Application of gastrointestinal modelling to the study of the digestion and transformation of dietary glycidyl esters.

Glycidyl esters (GEs) are known to be formed during vegetable oil processing. Because of their structure, it has been hypothesised that GEs, like fatty acid esters of chloropropanols (MCPD esters), may be accepted as substrates by gut lipases to release the epoxide glycidol. If confirmed such a hypothesis would be important for risk assessment since glycidol is considered as a genotoxic carcinogen. In the present study, biotransformation was investigated using static and dynamic gastrointestinal models. During the experiments, aliquots were analysed for non-digested GEs using liquid chromatography-time-of-flight-mass spectrometry (LC-ToF-MS). In the static model, a fast hydrolysis of GEs was observed as a result of lipase action. Lipase was very efficient at pH 4.8, and totally inhibited at very low pH (1.7). In the absence of lipase, GEs were found to be relatively stable. The potential impact of food matrix was studied using milk in a dynamic model simulating human physiological conditions. The fast, pH-dependent hydrolysis of GEs was further confirmed. The possible transformation of the digestion products was then investigated using gas chromatography coupled to mass spectrometry (GC-MS), mainly the epoxide ring-opening to glycerol followed by additional reactions. In any conditions applied, neither 2- nor 3-mono-chloropropanediol (2- nor 3-MCPD) were formed, indicating that a ring-opening of the epoxide group of GEs or glycidol followed by a reaction with chloride was unlikely. A small transformation of glycidol into glycerol was observed after longer incubation time correlated with a low pH. This suggested that ring-opening and reaction with water is possible in strongly acidic conditions. Overall, it is concluded that GEs are rapidly digested by gut lipases to form glycidol. Consequently, GEs should be considered as sources of glycidol exposure. In addition, risk assessment of GEs can likely rely on hazard identification and characterisation data specific for glycidol.

Analytical approaches for MCPD esters and glycidyl esters in food and biological samples: a review and future perspectives.

Esters of 2 - and 3-monochloropropane-1,2-diol (MCPD) and glycidol esters are important contaminants of processed edible oils used as foods or food ingredients. This review describes the occurrence and analysis of MCPD esters and glycidol esters in vegetable oils and some other foods. The focus is on the analytical methods based on both direct and indirect methods. Methods of analysis applied to oils and lipid extracts of foods have been based on transesterification to free MCPD and determination by gas chromatography-mass spectrometry (indirect methods) and by high-performance liquid chromatography-mass spectrometry (direct methods). The evolution and performance of the different methods is described and their advantages and disadvantages are discussed. The application of direct and indirect methods to the analysis of foods and to research studies is described. The metabolism and fate of MCPD esters and glycidol esters in biological systems and the methods used to study these in body tissues studies are described. A clear understanding of the chemistry of the methods is important when choosing those suitable for the desired application, and will contribute to the mitigation of these contaminants.

Determination of fumonisins B1 and B2 in corn by LC/MS with immunoaffinity column cleanup: interlaboratory study.

An interlaboratory validation study was conducted to establish the method performance characteristics of an immunoaffinity column (IAC) cleanup procedure followed by LC/MS for the determination of fumonisins B1 (FB1) and B2 (FB2) and combined FB1 + FB2 in corn. The test portion is extracted with acetonitrile-methanol-water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an IAC. FB1 and FB2 are removed with methanol, followed by water, then directly determined by RPLC with MS detection using selected-ion monitoring of two characteristic ions in each case. Naturally contaminated corn samples were milled to a fine powder and mixed to produce three samples with target levels of combined FB1 + FB2 ranging from 350 to 4000 microg/kg. Of 15 initially participating laboratories, two failed to report results and another did not follow the prescribed method. Thus, valid results were obtained from 12 participants located in 11 countries. Statistical analysis of the results produced RSDr values of 4.6-11.9, 1.9-12.6, and 1.4-11.5% for FB1, FB2, and combined FB1 + FB2, respectively; the corresponding RSDR values were 19.8-23.8, 18.2-25.5, and 18.8-23.2%. The three concentration levels of combined FB1 + FB2 were 534, 1194, and 1954 microg/kg. HorRat values for r and R were all < 2.0, indicating that the method is suitable as a regulatory method for the enforcement of European Union limits for fumonisins in corn.

Esters of 3-chloro-1,2-propanediol (3-MCPD) in vegetable oils: significance in the formation of 3-MCPD.

3-Mono-chloropropane-1,2-diol (3-MCPD) is a contaminant that occurs in food in its free (diol) form as well as in an esterified (with fatty acids) form. Using a simple intestinal model, it was demonstrated that 3-MCPD monoesters and 3-MCPD diesters are accepted by intestinal lipase as substrates in vitro. Under the chosen conditions, the yield of 3-MCPD from a 3-MCPD monoester was greater than 95% in approximately 1 min. Release from the diesters was slower, reaching about 45, 65 and 95% of 3-MCPD after 1, 5 and 90 min of incubation, respectively. However, in human, the hydrolysis of 3-MCPD esters is unlikely to release 100% as 3-MCPD, as triglycerides and phospholipids are hydrolysed in the intestine liberating 2-monoglycerides. Assuming a similar metabolism for 3-MCPD esters as that known for acylglycerols in humans in vivo, the de-esterification in positions 1 and 3 would thus be favoured by pancreatic lipases. Therefore, 3-MCPD, and 3-MCPD-2 monoesters would be released, respectively, from the 1-/3-monoesters, and the diesters potentially present in food. Hence, information on the exact amounts of the partial fatty acid chloroesters, i.e. 3-MCPD mono- and diesters, is important to assess the contribution of foods to the bioavailability of 3-MCPD. Therefore, a rapid method for the determination of the ratio of 3-MCPD monoesters to diesters in fats and oils was developed using gas chromatography-mass spectrometry (GC-MS) and isotopically labelled 3-MCPD esters as internal standards. The analysis of 11 different samples of fat mixes typically employed in food manufacturing demonstrated that a maximum of about 15% of the total amount of 3-MCPD bound in esters is present in the monoesterified form. The potentially slower release of 3-MCPD from 3-MCPD diesters, and the mono- to diesters ratio suggest that 3-MCPD esters may in fact contribute only marginally to the overall dietary exposure to 3-MCPD. Further work on the bioavailability, metabolism and possible toxicity of chloroesters per se is warranted.

Occurrence of fumonisins in asparagus (asparagus officinalis L.) and garlic (allium sativum L.) from Germany.

Fusarium proliferatum is able to produce fumonisins and is considered a pathogen of many economically important plants (e.g. corn, rice, asparagus) [1]. The occurrence of fumonisin FB1 inF. proliferatum infected asparagus spears from Germany was investigated using a liquid chromatography/electrospray ionization-mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB1-d6 as internal standard. Asparagus samples were harvested in July 2000 and screened forFusarium species. AltogetherF. oxysporum, F. proliferatum and F. sambucinum were isolated from the spears. The samples infected with F.proliferatum were subsequently analyzed for fumonisins. FB1 was detected in 9 of the 10 samples in amounts ranging from 36.4 ng/g to 4513.7 ng/g (based on dry weight). Fumonisins FB2 and FB3 were found in six samples in lower concentrations. In asparagus spears of June 2002 we could findF. proliferatum in 6% of the samples, however no fumonisins were detectable.Furthermore the capability of producing FB1 by the fungus in garlic bulbs was investigated. Therefore garlic was cultured inF. proliferatum contaminated soil and the bulbs were screened for infection with F.proliferatum and for the occurrence of fumonisins by LC-MS. F.proliferatum was detectable in the garlic tissue and all samples contained FB1 (26.0 ng/g to 94.6 ng/g).This is the first report of the natural occurrence of FB1 in German asparagus spears and furthermore our findings suggest a potential for natural contamination of garlic bulbs with fumonisins. For detailed results and methods see Ref. [2].

Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites.

Fumonisin B1 (FB1) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB1, fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1) and N-palmitoyl-hydrolyzed fumonisin B1 (N-Pal-HFB1) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 micromol/L FB1 for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB1 was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis.

Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins.

Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H](+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.

Formation of fumonisin artefacts in thermal treated food.

In order to study the formation of fumonisin artefacts and the binding of fumonisins to matrix components (e.g. starch and protein) in thermal treated food, model experiments were performed by heating fumonisin FB1 with amino acid derivatives (protein model) and methyl-a-D-glucopyranoside (starch model). The reaction products were analysed by highperformance liquid chromatography-electrospray ionisationtandem mass spectrometry (HPLC-MS/MS). Using MS/MS experiments the formed reaction products were characterized as conjugates between fumonisin B1 and the used substrates. The reaction product between fumonisin B1 and methyl-α-D-glucopyranoside was purified and identified by nuclear magnetic resonance (NMR) spectroscopy as the diester of fumonisin B1 and methyl-α-D-glucopyranoside. These studies indicate that fumonisins can bind to matrix components via their TCA side chains.

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry.

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.

[Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites].

It is well-known that fumonisin B1 (FB1) stimulates apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been investigated. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB1, fumonisin B2 (FB2), hydrolyzed fumonisin B1 (HFB1) and N-palmitoyl-hydrolyzed fumonisin B1 (N-Pal-HFB1) and investigated caspase 3 activation and DNA fragmentation. Only exposure to 10 µmol/L FB1 for 24h led to a significantly increased activity of caspase 3 and to DNA fragmentation. All other compounds tested did not show any significant activation of caspase 3 activity. Further we examined wether a sphinganine accumulation is correlated with the induction of apoptosis in IHKE cells. Therefore we developed a liquid chromato-graphy/electrospray ionization-tandem-mass spectrometry(HPLC-MS/MS)-method using phytosphingosine as an internal standard to determine sphinganine- and sphingosine concentrations in incubated IHKE cells. Whereas a significant increase of sphinganine was observed with all substrates, sphingosine levels remained unchanged. This shows that FB1 exposure leads to apoptosis in a sphinganine-independent mechanism.

[Formation of Fumonisin artefacts in thermal treated food].

Although it is well-known that fumonisin B1 (FB1) in corn meal decreases during baking, frying, and cooking, little is known about the formation of fumonisin artefacts during thermal treatment. In model experiments FB1, hydrolyzed fumonisin B1 (HFB1) and N-tert-butoxycarbonyl-fumonisin B1 (N-BOC-FB1) were heated with D-glucose, respectively, and samples were analyzed by liquid chromatography/electrospray ionization-tandem-spectrometry (HPLC-MS/MS). Whereas in samples containing FB1 and HFB1 stable products with mass/charge ratio (m/z) 884 and m/z 568 could be detected, no compounds were found following the reaction of N-BOC-FB1 and D-glucose. The compound with m/z 884 was identified as the Amadori rearrengement product of Schiff base formed by the reaction of FB1 with D-glucose.