Thomas Simat

Formation of N-formylkynurenine suggests the involvement of apolipoprotein B-100 centered tryptophan radicals in the initiation of LDL lipid peroxidation.

Tryptophan oxidation products were determined in pronase E digests of apo B of delipidated, native and Cu2+ oxidized LDL using a sensitive and specific HPLC method. Oxidized LDL contained N‐formylkynurenine, kynurenine and tryptamine but no oxindolylalanine and 5‐hydroxytryptophan. N‐Formylkynurenine increased from an initial value of 0.21 to 1.67 mollmol apo B within 5 h. Apo B of native LDL also contained kynurenine (0.80 mollmol) and tryptamine (0.13 mol/mol). The results support the assumption that oxidation of Trp residues in apo B is an early event and possibly an elementary reaction involved in initiating LDL oxidation.

Synthesis and analysis of oxidation and carbonyl condensation compounds of tryptophan

Abstract Modified methods of synthesizing the oxidative degradation products N-formylkynurenine, oxyindolylalanine diastereomers and dioxyindolylalanine diastereomers (DiOia) and the carbonyl condensation products 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-pentyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid are described. These methods produce compounds with purities sufficiently high to allow them to be used as reference substances for analytical purposes and as samples for toxicological investigations. The obtained substances were characterized by 1H-NMR, 13C-NMR, IR and UV spectroscopy. The purities of the substances were verified by RP-HPLC and UV detection. An RP-HPLC method was developed which allowed the separation of the synthesized products, 5-hydroxytryptophan, 3-hydroxykynurenine and tryptophan. As an application, a sample of eosinophilia-myalgia syndrome (EMS)-related tryptophan was examined. Low contents of the oxidation products were found, together with the known peaks A–E.

Correlation between the level of the potential biomarker, heat-shock protein, and the occurrence of DNA damage in the dab, Limanda limanda : a field study in the North Sea and the English Channel

In the present study, heat-shock protein of M(r) 70 kDa (HSP70), a marker of cellular stress response, was validated as a potential biomarker under field conditions. The dab, Limanda limanda (female, size > or = 25 cm, spawning maturity stage 2) was used as the indicator organism. The data on HSP level were correlated with the occurrence of DNA damage, measured in the same specimens of L. limanda, to prove the usefulness of the method. The area under investigation was the North Sea. Four locations were selected: station N01, close to Heligoland, in the North Sea; station N04 at the Dogger Bank; station N06 at the Firth of Forth; and station G08 in the English Channel. Ten animals from each location were selected and their livers used for the experiments. The results show that the highest levels of HSP70 (consisting of two forms of M(r) 75 and 73 kDa) were in fish from station N04, while low values were measured in livers from L. limanda collected at station N01. Intermediate levels were seen in the animals from the two other locations. By application of a novel technique, it was found that the extent of DNA damage (single-strand breaks and alkaline labile sites) in fish liver parallels the levels of both HSP70 forms. Our results suggest that L. limanda may be a useful bioindicator and heat-shock proteins, a useful biomarker for monitoring of environmental pollution.

Migration from can coatings: Part 3. Synthesis, identification and quantification of migrating epoxy-based substances below 1000 Da

Bisphenol A-derived glycidyl ethers as well as its reaction products with other lacquer components can migrate into the packed food from epoxy-based can coatings. A sensitive and selective method is presented using high-performance liquid chromatography coupled with ultraviolet light, fluorescence and electrospray ionization-mass selective detection for the identification and quantification of all migrants with a bisphenol A backbone and a molecular weight below 1000 Da, an estimated boundary for the absorption in the gastrointestinal tract. The identification of migrants was confirmed by microreactions of technical bisphenol A diglycidyl ether with solvents and phenols, which provided the fragmentation pattern of the mass selective detection and relative retentions of 42 different bisphenol A-related substances. It was shown by calibration of different isolated and synthesized bisphenol A derivatives that the fluorescence response relies on the amount of bisphenol A moiety in the respective molecule. Therefore, all migrating bisphenol A-related substances below 1000 Da were determined as bisphenol A diglycidyl ether equivalents using a calibration (fluorescence detection) of the commercially available bisphenol A diglycidyl ether monomer. The limit of quantification was set at 5 μg bisphenol A diglycidyl ether equivalents kg−1 (or 0.8 μg dm−2). This method was validated for epoxy coatings (0.1 μg dm−2 limit of detection and 24 μg bisphenol A-related substances below 1000 Da dm−2 standard deviation, corresponding to 4.4% relative standard deviation). The quantification could be extended by combining the fluorescence response and structural information gained from the mass spectra, which provides more accurate results for each migrant. The calculation is based on the calibration of the bisphenol A chromophore content of the molecule. According to this method, the amount of migrating bisphenol A-related substances below 1000 Da in the acetonitrile extract (assuming a worst case) varied from about 0.4 to 0.7 mg dm−2 in the examined coatings. The determined amounts comply with about 50% of the total migrate below 1000 Da.

‘Untypical aging off-flavor’ in wine: formation of 2-aminoacetophenone and evaluation of its influencing factors

2-Aminoacetophenone (AAP) is known as the character impact compound responsible for the so called untypical ‘aging off-flavor’ (UTA) in Vitis vinifera wines. The formation of AAP is caused by an oxidative degradation of the phytohormone indole-3-acetic acid (IAA), triggered by sulfuration after fermentation. Using different radical generating systems (hydroxyl and/or superoxide radical generating systems) it could be shown especially that superoxide radicals are responsible for the formation of AAP. Hereby, a pyrrole ring cleavage of IAA yields 3-(2-formylaminophenyl)-3-oxo-propionic acid (FAPOP), N-formyl-2-aminoacetophenone (FAP), and AAP. Analysis of 32 grape musts and their corresponding wines revealed that wines with a higher superoxide radical scavenger activity are less prone to UTA formation. However, UTA is not correlated with the IAA content of the must or wine. Differences in the release of IAA during fermentation of musts derived from early and late harvested grapes indicate a correlation between the ripeness of the processed grapes, the IAA content at the time of sulfuration, and the UTA formation.

Induction of DNA damage and expression of heat shock protein HSP70 by polychlorinated biphenyls in the marine sponge Suberites domuncula Olivi

The effects of different polychlorinated biphenyls (PCBs) including toxic coplanar non-ortho (PCB77) and non-planar mono-ortho (PCB118) and di-ortho (PCB153) congeners on the extent of DNA damage as well as on the expression of heat shock protein 70 (HSP70) were investigated in the marine sponge Suberites domuncula Olivi (Porifera; Demospongiae). A time-dependent increase in the number of DNA single-strand breaks, expressed as strand scission factor (SSF), was found after injection of a single dose of 25 μg of PCB118 or PCB153 per gram wet mass of S. domuncula, using Fast Micromethod assay, which is based on the unwinding of DNA under alkaline conditions. The number of strand breaks induced by PCB153 was about two-fold higher than that observed for PCB118 after an incubation period of six days. Unexpectedly, a strong reduction in the rate of DNA unwinding compared to untreated control, that could be indicative for the formation of DNA crosslinks, was found following treatment of S. domuncula with PCB77, which is, in vertebrate systems, the most toxic among the PCB congeners studied. The three selected PCB congeners are able to induce the expression of both “constitutive” (HSP73; Mr 73 kDa) and “inducible” (HSP75; Mr 75 kDa) HSP70 proteins. The expression of HSP75 was markedly lower than that observed for HSP73, and varied considerably among different sponge individuals exposed to PCB77. The results in this work demonstrate that: (1) marine sponges (S. domuncula) respond to distinct PCB congeners by induction of different degrees of DNA damage, and (2) expression of HSP is a potential (but not specific) biomarker for PCB exposure in sponges.

Migration of mineral oil, photoinitiators and plasticisers from recycled paperboard into dry foods: a study under controlled conditions

Migration from recycled paperboard was monitored after 2, 4 and 9 months of storage for six test foods industrially packed in five configurations, four with internal plastic films. After 9 months, the migration of mineral oil saturated hydrocarbons into foods directly packed in the paperboard amounted to 30–52 mg/kg, which corresponded to 65%–80% of those of a volatility up to that of the n-alkane C24 in the paperboard. The concentration of the migrated aromatic hydrocarbons in the foods ranged from 5.5 to 9.4 mg/kg. More than half of this migration occurred in the first 2 months. Differences between the foods amounted to mostly less than a factor of 2 and seemed to be related to porosity or permeability more than fat content. Nine photoinitiators were detected in the paperboard, of which eight migrated into the packed food at up to 24%. Several plasticisers were present in the recycled paperboard, but only butyl phthalates showed significant migration. After 9 months, up to 40% of diisobutyl phthalate and 20% of dibutyl phthalate migrated into the food with direct contact. The internal polyethylene film hardly slowed migration, but the film and the tray absorbed approximately three times more mineral oil than the food, despite constituting merely 4% of the mass of the pack. Oriented polypropylene strongly slowed migration: The highest migration of saturated hydrocarbons measured after 9 months (2.3 mg/kg) corresponded to only 3% of the content in the paperboard and included migrated polyolefin oligomeric saturated hydrocarbons. Coating of polypropylene with an acrylate further slowed the migration, but the migration from the paperboard was still detectable in four of the six samples. Polyethylene terephthalate was a tight barrier.

Migration from can coatings: Part 2. Identification and quantification of migrating cyclic oligoesters below 1000 Da

Metal cans for food use can be coated with lacquers based on polyester resins. Recent research has focussed on the identification and quantification of migrants released by coatings that are potentially absorbable (below 1000 Da). The presented method describes a procedure that was optimized to hydrolyse the polyester migrants into their monomers, polyvalent acids and polyols. The polyols were identified by gas chromatography with flame ionization detection GC-FID and the acids by high-performance liquid chromatography (HPLC) coupled with an ultraviolet and an electrospray ionization-mass selective detector (HPLC-ESI-MSD/UVD), respectively. With the knowledge of the polyester monomers, it was possible — at least tentatively — to identify the main components in the migrate as cyclic oligoesters by HPLC-ESI-MSD/UVD. A cyclic oligomer, CYCLO [3IPA (isophthalic acid) 3EG (ethylene glycol)] was synthesized and characterized by infrared, nuclear magnetic resonance and mass spectrometry as well as by elementary analysis for further confirmation. To determine the amount of migrating cyclic oligoesters, the response of the migrating substances was compared using different detectors, UVD, MSD and evaporative light scattering detector (ELSD). The response of the ELSD was dependent on the molecular weight of the analytes that reduced the accuracy of this detection type. The wavelength with the same absorption coefficient for IPA and terephthalic acid (TPA) was obtained at 232 nm. The UV232nm response of an oligoester is proportional to the number of its IPA/TPA moieties, which was verified for several TPA/IPA esters. The amount of the migrating oligoesters was determined using an UV232nm calibration of a commercially available TPA ester and the number of IPA/TPA moieties molecules gained from the ESI-MSD spectra. According to this method, the amount of migrating oligoesters below 1000 Da in the 95% ethanol migrate varied from 0.1 to 0.6 mg dm−2 (0.6–3.6 mg kg−1 food) in the examined coatings. The determined amounts account for about 50% of the total migrate below 1000 Da.

Migration of polyolefin oligomeric saturated hydrocarbons (POSH) into food

POSH are polyolefin oligomeric saturated hydrocarbons, such as oligomers from polyethylene or polypropylene. POSH that have migrated into foods are easily mistaken for mineral oil-saturated hydrocarbons (MOSH). In fact, both POSH and MOSH largely consist of highly isomerised branched and possibly cyclic hydrocarbons, both forming humps of unresolved components in gas chromatography. Chromatograms are reported to show typical elution patterns of POSH and help analysts distinguishing POSH from MOSH as far as possible. Since the structures of the POSH are not fundamentally different from those of the MOSH, it would be prudent to apply the evaluation of the MOSH. However, the migration is frequently beyond that for which safety has been demonstrated. This is shown for a few examples, particularly for powdered formula for babies.

Mass transfer ways of ultraviolet printing ink ingredients into foodstuffs.

The case of isopropylthioxanthone (ITX) showed conclusively that the ingredients of ultraviolet printing inks may migrate into packaged foodstuffs. For multilayered materials like beverage cartons, the only way that mass transfer can occur is by the so-called set-off effect. In contrast, in the case of rigid plastics like yoghurt cups, two other methods of mass transfer, permeation and gas phase, have to be considered. In cooperation with producers of ink, plastic cups and yoghurt, a project was conducted in order to elucidate the mass transfer of ink ingredients. In addition, the influence of storage time and the age of ultraviolet lamps on the migration level was examined. The suitability of 50% ethanol as a simulant for yoghurt was also tested. ITX was chosen as a model migrant, as it is easily detectable. Furthermore, the migration of two other substances, the photo-initiator 2-methyl-4′-(methylthio)-2-morpholinopropiophenone (MTMP) and the amine synergist ethyl-4-(dimethylamino)benzoate (EDAB), which may be used in combination with ITX, was studied. Before being filled with yoghurt or 50% ethanol, the printed cups were stored under different contact conditions, with and without contact between the inner layer and the printed surfaces, in order to distinguish between the possible mass transfer ways. All analyses were performed by means of high performance liquid chromatography with diode array and fluorescence detection (HPLC-DAD/FLD). It was shown that contamination with ITX and EDAB occurs via set-off and that the degree of migration increases with lamp age and storage time of the unfilled cups. Migration of MTMP was not detectable. The results show that besides the careful selection of the appropriate raw materials for printing ink, a close monitoring of the process also plays a major role in migration control. In addition, the results proved that 50% ethanol is a suitable simulant for yoghurt.