % ULS).

  Norwalk, CT, USA), under the optimized operating conditions shown in table II. Transversely-heated graphite tubes with end caps supplied by Perkin Elmer were used. A single element hollow cathode lamp (Perkin Elmer) was operated at 25 mA.

	Chromium concentrations were determined in sample digestions by Zeeman background
	correction graphite furnace atomic absorption spectrometry (ZGF-AAS, Perkin Elmer
	Aanalyst 800,
	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	6

  table VIII about here]

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

  are required in the future to research both, exogenous or intrinsic chromium source of origin and valence state of chromium released from stainless steel surfaces[START_REF] Lameiras | Quantification of total chromium and hexavalent chromium in UHT milk by ETAAS[END_REF] with the objective of establish the total harmlessness and safety of infant formulae.

	EVM, Expert Group on Vitamins and Minerals, 2003, Safe upper levels for vitamins and
	minerals. Food Standards Agency, United Kingdom, May 2003. Available at:
	http://www.food.gov.uk/multimedia/pdfs/vitmin2003.pdf
	FNB, Food and Nutrition Board, Institute of Medicine, 2001, Chromium. Dietary
	F o r F o r F o r F o r F o r F o r
	P P P P P P
	e e e e e e
	e r R e v i e e r R e v i e e r R e v i e e r R e v i e e r R e v i e e r A F o l d a l o p t w -u p e d F R e e i v F u u n c t n c i o t i o n a l n a 2 l 1
	w w w w w w
	O n l O n l O n l O n l O n l O n l
	y y y y y y
	16

Table I .

 I Percentages of Upper Limit Safety for chromium estimated from infant formulae. Chromium content (µg l -1 ) of mature human milk from different countries

	Page 21 of 32	Food Additives and Contaminants
	30
	Human milk (lower limit) Adequate intak e (AI-CPS) Drinking water Spanish human milk Human milk (higher limit) Preterm formula o F r P e e r 5 10 15 20 25 Figure 1. Box plots of determined chromium content in different types of infant formulae Adapted F. Lactose-free F. Hypoallergenic F. Soya F. Follow-up F. Toddler F. studied (µg l -1 ) Figure 2. Daily dietary chromium intake (µg day -1 ) for infant fed on premature infant formulae, human milk (Spanish and Reference levels) and drinking water used in powder Country Reference Cr (µg L -1 ) n Range Austria Krachler et al. 2000 24.3 27 < 0.8 -163 Belgium Deelstra et al. 1988 0.14 ± 0.054 10 0.10 -0.23 Finland Kumpulainen et al., 1983 0.39 ± 0.15 20 0.20 -0.53 % Upper Limit Safety (ULS-CRN) F o r formula reconstitution. Figure 3. F o r Philippines WHO/IAEA 1989 3.46 10 0.97 -6.13 Germany Wappelhorst et al. 2002 10.8 19 3.1 -19.4 Guatemala WHO/IAEA 1989 1.17 9 0.34 -2.52 Hungary WHO/IAEA 1989 0.78 11 0.23 -2.24 F o r 0 0 -2 week 3 -4 week 2 month 3 month 4 -5 months 6 month 7-12 months 2 year 3 year Age P e e r P e e r Italy Aquilio et al. 1996 Coni et al. 1990 Muzzarelli et al. 1983 1.2 ± 0.5 9 -110 9 8 -2 -59 P 0.5 -3.6 Nigeria WHO/IAEA 1989 4.35 4 e 3.44 -8.18 Spain Cocho et al. 1992 Rivero et al. 2001 1.56 ± 0.78 0.8 ± 0.3* 21 1 0.27 -3.00 -e r
	R R R WHO/IAEA 1989 United Kingdom MAFF 1996 Sweden R	1.48 -	6 4	0.61 -4.34 < 16 -40
	e v i e w e v i e w e v i e w Casey and Hambidge 1984 0.27 ± 0.11 49 0.06 -1.56 Anderson et al. 1993 e 0.18 ± 0.02 17 -Mohamedshah et al. 1998 0.22 20 0.09-0.46 Gunshin et al. 1985 6.5 26 1.40 -21.0 WHO/IAEA 1989 1.07 v i e 11 < LOD -3.99 *deviation standard from five replicates USA Japan Zaire w
			O n l O n l O n l O n l
				y y y y
					3 4 2

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table II .

 II Graphite furnace program for the determination of chromium in infant formulae.

	Page 25 of 32	Food Additives and Contaminants
	Step	Temperature (ºC)	Ramp (s)	Hold (s)	Argon flow (ml min -1 )	Read on
	Drying	130	15	40	250	-
	Charring	1400	10	20	250	-
	Atomization	2300	0	5	0	Yes
	Cleaning Cooling F o r 2500 20	1 -	5 -	250 250	--
		P
		e
		e r
		R
		e
			v i e
				w
				O n l
					y
						3

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table III .

 III Chromium concentrations in different types of infant formulae investigated (µg l - 1 ).

	Infant formulae	n Median Media ± SD*	Range
	Adapted Formula	34	6.66	6.86 ± 1.09 2.62-16.15
	Type 1	21	5.89	6.29 ± 1.21 2.62-14.09
	Functional 1 Follow-up Formula 30 13 Type 2 20 F o r	6.75 8.05 8.24	7.77 ± 2.22 2.87-16.15 8.60 ± 1.47 2.06-20.33 8.73 ± 2.17 2.06-20.33
	Functional 2 Toddler Formula	10 6 P e 7.85 8.12	8.35 ± 1.55 5.96-13.10 9.68 ± 6.71 2.91-21.10
	Preterm Formula Hypoallergenic F.	7 10	12.94 15.02 e r 11.48 ± 3.15 4.83-14.61 18.16 ± 7.89 8.22-46.50
	Lactose-free F.	10	11.67 R 11.37 ± 3.07 5.29-18.28
	Soya Formula	7	9.55	10.43 ± 4.05 5.82-19.15 e
	Total *SD at 95 % interval	104	8.15	9.60 ± 1.13 2.06-46.50 v i e
				w
					O n l
					y
					4

Table IV .

 IV Chromium levels found from different types of infant formulae (µg l -1 ).

		Deelstra et al. 1988	3.3 -19.6	11	Milk-based
			12.1	1	Soy-based
			5.7	1	Lactose-free
	Country	Reference	Cr (µg L -1 ) n 7.5-8.1 2	HA	Description
	Austria Belgium France Italy *Recalculated chromium concentration and expressed in µg/L Krachler et al. 2000 Deelstra et al. 1988 Abbot 1998 Biego et al. 1998 Muzzarelli et al. 1983 F o r F o r	< 4.0 -14.5 4 18.0-23.4 3 6.2 ± 2.2 12.1 14.2 ± 7.3 7 1 6 7.7 -20 -40 -142 -174 -45 ± 8 26 ± 5 5 4 1.9 ± 1.8 3.9 ± 0.6 4.2 7 2 1 8 -22 -	Therapeutic F. Adapted formula Milk-based Adapted formula Soy-based Soy-based Casein Hydrol. F. Special formula Milk-based Soy-based Adapted formula Lactose-free Soy-based Preterm
	Nigeria Spain	Ikem et al. 2002 Cocho et al. 1992 P P e e e r e r	6 ± 3 5.19 ± 3.26 3.83 ± 3.29 4.19 ± 1.42 13.92 ± 4.46 12.03 ± 7.58 21.12 ± 8.67	2 17 7 3 3 8 9	Powder Milk-based Adapted formula Follow-up Preterm Soy-based Special formula Metabolic formula
	Viñas et al. 1997 United Kingdom Food Standards Agency 2003 R R Ikem et al. 2002 MAFF 1999 e e v i 27.0 ± 6.7* 10.4* 5.3* 3.9* 8.9* 10.8* 40 5 ± 5 15 ± 4 15.0 e v i e w w	1 1 1 18 3 3 4 6 16 4	---Milk-based Soy-based Ready to use Powder milk-based Ready to use Powder milk-based Ready to use
		MAFF 1996 MAFF 1995	< 16 -20 20 < 16 -20 < 18 20 -30 < 16 -20 2.16 -99.22 O n 5 5 5 5 4 4 51 l Powder Whey-bas. Liq. Whey-based Powder Casein-bas. Liq. Casein-bas. O n l Follow-up Soy-based Powder Milk-based y y
	USA	SHS 2005	11.2*	-	Free aa
		Ikem et al. 2002	7 ± 9	3	Powder Milk-based
			11 ± 7	2	Powder Soy-based
		Patterson et al. 1985	7.5	1	Preterm formula
			18	1	Preterm formula
						5 6

Table V .

 V Chromium content in infant formulae according to the main protein contained (µg l - 1 ).

		Food Additives and Contaminants
	Infant formula	n Median	Media ± SD	Range
	Whey-based	23 6.66	6.41 ± 1.08 a	2.62-13.53
	Skim-milk-based 41 7.54 Whole-milk-based 4 7.25 Soy-based 7 9.55 F o r F o r	8.20 ± 1.27 a, b 9.12 ± 8.54 a, b, c 10.43 ± 4.05 b, c, d	2.06-21.10 5.21-16.77 5.82-19.15
	Casein-based Whey hydrolysed 13 12.16 10 11.67 P P Casein hydrolysed 5 14.08 e e Free aminoacids 1 -e r 11.37 ± 3.04 c, d, e 13.03 ± 2.82 c, d, e 15.03 ± 5.52 e 46.50 f e r	5.29-18.28 7.03-20.68 9.74-22.01 -
			R R
			e e
			v i e v i e
			w w
			O n l O n l
				y y
				8

a, b, c, d, e, f Results with the same superscript letter were not significantly different (p > 0.05) Page 29 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table VII .

 VII Chromium levels (µg l -1 ) from different container types of powder or liquid formulae.

		n Median Media± SD	Range
	Powder F.	97	8.22	9.83 ± 1.19	2.06-46.50
	Canned F.	61	8.39	10.00 ± 1.69 2.06-46.50
	Al. bag F. Ready-to-use Tetra brick F o 36 7 4 r	7.82 7.43 5.82	9.55 ± 1.56 6.35 ± 2.90 6.30 ± 5.78	2.87-22.01 2.70-10.87 2.70-10.87
	Glass bottle Plastic bottle	2 1 P 8.18 -e	8.18 ± 1.05* 2.91	7.44-8.92 -
			e r
				R
				e
				v i e
					w
					O n l
					y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

The authors are grateful to the Departamento de Industria y Tecnología, Comercio, Turismo y Trabajo (Gobierno de Navarra) and Plan de Investigación de la Universidad de Navarra (PIUNA) for financial support.


Jacqueline ; Van Der Wielen-Hustinx
email: fac@tandf.co.uk

Keywords: BaP, routine LC-method, fluorescence detection, validation, quantification, oils, food supplements

Determination of the level of benzo[a]pyrene

* corresponding author -jacqueline.van.der.wielen@vwa.nl A routine method was developed for the quantification of benzo[a]pyrene (BaP) in edible oils and food supplements. BaP is often taken as an indicator of the presence of polycyclic aromatic hydrocarbons. The method consists of on-line LC-clean up followed by injection to an HPLC-system with fluorescence detection. The method has good performance characteristics and gave good results in proficiency tests.

From 2002 to 2004 about 1350 samples of oils and food supplements, were analysed using this method to test the level of benzo[a]pyrene. About 20% of the edible oils contained more than 1.2 µg/kg BaP (which is the limit applied by the Dutch Food and Consumer Product Safety Authority until 1 April 2005, and includes measurement uncertainty). In the case of food supplements more then 30% contained too high levels of BaP, ranging from 1.2 to 135 µg/kg.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, a number of which are considered to be human carcinogens. In foodstuffs, PAHs can be formed directly as a result of some heat processes (charcoal grilling, roasting, smoke drying) or by addition of smoke flavouring additives [START_REF] Garcia-Falcon | Analysis of BaP in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples[END_REF][START_REF] Simko | Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives[END_REF]; [START_REF] Barranco | Solid-phase clean-up in the liquid chromatographic determination of polycyclic aromatic hydrocarbons in edible oils[END_REF]. It has been estimated that human intake of PAHs from food is considerably higher than that from air or drinking water, edible oils and fats being the most significant sources because of their lipophilic nature [START_REF] Garcia-Falcon | Analysis of BaP in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples[END_REF][START_REF] Barranco | Solid-phase clean-up in the liquid chromatographic determination of polycyclic aromatic hydrocarbons in edible oils[END_REF]. PAHs can be removed from edible oils by treatment with activated charcoal, a practice that has been widely used for a long time [START_REF] Weisshaar | Table II: Wavelenghts for fluorescence detection of PAHs after elution of LC system Time (min) Component Excitation (nm) Emission (nm)[END_REF]. More than 100 PAHs have been found in nature; however, only 16 have been selected as priority pollutants by the United States Environmental Protection Agency (EPA) on the basis of occurrence and carcinogenicity [START_REF] Barranco | Solid-phase clean-up in the liquid chromatographic determination of polycyclic aromatic hydrocarbons in edible oils[END_REF][START_REF] Teixeira | PAHs content in sunflower and soybean oils: Evaluation in commercial samples and during ripening p558-561[END_REF] Benzo[a]pyrene is often used as an indicator of the presence of PAHs in water and foodstuffs, even though it is well known that this compound is usually generated together with many other PAHs which contaminate the food [START_REF] Garcia-Falcon | Analysis of BaP in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples[END_REF][START_REF] Simko | Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives[END_REF][START_REF] Bogusz | Rapid determination of BaP in olive oil samples with solid-phase extraction and lowpressure, wide-bore gas chromatography-mass spectrometry and fast liquid chromatography with fluorescence detection[END_REF]). The Scientific Committee on Food (SCF 2002) concluded that BaP could be used as a marker for the occurrence and effect of carcinogenic PAH in food. However further analysis of the relative proportions of these 16 PAHs (15 EU + 1 JECFA) in foods is necessary, to review the suitability of maintaining BaP as a marker (SCF 2002, Commission of the European Communities 2005).

The International Olive Oil Council recommended in 2001 a value of 2 µg/kg as a maximum tolerable concentration of BaP as well as other PAHs in olive-pomace oil (International Olive Oil Council 2001). It is generally recognised that total PAHs in a refined oil or fat should not exceed 25 µg/kg, of which not more than 5 µg/kg should be heavy PAHs (5-and 6-nuclear PAHs) [START_REF] Fediol | Code of practise on polycyclic aromatic hydrocarbons (PAHs)[END_REF]. The SCF recommended, in their opinion of 2002, that exposure to PAHs from food should be as low as reasonably achievable (ALARA principle), due to the carcinogenicity and genotoxicity, the most critical effects of PAHs. Prior to April 2005 the majority of European countries set independently limits . The Dutch Food and Consumer Product Safety Authority (VWA) had set a limit of 1 µg/kg for BaP in oils and food supplements in 2002. Based on the ALARA principle a level of 1 µg/kg was used, as the presence of BaP is avoidable, due to processing steps which can reduce the content of PAHs. Based on a report by [START_REF] Kroese | Tumorigenic effects in Wistar rats orally administered benzo[a] pyrene for two years (gavage studies). Implications for human cancer risks associated with oral exposure to polycyclic aromatic hydrocarbons[END_REF], which set the virtual sate dose at 0.5 ng/kg bodyweight, an Acceptable Daily Intake (ADI) was calculated based on an average of people weighing 60 kg. This ADI of 30 ng was taken as a 2 µg/kg for oils and fats for human consumption as well for those used as ingredients in foods and food supplements. In order to obtain more information about the patterns of PAH distributions (profiles) relative to BaP in various foods, the Commission recommended that monitoring studies be carried out on 16 PAHs (15 EU and 1 JECFA) in various foods . This dataset can then be used to evaluate if maximum levels should be set for other PAHs and the accuracy of using BaP as a marker for all PAHs.

The principal problems associated with the determination of BaP in foods are the low detection levels (µg/kg) and the diversity of potential interferences present in food and food supplements [START_REF] Garcia-Falcon | Analysis of BaP in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples[END_REF]. Although all sample preparation steps are important, chromatographic separation is considered as most significant for correct results [START_REF] Simko | Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives[END_REF]. There are several HPLC methods for the determination of PAH in edible oils, using on-line sample clean-up followed by injecting to an analytical C18-column and fluorescence detection [START_REF] Garcia-Falcon | Analysis of BaP in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples[END_REF][START_REF] Moret | A rapid method for polycyclic aromatic hydrocarbons determination in vegetable oils[END_REF][START_REF] Bogusz | Rapid determination of BaP in olive oil samples with solid-phase extraction and lowpressure, wide-bore gas chromatography-mass spectrometry and fast liquid chromatography with fluorescence detection[END_REF]). Fluorescence detection is often used due to its selectivity and sensitivity [START_REF] Barranco | Solid-phase clean-up in the liquid chromatographic determination of polycyclic aromatic hydrocarbons in edible oils[END_REF]. GC-MS methods as well as HPLC methods are regarded as complementary in the analysis of PAHs [START_REF] Bogusz | Rapid determination of BaP in olive oil samples with solid-phase extraction and lowpressure, wide-bore gas chromatography-mass spectrometry and fast liquid chromatography with fluorescence detection[END_REF]. The objectives of this study were (1) to develop a routine method for the determination of BaP and (2) to perform a monitoring study on edible oils and oilbased food supplements. The development of the method was based on the methodology of [START_REF] Stijn F Van | Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection[END_REF], in which PAHs were under investigation. In this method, a donor-acceptor complex chromatography (DACC) technique was been used for the on-line sample clean-up. PAHs are electron donors (π-electrons) and the strong interaction of the PAHs with an electron acceptor stationary phase results in retention of the PAHs and elution of (the bulk of) the other compounds in the oil.

By coupling the DACC-column automated to the analytical column for separation of the PAHs (a LC-LC coupling), the method can be applied easily for routine analysis.

In 2002 a monitoring study was initiated to investigate the amounts of BaP in olive oils, of which several rapid alerts (a notification of a direct or indirect risk to human health deriving from Food or Feed, for which EU has set up a system) were reported.

Soon it was decided to broaden the monitoring study to other kind of edible oils, for example corn oil and sunflower oil as well as Evening Primrose oil (Oenothera biennes). This latter oil is often used as an ingredient for food supplements, e.g. for fat burners. Because it was found that "special oils", which are being used as a raw material for food supplements quite often exceeded the limit of 1.2 µg/kg BaP (the Dutch limit including measurement uncertainty) the research was broadened to further analyse food supplements. The food supplements of interest were oil capsules, e.g. fat burners, vitamin supplements. The method reported here is based on donor-acceptor complex chromatography (DACC) [START_REF] Stijn F Van | Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection[END_REF]. First, the samples were homogenized, then 10 ml isopropyl alcohol and 200 µl benzo[b]chrysene (10 ng/ml, Dr. Ehrenstorfer) were added to 500 mg of sample. The PAHs were extracted from the sample by shaking for 30 min. Benzo[b]chrysene was added for quality control purposes, to check the stability of retention times and recovery -it was not used as an internal standard for the correction of recovery. After filtration, the extract was injected on a DACC column (Chromspher-phi 7µm 80 mm). The DACC column was connected to an HPLC system (Agilent) with a switch system. The DACC column was first washed with isopropyl alcohol, then the PAHs are eluted from the column with 30% acetonitril/70% ethylacetate and finally the DACC column was cleaned with isopropyl alcohol. Only the eluted PAHs were injected onto the HPLC column by the switch system. The analytical column was a reversed phase column (Lichrocart 250-4 Lichrospher PAH RP-18 5 µm, 2* in series), held at 30°C. The HPLC conditions are listed in table I.

Method

Detection of the eluted PAHs was carried out by application of a variable wavelength fluorescence detector (Agilent). Excitation and emission wavelengths are given in Table II. A standard mix of PAHs was applied by Dr. Ehrenstorfer (PAH Mix 9, 10 ng/µl of each PAH in acetonitril). This standard mixture contains the 16 EPA PAHs, however, not all 16 EPA PAHs can be detected with the method.

[INSERT TABLE I ABOUT HERE] [INSERT TABLE II ABOUT HERE]

Sample collection

All samples were collected by inspectors of the Dutch Food and Consumer Product Safety Authority (VWA). Several kinds of oils for human consumption (e.g. sunflower oil, olive oil) were taken from shops (e.g. supermarkets) for analysis. When these samples appeared to be above the limit, samples of the same charge and also of other charges were collected at producers and/or importers before actions, like discarding a whole charge from the market, were taken. All food supplements collected were supplements on an oil base (oil capsules). For these samples the same strategy was followed as for the oil samples; thus if a food supplement from a shop appeared to be above the limit, same and other charges at producers or importers side were collected and analysed.

Results and Discussion

Method characteristics

The method of [START_REF] Stijn F Van | Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection[END_REF] 1996) uses both water/acetonitrile (15%/85%) and acetonitrile/ethyl acetate (30%/70%) to strip the PAHs off the DACC column, while in this method the PAHs were stripped off the DACC column by only 30% acetonitrile / 70% ethyl acetate. The method of [START_REF] Stijn F Van | Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection[END_REF] shows peak identification of 17 PAHs. Applying the method described in this study, 12 PAHs of the 16 EPA PAHs were identified,. 7 of these 12 identified PAHs are also mentioned in the list of the 15 'priority PAHs' by the SCF. Figure 1 shows an example of a chromatogram of a standard containing 12 PAHs. Because BaP was considered a key component, the method has been especially validated for this component, however the other PAHs identified here, can also be quantified.

[INSERT FIGURE 1 ABOUT HERE]

For the in-house validation of the method, first the limit of detection and quantification (LOD and LOQ) were determined. The LOD was defined as the concentration which is three times higher than the standard deviation of the chromatographic background.

The limit of detection determined in this way was 0.07 µg/kg. The limit of quantification has been defined as twice the limit of detection and was thus 0.14 µg/kg. The calibration curve showed linearity with a correlation coefficient of at least 0.9995 in the range of 0.14 up to 8.0 µg/kg. Therefore the range of application has been determined as 0.14 to 8.0 µg/kg. Samples containing higher values of BaP have to be diluted with isopropyl alcohol. The validity of the calibration was checked with each series of samples. The recovery of BaP from a spiked blank sunflower sample (determined from 6 replicate measurements under repeatability conditions) was 99.9% with a relative standard deviation (RSD) of 2.8%. The reproducibility, determined with different operators and different intervals of time and using a standardised control material (SCM, sunflower oil) at a level of 1 µg/kg BaP was 0.18 µg/kg. The measurement uncertainty at the level of 1 µg/kg was established at 0.2 µg/kg. The recovery and the RSD were also determined under repeatability conditions in a herbal food supplement (oil capsules), where they were 100.1% and 4.1% respectively. The reproducibility of using a food supplement at a level of 1.4 µg/kg was 0.40 µg/kg, slightly higher than for oil. The method performed well in proficiency tests with oil samples (Central Science Laboratory 2003, FAPAS Proficiency Testing 2004). The Z-scores obtained applying this method in these proficiency tests were -1.3 respectively +0.8 for benzo[a]pyrene.

Samples analysed

For every series of samples the analytical protocol included several quality control (QC) measurements, e.g. check of validity of calibration, and the analysis of QC samples. A reference material (oil) was used as QC material for oils, whereas a herbal food supplement in form of oil capsules was applied for the measurements of food supplements. The mean values and the 95% confidence interval of these QC materials are 2.21 ± 0.27 and 1.28 ± 0.30 respectively. The results of analysis of the QC samples had to be within these limits. When all quality checks for a series of samples are in line with the performance expected, samples can be evaluated on their level of BaP.

Ten oil samples, taken from the market (shops), analysed with this DACCmethod were sent to a national research laboratory in the Netherlands, where they were analysed with another LC-method and a GCMS-method. These samples contained BaP levels ranging from 1 to 30 µg/kg.The results of each of the samples obtained by these three methods were similar, all results were within the measurement uncertainty of the level of BaP in the sample.

In the monitoring study about 1350 samples were analysed between 2002 and 2004 for their BaP content , this being around 170 olive oils, 170 other oils fpr human consumption and 950 supplements . Additionaly 60 further oils (raw materials), used as ingredients for food supplements, were also analysed. These latter oils were different kind of oils, collected at producers. The results are shown in figure 2.

[INSERT FIGURE 2 ABOUT HERE]

As can be seen in figure 2 about 80% of the olive oils and other oils for human consumption (eg. Sunflower oil) contained less than 1.2 µg/kg BaP, however there were some olive oils which contained up to 50 µg/kg BaP and one with 85 µg/kg BaP.

The UK Food Standards Agency (2001) reported similar results with levels of BaP ranging from 9 to 43 µg/kg in some olive-pomace oil samples, originating from Greece, Spain and Italy. In the other oils for human consumption the amounts of BaP were much lower, the highest level rising to 9 µg/kg BaP. At inspections of producers and importers of oils for human consumption in the Netherlands by the VWA, it was noticed that producers and importers are aware of the possibility of contamination of PAHs and are taking action to reduce the amount of BaP in their oils. During the research over the last three years a slight decrease in the number of oil samples exceeding the limit of 1.2 µg/kg BaP was seen. Nearly half of the raw materials (oils) which are used as a basis for food supplements exceeded the limit of 1.2 µg/kg BaP, containing up to 80 µg/kg BaP, with 2 exceedances of 200 and 275 µg/kg BaP. During inspection of the producers of food supplements it was found that some of them were not aware of the fact that BaP could be present in their raw (oil) materials. These special oils were not controlled for PAHs or other contaminants before dissolution of the supplements and encapsulation of the products. Most of the food supplements under investigation were fat burners (herbal extracts) and fat-soluble vitamin preparations. A lot of supplements with tocopherols (vitamin E) were analysed. Tocopherols may interfere in the determination of PAHs (Joint DG JRC/DG SANCO/EFSA workshop, 2005). However with this DACC-method such problems were not found. Since the end products contain additional substances apart from oils, which probably do not contribute to the PAH contamination, the actual concentration of PAHs in those products, expressed as a fraction of the sample weight, has to be lower than that of the oil itself. Of the food supplements which were analysed 30% contained too much BaP with values of 140 and even 200 µg/kg. The percentage of food supplement samples that exceeded the limit for BaP was the same in 2004 as in 2002. This suggests that companies producing these type of products regularly ignore or are not aware of the presence of PAHs in their raw materials (oils) and/or products. Thus, action on the part of the producers as well as of the authorities is still needed to monitor this situation.

Conclusions

The method developed for the determination of BaP in edible oils and oil based food supplements is suitable to be applied as routine laboratory analysis and is relatively fast (about 90 min per analysis). The validation characteristics of the method are in line with international standards, as can be expected. The range of application is from 0.14 up to 8.0 µg/kg BaP, so even low levels of BaP can be determined. The recovery in oil was 99.9% with an RSDr of 2.8%, in a food supplement the recovery had a value of 100.1% with an RSDr of 4.1%. The performance of the method in proficiency tests was good and samples analysed with other methods (LC and GC-MS) showed similar results . With this method other PAHs can also be identified and quantified. However, the method is not validated for these components at present.

For the time being, the method is single laboratory validated for BaP only. It is intended to expand the validation to cover various PAHs, by a collaborative method validation study to become an ISO standard (ISO/TC34/SC11 2005). About 1350 samples were analysed for their BaP content, covering both oils for human consumption and food supplements. In the oils about 20% exceeded the limit of 1.2 µg/kg BaP applied by the VWA. In food supplements over 30% exceeded the legal limit, which might be due to the raw materials (oil) which is being used as basis for food supplements. Producers of food supplements need to become aware of the presence of BaP in oils/oil extracts. More attention must be paid in the coming years to the presence of BaP and other PAHs in food supplements. In addition, oils for human consumption have to be checked, although to lesser extent than with food supplements.

  Determination of the level of Benzo[a]pyrene in fatty foods and food supplements J.C.A. van der Wielen*, J.T.A. Jansen, M.J. Martena, H.N. de Groot and P.H. in 't Veld Food and Consumer Product Safety Authority (VWA) -Region South, V. Montgomerylaan 500 / PO Box 2168 5600 CD Eindhoven, The Netherlands.

  . In Commission Recommendation 2005/108/EC, the Commission of the European Communities (2005) also selected 15 priority PAHs, based on their carcinogenicity (some being different from the EPA PAHs). At a special workshop organised jointly by the European Commission (Directorate Joint Research Centre and Directorate Health and Consumer Protection) and the European Food Safety Authority (EFSA), it was decided to add one more PAH (benzo[c]fluorene) to this list, the later also recommended by JECFA.

  additional measures were required in order to minimise the risk to public health. Due to the fact that several member states of the EU have set their own legal limits, the Commission of the European Communities (2005) issued a Regulation (No 208/2005) amending Regulation (EC) No 466/2001 with regard to polycyclic aromatic hydrocarbons. This regulation sets the maximum level for BaP at

  was optimised giving special attention to fluorescence detection. The following parameters were optimised: injection volume of samples on DACC-column, back flush of the components of the DACC-column, removal of isopropylacohol from DACC-column before injection on the analytical column, the level of concentration of acetonitrile necessary to strip off the PAHs from the DACC column, column temperature of the analytical column, gradients of the analytical column and wavelengths of the fluorescence detection. In addition, benzo[b]chrysene was added to each sample for quality control purposes. If the column switching system does not work correctly, retention times change and/or there is no evidence of benzo[b]chrysene. The method of Stijn et al. (1996) differs from the developed method in the injection volume (250 µl compared to 100µl), column temperature of the HPLC-columns (20°C compared to 30°C), the excitation and emission wavelengths of the fluorescence PAHs and the back flush of the PAHs from the DACC column. The method of Stijn et al. (

  . We, the authors of this study, would like to thank the team who managed this project, being organisationally very complicated. A special thanks is due to the chemistry laboratory of VWA -region South, which performed all the analyses, and to the inspectors of the VWA who visited the companies to gather all samples. The authors would also like to thank the National Institute of Public Health and the Environment (RIVM, The Netherlands), who carried out analysis using other methods, which enabled us to confirm our results.

Figure 2 :

 2 Figure 1: Chromatogram of a standard containing 12 PAHs

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Scientific Committee on Food. 2002. Polycyclic Aromatic Hydrocarbons -Occurrence

Acknowledgement

This study resulted from the realisation that the levels of BaP were far in excess of those which are acceptable. It was necessary to inform and inspect a very large