Nikolaj S Nytofte

Maria A Serrano

Maria J Monte

Zeynep Tumer

Karin Ladefoged

Jose J G Marin
email: jjgmarin@usal.es

Ester Gonzalez-Sanchez

Oscar Briz

A homozygous nonsense mutation (c.214C>A) in biliverdin reductase alpha gene (BLVRA

Keywords: Bilirubin, Biliverdin, Cholestasis, Cholelithiasis, Heme, Liver BLVRA, Biliverdin reductase alpha gene symbol, BVRα, Biliverdin reductase alpha, ICP, intrahepatic cholestasis of pregnancy, SNP, single nucleotide polymorphism

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INTRODUCTION

Biliverdin is generated, together with Fe and CO, in heme catabolism, in a reaction catalyzed by heme oxygenase [START_REF] Ryter | The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro-and antioxidant properties[END_REF]. The main product is biliverdin IXα, although, depending on the protoporphyrin IX bridge cleaved, smaller amounts of the other three isomers (IXβ, IXγ and IXδ) are also generated in humans and other mammals [START_REF] Yamaguchi | Changes in the composition of bilirubin-IX isomers during human prenatal development[END_REF]. Biliverdin IXα is converted into Bilirubin IXα by biliverdin reductase alpha (BVRα), which is expressed in many organs, mainly liver, brain, lung, pancreas, kidney, spleen and placenta [START_REF] Mccoubrey | The structure, organization and differential expression of the rat gene encoding biliverdin reductase[END_REF][START_REF] Komuro | Cloning and characterization of the cDNA encoding human biliverdin-IX alpha reductase[END_REF][START_REF] Briz | Excretion of fetal biliverdin by the rat placenta-maternal liver tandem[END_REF]. The overall reduction of biliverdin to bilirubin is very efficient, and under physiological circumstances the level of serum biliverdin is low [START_REF] Fevery | Bilirubin in clinical practice: a review[END_REF]. Subsequently, bilirubin is taken up by hepatocytes and conjugated with glucuronic acid by bilirubin uridine diphosphate-glucuronosyl transferase-1A1 [START_REF] Bosma | Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man[END_REF] prior to its secretion into bile [START_REF] Fevery | Bilirubin in clinical practice: a review[END_REF]. Thus, cholestasis normally results in hyperbilirubinemia.

Although for many years it has simply been considered a mere mechanism for the elimination of waste products, recently the physiological advantages of the transformation of heme into biliverdin and this into bilirubin have been recognized. Thus, generation of endogenous CO, a potent vasodilator, may play a role in the control of local vascular tone [START_REF] Mclean | Expression of the heme oxygenase-carbon monoxide signalling system in human placenta[END_REF]. Fetal biliverdin can be taken up by the placenta, where it is converted into bilirubin by BVRα prior to its transfer to the maternal compartment [START_REF] Briz | Excretion of fetal biliverdin by the rat placenta-maternal liver tandem[END_REF]. Moreover, bilirubin has beneficial antioxidant properties, although they are limited to low concentrations of this potentially toxic pigment [9]. An explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin would be oxidized to biliverdin and then recycled back to bilirubin by BVRα [START_REF] Baranano | Biliverdin reductase: a major physiologic cytoprotectant[END_REF]. However, recent in vitro studies argue against the BVRα-mediated redox cycle playing a general or important role as a cellular antioxidant defense mechanism [START_REF] Maghzal | Limited role for the bilirubinbiliverdin redox amplification cycle in the cellular antioxidant protection by biliverdin reductase[END_REF].

Under physiological circumstances the levels of biliverdin are much lower than those of bilirubin [START_REF] Fevery | Bilirubin in clinical practice: a review[END_REF]. However, in rare cases impairment in the biliverdin/bilirubin pathway has been reported to result in green jaundice and a green discoloration of body fluids [START_REF] Greenberg | Green jaundice. A study of serum biliverdin, mesobiliverdin and other green pigments[END_REF][START_REF] Larson | A study of the serum biliverdin concentration in various types of jaundice[END_REF][START_REF] Prichard | Biliverdin appearing in a case of malnutrition[END_REF][START_REF] Gafvels | A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice)[END_REF]. This has been ascribed to biliverdin accumulation, but identification of the pigment responsible has been hampered by the use of non-specific biliverdin assays [START_REF] Greenberg | Green jaundice. A study of serum biliverdin, mesobiliverdin and other green pigments[END_REF]. Clinically, the appearance of green jaundice has been considered to be a sign of a poor prognosis [START_REF] Larson | A study of the serum biliverdin concentration in various types of jaundice[END_REF].

In the present study we describe two cases of green discoloration of the skin and biliverdin accumulation in plasma and urine in two unrelated female Inuit patients from different geographic areas of Greenland who had suffered from episodes of obstructive cholestasis. The biliverdin concentrations in their body fluids were completely restored to normal levels after surgical correction of the cholestasis. The genetic bases for this alteration have been elucidated.

MATERIALS AND METHODS

Patients

The research protocol, which conformed to the ethical guidelines of the 1975 Declaration of Helsinki, was reviewed and approved by the Human Ethics Committees of the University of Salamanca (Spain) and the Queen Ingrid's Hospital (Nuuk, Greenland, Denmark). Written consent was obtained from the patients in all cases.

Case #1, Patient PJ

First episode: A 22-years-old pregnant woman from southern Greenland contacted the local Health Centre with complaints of back pain, nausea, vomiting and headache. The patient had previous been in good health. The local doctor noted that the urine was green. Urine microscopy revealed that the patient had a urinary infection. The patient developed contractions and was referred to the Queen Ingrid's Hospital where she was admitted to the Gynaecological Department on April 20 th 2007. The contractions and the back pain had remitted, but the patient still complained of nausea and bilious vomiting. Apart from penicillin, the patient did not receive any medication before admission.

The patient was at gestation stage of 35(+1) weeks on the day of admission. The patient had a body mass index of 39.5 kg/m 2 . Blood pressure was 143/84; pulse 100/min, and temperature 37.0ºC. An abdominal examination revealed a soft and initially non-tender abdomen. The skin of the patient presented a greenish hue. Both urine and plasma were green, the urine somewhat darker than the latter (Figure 1). Laboratory tests from the two days prior to the surgical intervention afforded normal values, except for serum alkaline phosphatase (282 U/l) and transaminases (ALAT-GPT: 58 U/l; ALAT-GOT: 54 U/l). Total bilirubin was only moderately elevated (18 µM). On April 22 nd the patient complained of abdominal pain and her nausea and vomiting had worsened. Abdominal ultrasound imaging revealed a living foetus, signs of obstructive cholestasis and an enlarged, thin-walled gallbladder with multiple gallstones. Her blood pressure had risen (154/100 mmHg), a discrete oedema of the lower extremities was noticed, and the patient had mild proteinuria.

On April 23 rd a healthy-looking baby was delivered by Caesarean section. Neither the skin of the baby nor the amniotic fluid was green. A cholecystolithotomy was performed and very green bile was drained from the thin-walled gallbladder, after which multiple gallstones were removed. During the operation, a cholangiography revealed a very distended biliary tree without drainage to the duodenum. One week after the operation, a second cholangiography disclosed normal drainage to the duodenum. Laboratory tests afforded normal values (data not shown). Moreover, all symptoms, as well as the green discoloration of the skin, urine and plasma (Figure 1), quickly regressed and the patient was discharged in good health.

Second episode: On October 15 th 2008 the patient noticed that her urine was dark green again. On the following day, she developed abdominal pain in the epigastric region accompanied by bilious vomiting. No discoloration of the skin was observed. On October 17 th the patient was admitted to the Queen Ingrid's Hospital. The patient was not febrile. The abdomen was soft but tender in the epigastric region. No skin discoloration was noted, but the urine and serum were green. Laboratory tests from October 16 th to the day of surgical intervention revealed normal values except for alkaline phosphatase (370 U/l) and transaminases (ALAT-GPT: 324 U/l; ALAT-GOT: 140 U/l). Bilirubin levels were within the normal range (10 µM). On October 21 st 2008 a cholocystectomy was performed. The patient was well after the operation and discharged in good health during the following week.

Case #2 Patient EA

A 33-years-old non-pregnant woman in previous good health contacted a Settlement Health Station in western Greenland complaining of epigastric pain, diarrhoea, nausea and vomiting on April 3 rd 2008. The patient was not febrile and the abdomen was soft but tender. On April 18 th 2008 the patient developed a green colour of the skin. She also noted that her urine had turned dark green. On April 21 st 2008 the patient was transported to the Health Centre owing to a marked reduction (-8 kg) in body weight and she still complained of vomiting and diarrhoea, both described as greenish. The skin was visibly greyish green. The urine and serum were green. Laboratory tests afforded normal values except for serum bilirubin (56 µM), alkaline phosphatase (422 U/l) and transaminases (ALAT-GPT: 227 U/l; ALAT-GOT: 135 U/l). Leucocytosis was also observed (12.0 x10 9 /l). The following day an ultrasound examination of the abdomen revealed that the gallbladder had increased in wall thickness and contained multiple gallstones. On May 9 th , the patient was admitted to the Queen Ingrid's Hospital. The urine was still green and the skin had a greenish hue. Abdominal ultrasound showed cholelithiasis and cholestasis. On May 20 th , a cholecystectomy was performed, gallstones were removed, and a drain was inserted. Postoperatively, the discoloration of the skin and body fluids normalized and a cholangiography now showed normal drainage to the duodenum. The patient was discharged in good health during the following week.

Analytical Methods

Adaptations of previously described methods for biliverdin [START_REF] Niittynen | 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced accumulation of biliverdin and hepatic peliosis in rats[END_REF], bilirubin [START_REF] De Matteis | Bilirubin degradation by uncoupled cytochrome P450. Comparison with a chemical oxidation system and characterization of the products by high-performance liquid chromatography/electrospray ionization mass spectrometry[END_REF] and bile acid [START_REF] Ye | High-performance liquid chromatographytandem mass spectrometry for the analysis of bile acid profiles in serum of women with intrahepatic cholestasis of pregnancy[END_REF] measurements by HPLC-MS/MS were used in a 6410 Triple Quad LC/MS (Agilent Technologies, Santa Clara, CA), using MS2Scan to select the precursor ions. For biliverdin IXα and bilirubin IXα we used 583.3 and 585.3 m/z as the precursor ions, respectively, whereas for bile acids the selection was based on the molecular ion in each case. Positive (biliverdin IXα and bilirubin IXα) or negative (bile acid) electrospray ionization (ESI) was carried out. Precursor ions were then filtered and further fragmented in multiple-reaction monitoring (MRM) mode for using the product ions (297.2 m/z for biliverdin IXα, 299.2 m/z for bilirubin IXα, 80.2 m/z for taurine-conjugated bile acids, 74.0 m/z for glycine-conjugated bile acids and their own precursor ions for unconjugated bile acids) to measure the abundance of the indicated compounds.

HPLC was carried out using a Zorbax C18 column (30 mm x 2.1 mm, 3.5 µm) or Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 µm) for biliverdin/bilirubin and bile acids, respectively. The chromatographic conditions for biliverdin/bilirubin analyses were 85:15 methanol/water, both containing 5 mM ammonium acetate and 0.1% formic acid, pH 3.2.

Flow rate was 0.3 ml/min at 35ºC. For bile acids, solvents were also methanol and water, but containing 5 mM ammonium acetate and 0.01% formic acid, pH 4.6, flow rate was 0.5 ml/min, the chromatography was started at 80:20 methanol/water, and the proportion of methanol was increased linearly up to 97% over 9 min. Standard biliverdin IXα bilirubin IXα and bile acids (purity higher than 95%) were purchased from Porphyrin Products, Inc.

(Logan, Utah, US) and Sigma-Aldrich (Madrid, Spain), respectively. All other reagents were of analytical grade.

Genetic Analyses

Total RNA-free DNA from blood cells was obtained using a commercial kit (QIAamp DNA Mini, Spin Protocol, Qiagen) after treatment of blood samples with proteinase K and RNAse A. DNA from oral epithelial cells was obtained using sterile buccal swabs (Omni Swabs, Whatman). The coding region of the BVRα gene was amplified by PCR using primers whose nucleotide sequence was located in the intronic regions of the gene. The amplicons contained both the exons and the exon-intron boundaries. The PCR products from at least three reactions of PCR per exon were purified by agarose gel electrophoresis followed by extraction of DNA from the gel bands using the QIAquick Gel Extraction kit (Qiagen). The amplicons were sequenced in both directions using forward and reverse primers. Thus, each exon was sequenced at least six times. To develop a PCR multiplex test to identify the c. 214C>A mutation found in exon 3, the two variants of this exon were cloned in pGEM-T vector and amplified in transfected Escherichia coli DH5α. TaqMan probes and multiplex PCR were used for allelic discrimination (forward primer: CCTTCCTCAGCGTTCCTGAA;

Expression of BVRα in cells

Synthesis of cRNAs for injection into oocytes was performed using the plasmids containing the CDS cDNA of both wild-type and mutated BVRα. These were linearized with the restriction enzyme PmeI (Promega). Capped and poly(A)-tailed cRNAs were synthesized using the T7 mMessage mMachine Ultra kit (Ambion; Applied Biosystems). The harvesting and preparation of oocytes were carried out as described elsewhere [START_REF] Briz | Carriers involved in targeting the cytostatic bile acid-cisplatin derivatives cis-diammine-chloro-cholylglycinate-platinum(II) and cis-diammine-bisursodeoxycholateplatinum(II) toward liver cells[END_REF].

Mature female frogs (Xenopus

Oocytes were injected with 9 ng of cRNA and were subsequently cultured at 18°C for 2 days.

The PLC/PRF/5 hepatoma cell line (Alexander, ATCC number CCL 8024) was purchased from the ATCC (LGC Standards, Barcelona). Cells growing on glass cover slips were transiently transfected with lipofectamine LTX (Invitrogen) and cultured for 3 days. V5-tagged BVRα was detected by immunostaining by using an anti-V5 monoclonal antibody (Invitrogen)

and an anti-mouse FITC-conjugated secondary antibody (Invitrogen). DAPI (diamidino-2phenylindole) was used to counterstain nuclei. Fluorescence photographs were obtained using a Nikon Eclipse TE 2000-S microscope.

Cell lysates (25 μg protein as measured determined using a modification of the Lowry method [START_REF] Markwell | A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples[END_REF]) were used for immunoblotting analyses. Blots were probed with primary monoclonal antibodies against V5 epitope (Invitrogen). The appropriate horseradish peroxidase-linked secondary antibody was from Invitrogen.

An enhanced chemiluminescence detection system (Hybond ECL; GE Healthcare) was used to visualize the bands.

Functional study of BVRα activity

BVRa activity was assessed by studying conversion of biliverdin IXα into bilirubin IXα by oocytes expressing the enzyme. Oocytes were washed with substrate-free medium (100 mM sodium chloride, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, pH 7.5) and microinjected with 50 nl of similar medium but containing 300 pmol biliverdin IXα and then incubated at 25°C for 1 h. The oocytes were washed with 4 ml ice-cold medium before being placed individually in vials. Production of bilirubin IXα was measured by HPLC-MS/MS as described above after lysing the oocytes with 90% methanol.

RESULTS

Analysis of serum samples collected during green jaundice episodes in both patients

revealed profiles of bile acid levels typically associated to biliary obstruction, i.e., high levels of conjugated bile acids (mainly taurocholic, glycocholic, taurochenodeoxycholic, glycocholic and tauro-sulfo-lithocholic acids) and low levels of unconjugated bile acids and secondary bile acids (data not shown).

To elucidate whether the greenish tinge of the skin, serum, urine and milk (Figure 1) during the episodes of cholestasis in both patients was actually due to biliverdin, HPLC-MS/MS was carried out. This permitted us to identify and measure the levels of biliverdin and to distinguish between the two major isomers: biliverdin IXα and biliverdin IXβ (Figure 2A). By filtering and following the specific ions (Figure 2B and 2C), it was possible to identify the major form of biliverdin in body fluids, which in both patients was biliverdin IXα, and to determine that this was markedly accumulated (Table 1). The time course of serum biliverdin

IXα concentrations indicated that a marked reduction had occurred after surgical removal of the biliary obstruction (Table 1). An accumulation of biliverdin was also seen in urine and milk (Table 1). Interestingly, the bile of patient PJ collected after biliary drainage had been restored contained high amounts of biliverdin IXα (Table 1).

Since our findins suggested that patient PJ might have a deficient transformation of biliverdin

IXα into bilirubin IXα we screened the coding region of the BVRα gene (BLVRA) for sequence variations. We detected three changes: c.90G>A (p.Ala3Thr, exon 2), c.214C>A (p.Ser44X, exon 3), and c.743A>C (p.Gly220Gly, exon 8). No changes in the intron-exon boundaries were observed (data not shown). Accordingly, the p.Ser44X mutation identified in this study, is predicted to generate a truncated protein containing only the 43 N-terminal amino acids: MNTEPERKFGVVVVGVGRAGSVRMRDLRNPHPSSAFLNLI GFV.

Using TaqMan probes in PCR multiplex analyses, both patients PJ and EA were found to be homozygous for the c.214C>A mutation. Material (oral epithelial cells) was available from only members of PJ's family. A pedigree analysis of c.214C>A in this family was carried out.

This revealed that both PJ's parents were heterozygous for this mutation, whereas her brother was homozygous for the normal allele. In contrast, her sister was homozygous for the c.214C>A mutation. Although she had never suffered any episode of green jaundice, ultrasound investigation revealed a solitary stone in her gallbladder but no signs of biliary obstruction. Values of bile acid concentrations in the serum of PJ patient's sister were within the normal range (data not shown) and no biochemical signs of cholestasis were found (data not shown). Values of biliverdin concentrations in the serum of PJ patient's sister were low and similar to that found in her sister in absence of cholestatic crisis but about two-fold higher than that found in control women (Table 1).

Expression in both human hepatoma cells and Xenopus laevis oocytes of the wild-type BVRα tagged with the V5 epitope at the COOH end of the protein resulted in a detectable band of the expected size in Western blot (Figure 3) and clear labelling of the cytoplasm of the cells with green florescence in immunostaining assays (Figures 4A, 4C and4E). In contrast, when the construct containing the CDS of the mutated (c.214C>A) BVRα variant was used in the transfection of hepatoma cells or to obtain the mRNA that was injected in Xenopus laevis oocytes the result was a lack of detection of the V5 epitope both in Western blot (Figure 3) and immunofluorescence (Figures 4B, 4D and4F).

HPLC-MS/MS was used to analyze in Xenopus laevis oocytes the biotransformation of biliverdin into bilirubin by chromatographically separating them and following the disappearance and appearance, respectively, of characteristic product ions (Figure 5A). The ability to generate bilirubin from endogenous biliverdin was significantly higher in oocytes expressing BVRα (Figures 5B and5D). This enhanced ability was more clearly seen when the oocytes were microinjected with a bolus of exogenous biliverdin (Figures 5C and5E). In this case, detection of a marked decrease in biliverdin content was accompanied by a similar increase in the amount of bilirubin in the cells 1 h after microinjection of biliverdin. Catalytic activity of BVRα either on endogenous or microinjected biliverdin could not be observed in oocytes expressing the mutated (c.214C>A) BVRα variant (Figures 5B-5E).

DISCUSSION

Serum biochemical markers, and low levels of unconjugated bile acids and secondary bile acids were consistent with obstructive cholestasis, i.e., a reduced transit of bile acids through the intestine, and poor biotransformation of them by intestinal bacteria [START_REF] Ridlon | Bile salt biotransformations by human intestinal bacteria[END_REF]. Obstructive cholestasis was confirmed by additional analyses and subsequent surgery. When the drainage of bile to the duodenum was re-established, the values of laboratory tests and serum bile acids returned to within the normal range (data not shown). These findings support the hypothesis that biliary obstruction was the primary event and green jaundice appeared as a consequence. The marked accumulation of biliverdin, together with a moderate or absent hyperbilirubinemia, was the most interesting sign during crises of cholestasis in these patients. Accumulation of biliverdin has been already described in rare cases associated with biliary obstruction [START_REF] Greenberg | Green jaundice. A study of serum biliverdin, mesobiliverdin and other green pigments[END_REF][START_REF] Larson | A study of the serum biliverdin concentration in various types of jaundice[END_REF], but also with malnutrition [START_REF] Prichard | Biliverdin appearing in a case of malnutrition[END_REF], and several liver diseases [START_REF] Fenech | Hepatitis with biliverdinaemia in association with indomethacin therapy[END_REF], in which the appearance of green jaundice has been considered as a sign of poor prognosis [START_REF] Larson | A study of the serum biliverdin concentration in various types of jaundice[END_REF]. This is because the biliverdin-induced greenish colour of the skin, serum and urine probably masks the also enhanced levels of bilirubin, which might be accounted for by profound impairment of liver function. Indeed, in previously described cases of patients with enhanced biliverdinemia at levels similar to those found in patients studied here (30-40 µM) the levels of serum bilirubin were 10-fold higher (300-400 µM) [START_REF] Tickner | A simple colorimetric method for the estimation of plasma biliverdin[END_REF]. In the patients included in the present study, pure hyperbiliverdinemia was found in one of the cases (PJ), and a moderate degree of hyperbilirubinemia accompanying hyperbiliverdinemia was detected in the other (EA).

Using a candidate gene approach to investigate green jaundice episodes, three single nucleotide polymorphisms were found, which result in: i) p.Ala3Thr, a variant that has already been described in this gene both in human [START_REF] Komuro | Cloning and characterization of the cDNA encoding human biliverdin-IX alpha reductase[END_REF] and rat [START_REF] Kikuchi | Crystal structure of rat biliverdin reductase[END_REF] BVRα. The functional repercussions of the change in this residue are not known. ii) p.Gly220Gly, i.e., a silent mutation. iii) p.Ser44X, which is expected to account for the synthesis of a truncated protein.

The normal BVRα protein consists of 296 amino acids and contains two conserved sites: i) the Rossmann-fold NAD(P)H/NAD(P)(+) binding (NADB) domain at amino acid position 18-101, which is needed for the interaction with the cofactor and ii) the catalytic active site at amino acid position 133-246, which is involved in the interaction with the substrate (Figure 3).

Both sites are necessary for the ability of BVRα to convert biliverdin to bilirubin. When the mutated sequence, to which a V5 epitope tag was linked at the COOH end, was transfected in human hepatoma cells and Xenopus laevis oocytes the complete protein was not synthesized. Moreover, the peptide synthesized before translation reaches the mutation-generated stop codon, would lack most of the NADPH-binding site as well as the whole of the active site [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF][START_REF] Maines | New insights into biliverdin reductase functions: linking heme metabolism to cell signaling[END_REF]. This was consistent with the lack of catalytic activity observed in functional assays with human hepatoma cells and Xenopus laevis oocytes transfected with the mutated construct.

Recently in a Swedish male patient with end-stage liver cirrhosis a heterozygous nonsense mutation in exon 3 (Arg18X) of BLVRA has been described [START_REF] Gafvels | A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice)[END_REF]. It is interesting that this heterozygous mutation resulted in biliverdin accumulation in spite of the presence of one normal allele. The cause of hyperbiliverdinemia in this patient was suggested to be the profound impairment in liver function due to advanced cirrhosis. The authors underline that human cases with a total deficiency of BVRα activity had not been described so far. The present study fills this gap and affords interesting information on the relevance of the complete lack of BVRα activity in humans.

In spite of the patients studied here having a complete lack of BVRα, they had no apparent signs of an abnormal phenotype in the absence of cholestasis. However, it cannot be ruled out that the complete absence of BVRα activity has any deleterious effect. For instance, it is not known whether a marked biliary secretion of this pigment might be indirectly involved in favouring the formation of gallstones that were present in both patients and in PJ's sister.

Moreover, one of the subjects investigated here (PJ) was pregnant and gave birth during the first episode of green jaundice and biliverdin accumulation. The baby died a few months later of unknown causes. Thus, it cannot be ruled out that an accumulation of biliverdin during pregnancy may be harmful to the foetus.

In these patients, biochemical analyses of serum collected during green jaundice episodes revealed the presence of certain amount of total bilirubin (<20 µM and <60 µM in PJ and EA, respectively). As automated methods to determine serum bilirubin levels are based on the formation and colorimetric measurement of diazopigments and this method does not discriminate among bilirubin isomers [START_REF] Blumenthal | Changes in bilirubins in human prenatal development[END_REF], these finding were probably due to the generation in these patients of biliverdin/bilirubin isomers other than IXα.

It is interesting to note that even during cholestasis serum bilirubin levels were not markedly elevated. This, together with the presence of a large amount of biliverdin IXα in the bile of this patient, suggests that the main route for elimination of heme metabolites in individuals with complete BVRα deficiency could be the secretion into bile of biliverdin IXα. This maintains serum levels low as long as biliary function is not impaired. This may explain the interesting observation that PJ's sister, despite being homozygous mutation for the inactivating in BLVRA, did not have any episodes of green jaundice. The findings of heterozygous mutation in both PJ's parents who did not suffer any liver disease and had not had signs of green jaundice along their lives were in agreement with the concept that green jaundice occurs in heterozygous patients only when liver function is severely deteriorated [START_REF] Gafvels | A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice)[END_REF].

In conclusion, the present is the first report of a homozygous BLVRA inactivating mutation indicating that the complete absence of BVRα activity is a non-lethal condition, whose most evident phenotypic characteristic is the appearance of green jaundice accompanying cholestasis episodes. Detection was performed using a primary monoclonal antibody against the V5 epitope.

  ; C214A probe: VIC-GCTTCGTGTAGAGGT-MGB).Cloning and site-directed mutagenesis of BVRαUsing total RNA from human liver (Invitrogen) cDNA was synthesized by reverse transcription with random hexamers using the Cloned AMV First-Strand cDNA Synthesis kit (Invitrogen). The coding sequence (CDS) of BVRα including a Kozak sequence was then amplified by PCR, using high-fidelity AccuPrime Pfx DNA polymerase (Invitrogen) and two oligonucleotide primers specific to the sequence of GenBank accession number NM_000712 (forward: 5'-CCAAGATGAATGCAGAGCCCGA-3', reverse: 5'-CTTCCTTGAACAGCAATATTTCTGGATTTCTTCT-3') to which attB1 and attB2 sites were added, respectively, to obtain cDNA useful for Gateway cloning. PCR products were recombined with the attP-containing pDONR221 vector (Invitrogen) to generate Entry plasmids, which were further recombined with the pcDNA6.2/V5 destination vector (Invitrogen), containing the V5-tag downstream the insert, to generate an expression vector with the V5 epitope located at C-terminal end of BVRα. Complete sequencing of cloned BVRα CDS revealed the presence of the same silent polymorphism c.743C>A found in the patients. This construct (pcDNA6.2-BVRα-WT) was used to carry out homemade sitedirected mutagenesis of the whole plasmid [19] and obtain pcDNA6.2-BVRα-C214A containing the mutated BVRα CDS. The presence of the mutation c.214C>A was confirmed by complete sequencing of the BVRα CDS in this new plasmid.

  laevis), purchased from Regine Olig (Hamburg, Germany), were used. The animals received humane care as outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and were treated in accordance with the indications of current Spanish (RD 223/1988) and European Union (86/609/CEE) laws. Experimental protocols were approved and supervised by the Ethical Committee for Laboratory Animals of the University of Salamanca.

Figure 1 .

 1 Figure 1. Representative images of samples collected from patient PJ during the first episode of cholestasis and after surgical removal of her biliary obstruction. The milk sample is compared with milk collected from a healthy volunteer (Control).

Figure 2 .

 2 Figure 2. A. Schematic representation of the molecular structure of biliverdin IXα and biliverdin IXβ and the major fragments generated from these molecules in MS. B. Representative mass spectra of biliverdin IXα obtained in MS2-scan mode by HPLC-MS/MS using electrospray positive ionization to select the precursor ion (583.3 m/z), indicated with an asterisk. C. Mass spectra obtained in product ion mode by filtering the precursor ion at the first quadrupole showing the major product ion (297.25 m/z) generated in the second quadrupole, which was then used in MRM mode for quantitative analyses.

Figure 3 .

 3 Figure 3. Representative Western blots of wild-type and mutated (c.214C>A; p.Ser44X) V5-tagged biliverdin reductase-IXα (BVRα) carried out with lysates of Alexander cells (10 µg protein) and Xenopus laevis oocytes (0.5 µg protein). Alexander cells expressing V5tagged chloramphenicol acetyltransferase (CAT-V5) were used as a positive control.

Figure 4 .

 4 Figure 4. Representative phase-contrast (B,C) and fluorescence (D-G) microscopy photographs of human hepatoma Alexander cells expressing wild-type and mutated (c.214C>A; p.Ser44X) V5-tagged biliverdin reductase-IXα (BVRα). Nuclei were counterstained with DAPI (blue). Detection of BVRα (green) was performed using a primary monoclonal antibody against the V5 epitope and a FITC-conjugated secondary antibody.

Figure 5 .

 5 Figure 5. A. Representative total ion chromatograms (TIC) showing peaks corresponding to biliverdin IXα (product ion 297.2 m/z) and bilirubin IXα (product ion 299.2 m/z) obtained by injection in HPLC-MS/MS of methanol lysates from Xenopus laevis oocytes control (Non-injected with cRNA) or expressing wild type (WT-BVRα) or mutated (c.214C>A; p.Ser44X) human biliverdin reductase alpha (Mutated-BVRα). All oocytes were microinjected with exogenous biliverdin IXα (300 pmol/oocyte) 60 min before analysis. B-E. Functional activity of human biliverdin reductase alpha (BVRα) in Xenopus laevis oocytes. Oocyte contents of biliverdin IXα (B,C) and bilirubin IXα (D,E) were measured by HPLC-MS/MS in oocytes control and expressing wild type (WT-BVRα) or mutated

Table 1 .

 1 Biliverdin IXα concentrations in body fluids Surgical removal of biliary obstruction was carried out on day 0. Biliverdin IXα was determined in triplicate by HPLC-MS/MS. Control values were means±SD from samples collected from 7 volunteer women of ages similar to those of the probands. Four serum samples collected from the patient PJ's sister were also analyzed. Values are means±SD. Results above normal values are in bold case.

	Sample	Individuals	Day	Biliverdin
				(µM)
	Serum	Control		0.12±0.04
		PJ's Sister		0.36±0.03
		Patients
		EA	-1	51.5
		PJ	-2	13.1
			-1	25.7
			0	32.4
			+5	0.33
	Urine	Control		<0.05
		Patients
		EA	-1	14.6
		PJ	+5	3.13
	Milk	Control		0.10±0.01
		Patients
		PJ	+4	1.11
	Bile	Patients
		PJ	0	124.7

Acknowledgements

The authors thank the laboratory-managing work by E. Keck, secretarial help by M.I. Hernandez, technical help by A.I. Barrios-Fernandez. Revision of the English spelling, grammar and style of the manuscript by N. Skinner are also gratefully acknowledged.

This study was supported in part by the Instituto de Salud Carlos III, FIS (Grants PI070517 and PI080151); the Junta de Castilla y Leon (Grants GR75/08, SA033A08, SA03508, and SA036A08), Spain; the Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Grant BFU2006-12577 and and SAF2009-08493), Spain and the Fundacion Investigacion Medica, Mutua Madrileña (Convocatoria 2009), Spain. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grant BFU2007-30688-E/BFI) and belongs to the CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) Instituto de Salud Carlos III, Spain.

Licence Statement:

Jose J. G. Marin, the Corresponding Author of the article Ref: JMG/2009/074567 -"A homozygous nonsense mutation (c.214C>A) in biliverdin reductase alpha gene (BLVRA) results in accumulation of biliverdin during episodes of cholestasis", has the right to grant on behalf of all authors and does grant on behalf of all authors, a licence to the BMJ Publishing Group Ltd and its licensees, to permit this Contribution (if accepted) to be published in Journal of Medical Genetics (JMG) and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at:

(http://jmg.bmj.com/site/about/licence.pdf) I am one author signing on behalf of all co-owners of the Contribution.

Competing Interest: None declared.


Oliver Vendl

Colin Crews
email: colin.crews@fera.gsi.gov.uk

Susan Macdonald

Rudolf Krska

Franz Berthiller

Occurrence of free and conjugated Fusarium mycotoxins in cereal based food

Keywords: Beer, Bakery products, Bread, Cereals masked mycotoxins, deoxynivalenol, zearalenone, LC/MS, cereal based food, Fusarium

A collection of 84 cereal based food products in 25 composites, including beer, was screened for the presence of deoxynivalenol, zearalenone and their respective metabolites deoxynivalenol-3-glucopyranoside, 3-acetyl-deoxynivalenol, zearalenol-4-glucopyranoside, α-zearalenol, β-zearalenol, α-zearalenol-4-glucopyranoside, β-zearalenol-4-glucopyranoside and zearalenone-4-sulfate. The most abundant analyte was zearalenone-4-sulfate, which was found in 13 composites, albeit in low concentrations. Furthermore, deoxynivalenol was detected in eight, zearalenone in seven and deoxynivalenol-3-glucopyranoside in two composites. None of the remaining six analytes were found in any matrices, which suggests that, if at all present, concentrations of these latter metabolites are very low and hence do not impose any danger to the consumer. The highest mycotoxin content was found in bran flakes with 254 ng g -1 deoxynivalenol, 6 ng g -1 zearalenone-4-sulfate and 44 ng g -1 zearalenone.

Introduction

The mycotoxins deoxynivalenol (DON) and zearalenone (ZON) are common and well known contaminants of cereal products arising from infection of grain with Fusarium sp. These two compounds are produced by the fungus as part of its physiology of pathogenic invasion and, as the cereal ears are the location of infection, these mycotoxins have been found to contaminate cereal-based products [START_REF] Leonard | Fusarium Head Blight of Wheat and Barley[END_REF].

However, DON and ZON are partly metabolized by the infected host plant as well as by the producing fungus itself. Metabolism by the host plant generally aims at detoxification of these compounds which is often accomplished by attachment of hydrophilic groups, thus increasing their solubility in water [START_REF] Engelhardt | Metabolism of mycotoxins in plants[END_REF][START_REF] Karlovsky | Biological detoxification of fungal toxins and its use in plant breeding, feed and food production[END_REF]. In this manner, DON is converted to DON-3-glucopyranoside (D3G) (Sewald et al. 1992;[START_REF] Berthiller | Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry[END_REF], and ZON can be altered to ZON-4 glucopyranoside (Z4G) [START_REF] Engelhardt | Transformation of the Fusarium mycotoxin zearalenone in maize cell suspension cultures[END_REF]) by plants.

During fungal metabolism, ZON has been found to be partially converted to ZON-4-sulfate (Z4S) [START_REF] Plasencia | Isolation and characterization of zearalenone sulfate produced by Fusarium spp[END_REF] by various Fusarium sp. as well as to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) [START_REF] Böswald | Metabolism of the Fusarium mycotoxins zearalenone and deoxynivalenol by yeast strains of technological relevance[END_REF] by different yeast strains. Further glucosylation of the latter two compounds was observed to occur as part of the host plant´s metabolism, leading to α-ZOL-glucopyranoside (α-ZG) and β-ZOL-glucopyranoside (β-ZG), respectively [START_REF] Berthiller | Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination of phase II metabolites of the mycotoxin zearalenone in the model plant Arabidopsis thaliana[END_REF]. Despite their chemical alteration, there is evidence that the above mentioned metabolites have a similar toxic potential to those of their precursors when ingested with food, as attached functional groups like glycosylic or sulfate residues are likely to be enzymatically cleaved during digestion [START_REF] Gareis | Cleavage of zearalenoneglycoside, a "masked" mycotoxin, during digestion in swine[END_REF][START_REF] Plasencia | Isolation and characterization of zearalenone sulfate produced by Fusarium spp[END_REF]. Also D3G is at least partly cleaved to DON by various intestinal bacteria (F. Berthiller; manuscript in preparation). Furthermore, α-ZOL and β-ZOL have been shown to exhibit similar hyperestrogenic effects as their precursor ZON [START_REF] Fitzpatrick | Measurement of the relative binding affinity of zearalenone, alpha-zearalenol and beta-zearalenol for uterine and oviduct estrogen receptors in swine, rats and chickens: an indicator of estrogenic potencies[END_REF]). Analytical approaches targeted to the parental molecules DON and ZON, respectively, might not detect their metabolites, thus resulting in an underestimation of the inherent toxicity of a contaminated sample matrix. For these reasons, chemically altered metabolites of mycotoxins are also referred to as masked mycotoxins [START_REF] Gareis | Cleavage of zearalenoneglycoside, a "masked" mycotoxin, during digestion in swine[END_REF]. The formation, determination and significance of masked and other conjugated mycotoxins were recently reviewed (Berthiller et al. 2009a).

Maximum levels of DON and ZON have been set by European Commission regulations (European Commission 2007), ranging from 200-750 ng g -1 for DON and 20-100 ng g -1 for ZON in processed cereals, but masked metabolites have not yet been taken in account for an exposure assessment of total mycotoxins from cereal based foods. In order to provide simultaneous LC-MS/MS determination of all of the above mentioned metabolites together with DON and ZON in cereal based food, we developed and validated a method of sample preparation and measurement which has recently been published (Vendl et al. 2009). In the present paper we employ this method in order to determine the occurrence of the 10 target mycotoxins in a wide range of cereal based food products, purchased in the UK and Austria, consisting of 84 samples in 25 composites of product groups.

Materials and Methods

Chemicals

DON, D3G, 3ADON, ZON, α-ZOL and β-ZOL were purchased from Biopure Referenzsubstanzen GmbH (Tulln, Austria) as calibrants in acetonitrile. Z4G, α-ZG, β-ZG (Berthiller et al. 2009b) and Z4S [START_REF] Berthiller | LC-MS/MS von konjugierten Toxinen[END_REF] had previously been isolated by our group and were available as solutions in methanol. LC-grade acetonitrile was obtained from VWR (Leuven, Belgium). Water, which was used for the preparation of extraction solvents, Milli-Q plus system from Millipore (Molsheim, France).

Food and cereal samples

84 cereal products destined for food usage (either for direct consumption or for further processing) were purchased in retail markets in the UK and in Austria. For every product group, 3-4 commercial products from different manufacturers were collected and combined into one composite as described later. A total number of 25 product groups (composites) of cereal based food was chosen for initial screening. In case the screening results were positive, the samples were analyzed individually. Before further processing, samples were stored at room temperature in the darkness. Samples with short expiration period (particularly bread) were processed within 2 days of purchase, which was before the expiry date or best before date was reached. Other samples were stored at room temperature in darkness. Expiration dates were not exceeded, and the maximum storage time between purchase and processing was 3 weeks. Milled sample materials were stored in plastic bottles at room temperature in the dark.

Composite preparation

Dry samples (corn flakes, wheat flakes and raw pasta) were milled in a laboratory mill.

Samples containing higher amounts of fat or sugar were milled frozen with dry ice. This procedure was applied to the following composites: Extruded maize snacks, biscuits, muesli, crackers, popcorn, cereal snack bars, extruded oat snacks and some particular samples of the baby food composites. Bread samples were dried at 45 °C for 48 hours and subsequently milled. Milling was performed for every sample separately. For each product group, 100 g of each milled product were combined and homogenized in a laboratory blender. The milled 6 fractions of the non-combined individual samples were kept for optional separate analysis for the case that the respective composite proved to be contaminated. Canned maize was mashed in a blender together with an equal weight amount of 1% aqueous acetic acid (100 ml for 100 g sample), which corresponded to the water and acid content in the extraction solution. For the beer composite, equal volumes (100 ml each) of beer samples were combined and degassed in a sonic bath for 10 min.

Extraction

Extraction of samples was performed according to the procedure that has been published previously (Vendl et al. 2009). Briefly, for dry composites, 1.00 ± 0.01 g of ground and homogenized combined sample material was extracted with 4 ml extraction solvent (composition: acetonitrile/water/acetic acid in the ratio 79/20/1 v/v/v) for 90 min on a rotary shaker (Burgwedel, Germany) circulating at 190 rpm. Homogenized canned maize was extracted directly after mashing and homogenization by adding 400 ml acetonitrile/acetic acid (99/1) to 100 ml of the aqueous, acidic homogenate. The resulting mixture was distributed into four sealed 250 ml Erlenmeyer flasks and extracted for 90 min on a rotary shaker at 190 rpm. Beer was injected directly after degassing.

LC-MS/MS instrumentation and methodology

An 1100 Series HPLC System (Agilent, Waldbronn, Germany) linked to a QTrap 4000 LC-MS/MS System (Applied Biosystems, Foster City, USA) was used to analyze the extracts.

Electrospray ionisation (ESI) was used in negative mode at a 550°C source temperature.

HPLC and MS/MS parameters were set according to Vendl et al. (2009). Briefly, separation was performed on a Phenomenex Synergi Polar RP-18 column (150×3 mm, 4 µm) at 35 °C using an aqueous ammonium acetate (5mM) / acetonitrile gradient at a flow rate of 1 ml min -

Results and discussion

Twenty-five composites of cereal based food products were screened for the occurrence of DON, ZON and their respective major masked metabolites. Among the targeted analytes, DON, Z4S and ZON were the most abundant, as DON was detected in 8, Z4S in 13 and ZON in 7 composites (see table 1). The highest quantities of DON (254 ng g -1 ), Z4S (6.1 ng g -1 )

and ZON (44.2 ng g -1 ) were found in bran flakes. Relatively high levels of DON were also found in crackers (248 ng g -1 ) and wheat flour (237 ng g -1 ). Both latter matrices also showed Z4S contents of over 2 ng g -1 , although neither of them displayed significantly high levels of ZON. Due to its high ionization efficiency, Z4S is detectable at very low levels and could therefore be determined quantitatively in 10 composites, namely wheat flour, whole-meal wheat bread, maize meal, biscuits, wheat flakes, bran flakes, muesli, crackers, cereal snack bars and polenta. To our knowledge, a comparable wide range of food samples had never before been screened for the existence of Z4S, hence this study brings first evidence for a seemingly wide abundance of Z4S, although at low concentrations. ZON, which was quantifiable in wheat grain and bran flakes, was also detected in whole-meal wheat bread, extruded maize snacks, corn flakes, wheat flakes, crackers and cereal snack bars. Besides the three above analytes mentioned, D3G was detected in wheat bread and in whole-meal wheat bread but levels were below LOQ. the occurrence of D3G (along with DON) in unprocessed wheat and maize kernels after harvest was recently reported (Berthiller et al. 2009c). After bran flakes, wheat flower and crackers had the highest content of target analytes, therefore analyses of individual samples of these three matrices were carried out in addition to the composite screening. Although analysis of combined samples was expected to provide statistically more reliable results on the contamination of a particular product group, screening of individual samples gave a good idea of the actual mycotoxin distribution within one composite. Certain samples might be heavily contaminated and might not contain any mycotoxins at all, or, in contrast, the target analytes might be rather equally distributed among all samples of one product group. Table 2 shows the values of these additional measurements.

Within the composite "bran flakes", all three samples contained DON, Z4S and ZON within a relatively similar concentration range. In contrast, within both composites "crackers" and "wheat flour", two samples showed significantly higher mycotoxin concentrations than the third one of the composite. DON and Z4S were determined in all measured individual samples. ZON was found in all three samples of the bran flakes composite, but in only one out of three samples of wheat flour. In crackers, ZON was determined in two of the three samples. None of the remaining 6 analytes (3ADON, Z4G, α-ZOL, β-ZOL, α-ZG and β-ZG) were detected in any composite or sample, suggesting that, in the case of contamination, these metabolites occur at levels far below the parental compounds DON and ZON, respectively. Z4S has been found to be produced in fungal cell cultures in molar ratios from 1:12 to 1:1 compared to ZON [START_REF] Plasencia | Isolation and characterization of zearalenone sulfate produced by Fusarium spp[END_REF]. In this work, Z4S was present at levels between 9.7% of the ZON level in the wheat flour sample A and 17.9% of the ZON level in the crackers sample B2. In case of those composites, where ZON was detected but not quantified, values of Z4S contents suggested Z4G:ZON ratios of over 1:10 considering maximum ZON levels of 10 ng g -1 (=LOQ). For Z4G:ZON, correlations of contents have been reported in a study on wheat, stating Z4G values of about 10% of the ZON level in those samples in which both analytes were determined [START_REF] Schneeweis | Occurrence of zearalenone-4-β-Dglucopyranoside in wheat[END_REF]. On the other hand, Z4G could not be determined in 50% of those samples that contained ZON. In consideration With regard to food safety, the maximum levels for DON and ZON set by the European Commission were not exceeded in any product group, however, in the case of bran flakes the underestimation of toxicity imposed by masked mycotoxins becomes evident: The value of ZON concentration (44.2 ng g -1 ) lies below the EC limit, but an additional content of 6.1 ng g -1 Z4S has to be taken into consideration. Assuming that Z4S is totally hydrolyzed to ZON during digestion (slightly reducing its mass after cleavage of the sulfate group), the actual toxicity exposure corresponds to a ZON concentration of about 49 ng g -1 , which is closer to the maximum EC regulation level. This gives good evidence that cereal based food meeting regulatory levels for DON and ZON might not display the actual mycotoxin intake of the consumer.

A method to determine ZON and DON conjugates together indirectly after cleavage of the conjugates to the precursor toxins would be helpful and allow comparison of the total toxin values. However, alkaline hydrolysis would destroy the lactone ring of ZON and its derivatives. Acidic hydrolysis would be unlikely to work for D3G, as this compound is strongly resistant to acids even at pH values <1 at heightened temperatures. This leaves enzymatic hydrolysis as the only available option. β-glucosidases are not able to cleave D3G to DON, but cellulases have shown at least partial cleavage with pure D3G (data not shown).

Further studies will concentrate on indirect methods of analysis for conjugated mycotoxins. In none of 25 product groups of cereal based food did DON and ZON levels, exceed European Commission regulation limits. However, in the case of the bran flakes samples, if Z4S were to be completely hydrolyzed to ZON during digestion, the result would be an exposure of 49 ng g -1 ZON, which is close to the maximum regulation level. Out of eight major masked metabolites, six were not detected in any of the analyzed matrices.

Conclusion

  as a LC mobile phase, was purified successively by reverse osmosis and a

  z 355.1 -> 59.1; 295.1), 3ADON (m/z 397.1 -> 59.1; 337.0), Z4S (m/z 397.1 -> 317.1; 131.0), β-ZG and α-ZG (m/z 481.2 -> 319.0; 275.1), Z4G (m/z 479.2 -> 317.0; 131.0), β-ZOL and α-ZOL (m/z 319.1 -> 130.0; 160.0), ZON (m/z 317.1 ->131.0; 175.0).

  , values of Z4G would lie below limits of detection for those composites that exhibit ZON concentrations between LOD and LOQ.

  Lepschy von Gleissenthall J, Schuster M, Müller G, Aplin RT. 1992. Structure elucidation of a plant metabolite of 4-desoxynivalenol. Tetrahedron 3:953-960. Vendl O, Berthiller F, Crews C, Krska R. 2009. Simultaneous determination of deoxynivalenol, zearalenone and their major masked metabolites in cereal based food by LC-MS/MS. Anal Bioanal Chem. 395:1347-1354.

Table 2 .

 2 Contents of DON, Z4S and ZON in individual samples of the composites "wheat flour", "bran flakes" and "crackers" (ug/kg -1 ).

				Page 16 of 16
	Composites	DON Z4S	ZON
	Wheat flour sample A 307	1.8	18.2
	Wheat flour sample B 249	3.5	nd
	Wheat flour sample C 186	1.0	nd
	Bran flakes sample A 243	6.2	48.9
	Bran flakes sample B	241	5.5	40.1
	Bran flakes sample C	262	5.8	36.5
	Crackers sample A	324	2.2	14.5
	Crackers sample B	330	2.9	16.0
	Crackers sample C	132	0.7	< 10
	nd = not detected

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

Food Additives and Contaminants

. Two SRM transitions were recorded for each analyte: D3G (m/z 517.2 -> 457.2; 427.0),

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

Acknowledgements

The authors thank the UK Food Standard Agency, the Christian Doppler Society, the Lower Austrian government and the Austrian Science Fund (FWF) for financial support.


Tero ; Hirvonen

Harri Sinkko

Anja Hannele Hallikainen

Hannu Kiviranta

Pirjo Pietinen

Liisa Valsta

Jouni T Tuomisto

Liisa

Modelling the intake of polychlorinated dibenzo-p-dioxins and dibenzofurans: impact of energy under-reporting and number of reporting days in dietary surveys

Keywords: dioxins, energy under-reporting, intake assessment, dietary surveys 15

participants who were first interviewed with a 48-h recall and then filled in a 3-day food record twice. The concentrations of dioxins were obtained from previously published studies. The intake was estimated using a semi-parametric Monte Carlo simulation. The analyses were done separately for the whole study population and for the population excluding energy under-reporters. 10

To diminish the impact of intra-individual variation and nuisance effects, adjustment with software (C-SIDE ® ) was also done after Monte Carlo simulation. It was found that when C-SIDE ® was used, the 95 th percentile of intake and its confidence limit was higher with 2 reporting days than with a higher number of days. However, with a crude intake estimation (no adjustment), the confidence intervals of the 95 th percentile were also smaller with a higher number of days, but the 15 95 th percentiles were higher with a higher number of reporting days. When under-reporters were excluded the intakes increased, but the impact of energy under-reporting was smaller with 8 reporting days than with 2 days and smaller using C-SIDE ® than with a crude estimation. To conclude, adjustment for intra-individual variation and taking energy underreporting into account are essential for intake estimation of dioxins with food consumption data of limited number of 20 reporting days.

Introduction

Food of animal origin is the main source of dietary intake to polychlorinated dibenzo-p-dioxins and dibenzofurans (dioxins) (Liem et al. 2000). In Finland among foodstuffs the highest concentrations of dioxins are found in fish, especially Baltic Sea fatty fish [START_REF] Wiborg | EU maximum levels for 15 dioxins and dioxin-like PCBs -Impact on exposure and food supply in the Nordic countries[END_REF]) 5 (Fig. 1). The effects of dioxins on human health have been studied vigorously during past decades.

Based on animal studies and epidemiological human studies adverse health effects include cancer, immunotoxicity, developmental toxicity, and reproductive toxicity [START_REF] Who Temporary | Consultation on assessment of the health risk of dioxins; re-10 evaluation of the tolerable daily intake (TDI): executive summary[END_REF]. Public concern about the effects of dioxins on human health have lead to several risk assessments of these compounds, among others one carried out by the European Union Scientific Committee on Food 10 (SCF 2001). Furthermore, not only public concern was the cause of performing risk assessments of dioxins, but also scientific and political concern. Fish on the other hand, is an important source of polyunsaturated marine n-3 fatty acids, vitamin D, vitamin B 12 , and selenium (The National FINDIET 2007 Survey). For benefit/risk assessment of fish consumption, a precise estimation of intake of dioxins is essential. For dioxins -as for most of the contaminants -the chronic intake is 15 the most important issue in the risk assessment. Therefore, the usual (long term) intake of dioxins was estimated.

In order to be valid, the intake estimation of contaminants should be based on demographically representative food consumption data (Kroes et al. 2002). However, such data are usually not collected primarily for the purpose of intake assessment of contaminants, but for 20 nutritional epidemiology purposes or for dietary monitoring purposes. Nutrients usually have several sources of intake and their variation in concentration is relatively small. In addition, average intake, either for the population or at an individual level, is in most cases a sufficient level of information for nutrients. For contaminants the opposite is true: sources of individual contaminants are usually few and in food there is a great variance in contaminant concentration. In 25 the intake distribution should be estimated, including parameters like the 95 th percentile. The task is very challenging, as the dietary data usually cover only one or two reporting days. Furthermore, if only few reporting days are available, the intra-individual variation in food consumption is usually overestimated. However, software has been developed to take into account the intra-5

individual variation (removes the extra intra-individual variation caused by measurement error) and nuisance effects (e.g. day of the week, interviewer) in nutritional surveys [START_REF] Dodd | Statistical methods for estimating usual intake of nutrients and foods: a review of the theory[END_REF] and these could also be used for other substances than nutrients. The impact of the number of reporting days on contaminant intake has not, to our knowledge, been investigated earlier.

Energy under-reporting is a situation where energy intake calculated from food 10 consumption is not sufficient to cover the energy expenditure (Maurer et al. 2006). It is due either to misreporting of the diet or by restriction of the energy intake during the reporting period (e.g. food record). It is a serious problem in nutritional epidemiology that does not affect only the energy intake estimates, but all nutrients and foods [START_REF] Hirvonen | Underreporting does not distort dietary surveys[END_REF][START_REF] Stallone | Dietary assessment in Whitehall II: the influence of reporting bias on apparent socioeconomic variation in nutrient intakes[END_REF][START_REF] Cook | The problem of accuracy in dietary surveys: analysis of the over 65 UK national Diet and Nutrition Survey[END_REF]. The impact of energy under-reporting on contaminant intake has not, to our 15 knowledge, been investigated previously.

In this study, we assessed the impact of energy under-reporting and the number of reporting days in dietary surveys on the dioxin intake. In addition, we examined the impact of software that aims at decreasing the impact of intra-individual variation and nuisance effects (e.g. day of the week, interviewer). 20 (2) the city of Turku and some rural municipalities in South-Western Finland; and the provinces of (3) North Karelia, (4) North Savo and ( 5) Oulu. The participation rate was 63%, i.e. 6,259 subjects. Of these, 2,069 persons were invited to participate in the FINDIET Study. 5

Materials and methods

Data

Participants were interviewed using the 48-h dietary recall between January and March. The 48-h recall data consisted of all days except Fridays. A subsample of the participants filled in a 3-day food record twice: the first starting the day after the 48-h recall in January-March and the second in June-December (Fig. 2). The National Food Composition Database FINELI ® (www.fineli.fi) was used to calculate the food consumption. The content of dioxins of foods has 10 been determined in national food monitoring surveys during [START_REF] Hoffmann | Estimating the distribution of usual dietary intake by short-term measurements[END_REF][START_REF] Shetty | Energy requirements of adults[END_REF](Isosaari et al. 2006;;[START_REF] Wiborg | EU maximum levels for 15 dioxins and dioxin-like PCBs -Impact on exposure and food supply in the Nordic countries[END_REF]. The dioxin content in foods in these publications were expressed as PCDD equivalents.

The long-term intake of dioxins was estimated by multiplying the food consumption (g/day) by respective dioxin content (pg/g) for all reporting days. The multiplication was done 15 using bootstrapping (semiparametric Monte Carlo simulation), where food consumption data were treated as constant [START_REF] Iman | A Distribution-Free Approach to Including Rank Correlation Among Input Variables[END_REF]. Bootstraping was used in the intake estimation of both crude (C-SIDE not used) and adjusted (C-SIDE used) results. Therefore, bootstrapping was done for concentration data only. Bootstrapping was done only on 400 rounds because it was a timedemanding procedure. We used bootstrapping because concentration data has single foods with a 20 small number of values. In this case, estimation of theoretical distribution could be unreliable for a single food concentration. Medians produced from 400 point estimates and confidence intervals of parameters were calculated directly from the bootstrapped data when no adjustment was used (crude results) and from the usual intake distributions when adjustment (C-SIDE) was used. The impact of energy under-reporting on intake of dioxins was investigated by estimating the intakes both with under-reporters and without under-reporters. Energy underreporters were excluded using 1.00*BMR (basal metabolic rate) as a cut-off point [START_REF] Goldberg | 10 Critical evaluation of energy intake data using fundamental principles of energy physiology: 1. Derivation of cut-off limits to identify under-recording[END_REF].

The impact of intra-individual variation and nuisance effects on intake of dioxins 5 was assessed by comparing the results adjusted for intra-individual variation and nuisance effects with non-adjusted results. This adjustment was done after probabilistic (bootstrapping) intake estimation. The adjustment was done using the method of Nusser and co-workers (Nusser et al.1996). This method gives the long-run average of daily intakes (usual daily intake) by taking into account day-to-day correlation and nuisance effects (such as day-of-week and interview 10 sequence). It also allows exceptions from normality through grafted polynomial transformations and recognizes the measurement error associated with one-day dietary intakes. The estimations were done using the SAS based C-SIDE  program. All the analyses were done separately for men and for women, because men and women have different food consumption patterns (The National FINDIET Study 2007). 15

Results

The most important sources of intake of dioxins for both males and females were fish, especially Baltic herring and salmon (Table 1). The total daily intake of dioxins for men was 0.71 pg WHO-TEQ/kg bw and 0.47 pg WHO-TEQ/kg bw for women. The contribution of fish to the total intake 20 was 94% and 91% for men and women, respectively. Of all participants, 36% of men and 39% of women were identified as energy underreporters. The mean (standard deviation) energy intake in all males was 9000 kJ (2210 kJ) and in all females 7050 kJ (1610 kJ). Among under-reporters the mean energy intake was 6970 kJ (1110 kJ) in males and 5630 kJ (899 kJ) in females. The Spearman correlations between intake of 25 When no adjustments for intra-individual variation or nuisance effects were done (crude results), in males confidence limits of the 95 th percentile of intake of dioxins were higher for three reporting days than for two reporting days, but tended to diminish with higher number of 5 reporting days (Table 2). For females, the 95 th percentile of intake and their confidence limits tended to increase with increasing reporting days. The impact of excluding energy under-reporters among males was largest with three reporting days on intake of dioxins, but decreased with increasing number of reporting days for crude results. In females, the impact of under-reporters was negligible, but increased during the second record period. For median intakes the impact of 10 number of days or energy under-reporting was small.

When the adjustment for intra-individual variation and nuisance effects was done, the 95 th percentile of intake and its confidence limit was higher with 2 reporting days than with a higher number of days and both 95 th percentiles and their confidence limits diminished monotonically in men with an increasing number of reporting days for both males. In women, 15 however, the impact of number of reporting days was smaller (Table 2). The impact of excluding energy under-reporters diminished with increasing reporting days. In addition, for males the median intakes and their confidence limits diminished with increasing reporting days. In females, both energy under-reporting and number of reporting days had negligible impact on median intakes. 20

Discussion

In the present study, we found that the high number of reporting days had a great effect on the dioxin intake estimates, especially by narrowing the confidence intervals after adjustment for intra-individual variation and nuisance effects by software. In addition, adjusting for nuisance 25 effects and decreasing the intra-individual variation by the use of software, the impact of the number of reporting days on the confidence intervals of the intake estimates was bigger. In addition, by taking into account the nuisance effects and intra-individual variation, the impact of energy under-reporting was reduced. In addition, the intake of dioxin was smaller when the adjustment for intra-individual variance and nuisance effects was done than the non-adjusted 5 intakes.

It is clear that two consecutive reporting days is not enough to estimate the intake of dioxins or any other contaminant -especially if the higher end of the intake is of interest. However, it is not clear how many days are enough to estimate contaminant intake. There probably is a trade-off between the costs of additional days and accuracy of estimates. 10 Furthermore, the sufficient accuracy in turn depends on the margin of exposure of the contaminant, i.e. if exposure is likely to be near the tolerable intake level, then the accuracy should be higher than with a compound with higher margin of exposure. The number of reporting days depends also on the food consumption pattern, since sources of contaminants vary greatly culturally. If too few days are used, the intake of dioxins is overestimated and the risk for 15 excessive intake is also overestimated, especially when adjustment for intra-individual variation and nuisance effects are made. The number of reporting days is also a question of participant compliance: participation could drop dramatically if the work load of the participants is increased.

For example, in this study, 88% of the participants returned the first food record, but only 65% also returned the second food record (The National Findiet 2007Survey, 2008). 20

In addition to number of reporting days, it is also important that all days are not consecutive, since a low number of only consecutive days could result artificially to low intraindividual variation in food consumption and contaminant intake. This phenomenon was also seen in this study: only two consecutive days gave smaller confidence intervals than higher number of non-consecutive days. For nutrients two non-consecutive days are estimated to be sufficient for 25 [START_REF] Hoffmann | Estimating the distribution of usual dietary intake by short-term measurements[END_REF]. However, when the number of consecutive days is high enough (e.g. one week), the intra-individual variation is probably captured.

The fact that non-adjusted intakes were much higher than adjusted intakes when reporting days were increased is surprising and should be studied further. However, the confidence limits diminished monotonically in adjusted intakes, whereas in non-adjusted intakes the standard 5 error did not change. This is an expected result, since the software is able make use of the increased amount of measurements per individual. This and adds assurance that the programme functions properly. When these quite divergent results are taken together, it could be concluded that the software is useful in decreasing the intra-individual variation, but it is clear that the software cannot correct for insufficient number of reporting days. 10

Adjusting for intra-individual variation and nuisance effects by using software seems to be an efficient way to diminish the confidence limits of the 95 th percentiles and to decrease the impact of energy under-reporting. We did not, however, validate our results against a golden standard method (e.g. adipose tissue concentration of dioxins). Therefore, it is difficult to say whether a decrease in the intra-individual variation is in accordance with the reality. Therefore, 15 more research -both theoretical and practical -is needed to find out the role of intra-individual variation and energy under-reporting on contaminant intake estimation.

Energy under-reporting does not seem to be of great concern in the estimation of dioxin intake, especially if the number of reporting days is sufficient and if methods to adjust for intra-individual variation and nuisance effects are used. This is not surprising, since in a Finnish 20 study it was found that foods that are considered healthy are under-reported less or are even overreported [START_REF] Hirvonen | Underreporting does not distort dietary surveys[END_REF]. Fish in Finland is considered to be healthy and therefore it is probably not under-reported to a great extent. On the other hand, in an American study fish was not under-reported to a lesser extent than other foods (Krebs-Smith et al. 2000). In fact, in this study the qualitative differences in under-reporting were in all rather small. The differences in the 25 Finnish and the American study could be partly related to cultural factors and partly to the fact that in the Finnish study the method to measure food consumption was a 48-h recall and a 3-day food record whereas in the American study a food frequency questionnaire was used. The fact that under-reporting affected women less than men (at least in terms of absolute intake), even though women are in general more prone to under-report (Maurer et al.2006), is surprising. Perhaps this 5 has to do with higher health consciousness of women -they are more aware than men that fish is considered healthy and therefore, probably under-reported fish less or even overreport fish consumption.

However, it should be kept in mind that using 1.0*BMR as a cut-off point for underreporting is a rather low limit and therefore excludes only the extreme energy under-reporters. 10 Using a higher cut-off level, however, would not be justifiable, since due to high inter-individual variation in the basal metabolic rate [START_REF] Shetty | Energy requirements of adults[END_REF], part of the population has a very low basal metabolic rate and excluding them is not correct. As a consequence, the data still probably contain some bias caused by energy under-reporting.

The present study shows that the sources of intra-individual differences (e.g. 15 temporal variation) should be taken into account when food consumption studies that are used for intake estimation of contaminants are planned. This underlines the co-operation between those estimating contaminant intake and those who plan and carry out dietary surveys. In fact, precise calculations are needed to estimate the number of reporting days for the contaminants or other non-nutrient substances to be estimated prior to a dietary survey. The risk can only be estimated 20 when the high intake levels can be compared to reference levels, e.g. tolerable daily intake.

Could some other food consumption method be used to estimate contaminant intake in order to avoid the problems with 24/48-h recalls or food records? A food frequency questionnaire would be an appealing alternative because of its convenience both for the investigator and for the participant. However, for each group of contaminants a separate food 25 11 frequency questionnaire should be planned and validated separately. This is seldom feasible, since this process is time consuming and costly. In addition, food frequency questionnaires are not suitable for estimating absolute intakes, but are to group people into low and high intake groups (Willett 1998). The other problem with food frequency questionnaires is often energy overreporting rather than under-reporting. However, these problems can partly be overcome by asking 5 a small part of the participants (who fill also food frequency questionnaire) to fill in also dietary records. Dietary records could then be used to calibrate e.g. portion sizes and energy intake.

Another alternative to dietary recalls or food records would be dietary history. The problem with the low number of reporting days would be avoided, since dietary history aims at forming an overall view of the participant's diet. However, dietary history has probably also the problem of 10 energy under-reporting, since dietary history is usually started with a 24-h recall. In addition, dietary history is very time consuming both for study personnel and participants and therefore also costly.

Our results are not directly generalizable to other populations or to other contaminants or other substances, since energy under-reporting patterns and sources of 15 contaminants vary between populations and population groups (e.g. children). However, with a high number of reporting days the intake estimates of any contaminant in any population are probably more accurate than with a low number. In addition, estimating contaminant intake alsobut not solely -excluding under-reporters is useful and gives a rough estimate of the impact of under-reporting on results. 20

In conclusion, intra-individual variation and nuisance effects may have a large impact on contaminant intake estimates. In addition, by estimating the intake both with underreporters and without under-reporters the impact of energy under-reporting could be assessed.

Furthermore, one 48-h recall is not sufficient to estimate the intake of a contaminant. However,

  -term intake estimation of dioxins was carried out using food consumption 5 data obtained from the National FINDIET 2007 Survey. The study population consisted of 606

  on food consumption were obtained from the National FINDIET 2007 Survey, carried out as part of the FINRISK 2007 Study which monitors cardiovascular risk factors (Fig. 2), (The National FINDIET 2007 Survey ). A random sample of 9, 958 persons aged 25-74 years in five 25 by sex, area, and 10-year age groups, was taken from the population register. The study areas were: (1) Helsinki area;

  were in all males 0.15 and in females 0.02. Among under-reporting males the correlation was -0.04 and in females 0.02.

Figure 1 . 5 Figure 2 .

 152 Figure 1. Concentration of dibenzo-p-dioxins and dibenzofurans (WHO-TEQ) in foodstuffs. BS=

Table 1 .

 1 Sources of PCDD/F compounds (intake of WHO-TEQ) by sex.

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Table 2 .

 2 Intake of PCDD/F compounds (WHO-TEQ as pg/kg bw/day) in crude and adjusted estimations by sex, number of reporting days, and under-reporting,

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

This study was funded by the EU-funded project BENERIS. ://mc.manuscriptcentral.com/

All (n=606)

Under-reporters excluded (n=365) days Median (CI) 95% (CI) Median (CI) 95% (CI) 2 0.05 (0.05-0.06) 2.06 (1.65-2.62) 0.06 (0.05-0.06) 2.25 (1.65-3.09) 3 0.07 (0.07-0.08) 2.78 (2.04-3.84) 0.09 (0.08-0.10) 3.88 (2.64-5.71) 4 0.09 (0.08-0.10) 2. 55 (1.93-3.58) 0.13 (0.11-0.14) 3.44 (2.39-4.95) 5 0.11 (0.10-0.12) 2.58 (2.03-3.34) 0.16 (0.14-0.18) 2 0.30 (0.21-0.45) 1.90 (1.34-2.54) 0.30 (0.21-0.46) 2.47 (1.58-4.25) 3 0.31 (0.24-0.39) 1.13 (0.83-1.49) 0.33 (0.26-0.45) 1.54 (1.00-2.47) 4 0.30 (0.25-0.37) 1.07 (0.83-1.34) 0.31 (0.24-0.42) 1.37 (0.88-1.99) 5 0.30 (0.23-0.37) 1.04 (0.81-1.28) 0.31 (0.25-0.39) 1.19 (0.90-1.51) 6 0.29 (0.23-0.37) 0.92 (0.73-1.12) 0.31 (0.25-0.41) 1.05 (0.80-1.28) 7 0.28 (0.23-0.36) 0.87 (0.69-1.09) 0.30 (0.23-0.41) 1.03 (0.79-1.26)

Men

Adjusted estimation 8 0.28 (0.23-0.34) 0.86 (0.69-1.05) 0.29 (0.23-0.40) 0.99 (0.79-1.21) 2 0.05 (0.05-0.05) 1.27 (1.08-1.47) 0.06 (0.05-0.07) 1.38 (1.08-1.76) 3 0.07 (0.06-0.08) 1.44 (1.20-1.78) 0.08 (0.07-0.09) 1.56 (1.28-1.90) 4 0.09 (0.08-0.10) 1.74 (1.42-2.07) 0.10 (0.09-0.11) 1.81 (1.37-2.25) 5 0.11 (0.10-0.12) 1.78 (1.50-2.11) 0.13 (0.12-0.14) 1.74 (1.47-2.26) 6 0.13 (0.12-0.14) 1.95 (1.62-2.46) 0.14 (0.13-0.16) 1.70 (1.32-2.22) 7 0.14 (0.13-0.15) 1.96 (1.56-2.37) 0.15 (0.13-0.16) 1.66 (1.34-2.22) Crude estimation 8 0.16 (0.16-0.18) 2.09 (1.72-2.60) 0.17 (0.16-0.18) 1.88 (1.37-2.55) 2 0.23 (0.20-0.27) 1.03 (0.65-1.82) 0.30 (0.23-0.39) 1.15 (0.75-1.65) 3 0.24 (0.21-0.29) 0.81 (0.61-1.12) 0.32 (0.27-0.38) 0.94 (0.64-1.42) 4 0.28 (0.24-0.36) 0.83 (0.64-1.07) 0.35 (0.29-0.43) 0.97 (0.68-1.33) 5 0.27 (0.23-0.32) 0.82 (0.66-1.06) 0.33 (0.28-0.40) 0.92 (0.72-1.20) 6 0.29 (0.25-0.33) 0.80 (0.66-1.02) 0.35 (0.29-0.42) 0.94 (0.73-1.21) 7 0.30 (0.26-0.35) 0.82 (0.68-1.04) 0.37 (0.31-0.44) 0.98 (0.76-1.27) Women Adjusted estimation 8 0.30 (0.26-0.35) 0.80 (0.65-1.04) 0.38 (0.32-0.45) 0.99 (0.77-1.28)