Janique Richoz

Quantitative analysis of mutagenic heterocyclic aromatic amines in cooked meat using liquid chromatography–atmospheric pressure chemical ionisation tandem mass spectrometry

Five mutagenic heterocyclic aromatic amines (HAAs) were quantified from meat extracts, and grilled and pan fried bacon samples using stable isotopically labeled internal standards. These compounds were isolated from the matrices by a tandem solid-phase extraction procedure, followed by separation on reversed-phase liquid chromatography (HPLC) and quantified by atmospheric pressure chemical ionization tandem mass spectrometry (APCIMS-MS). Tandem mass spectrometry (MS-MS) acquisition was done in selected reaction monitoring (SRM) mode to provide a high degree of sensitivity and selectivity for accurate quantification of HAAs. The detection and quantification limits of these HAAs approached 0.015 and 0.045 microg/kg (part-per-billion), respectively, with only 4 g of meat. The HAA levels ranged widely from 0.045 to 45.500 microg/kg, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was the predominant HAA found in these samples. The amount of HAAs formed was highly dependent upon the type of meat and method of preparation. An intralaboratory comparison of the extraction procedure showed that estimates of these HAAs obtained by three different individuals at HAA levels below 2 microg/kg were within 5% with coefficients of variation below 19%, indicating the robustness of the analytical method. Moreover, because all of these HAAs from this class of molecules undergo facile cleavage at the N-methylimidazole moiety under collision-induced dissociation (CID) conditions, MS-MS analysis in the constant neutral loss mode of [M+H]+-15 enabled the identification of two other HAAs, 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) and 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), which have rarely been reported in cooked meats.

Nε-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

The effects of coffee on enzymes involved in metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine in rats

The effects of coffee on the metabolism and genotoxicity of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were investigated. Coffee diminished the bacterial mutagenicity of PhIP in the Ames reversion assay through inhibition of cytochrome P450 1A2 (CYP1A2), a key enzyme involved in the metabolic activation of PhIP. When given as part of the diet (0, 1 or 5% w/w) to male Fischer-344 rats for 2 weeks, coffee affected the expression of hepatic enzymes involved in PhIP metabolism. Coffee increased the expression of CYP1A2 by 16-fold in the 5% coffee-treated group, and approximately half of this inductive effect was attributed to caffeine. Coffee also increased the expression of enzymes involved in the detoxication of PhIP. A 2-fold increase in expression of glutathione S-transferase alpha was observed, UDP-glucuronosyl transferase (UGTs) activities of p-nitrophenol increased 2-fold, while N(2)-and N3-glucuronidation of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP) increased by 1.3-fold in the 5% coffee-treated over the control group. The amount of PhIP (0.75 mg/kg, 24 h) eliminated in urine as the N(2)-and N3-glucuronide conjugates of HONH-PhIP increased by 1.8- and 2.5-fold, respectively, in the 5% coffee-treated group over control rats, suggesting either increased rates of N-oxidation of PhIP or N-glucuronidation of HONH-PhIP. Despite the strong induction of CYP1A2, there was no increase in PhIP-DNA adduct formation in colon and pancreas while liver adducts decreased by 50% over control animals. These data suggest that the effect of coffee on inhibition of PhIP N-oxidation and ensuing DNA damage is more important in vivo than its effect on induction of PhIP N-hydroxylation.

Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations.

Caffeine and related methylxanthines were subjected to free radical mediated oxidation by incubation with Fe(3+)-EDTA/ascorbate and Fe(3+)-EDTA/polyphenolics. The reaction mixtures were analysed by reverse-phase HPLC, revealing the corresponding C-8 hydroxylated analogues as the major products of hydroxyl radical mediated attack. Further oxidation products of caffeine, analysed by liquid chromatography-mass spectrometry (LC-MS), were the N1-, N3- and N7-demethylated methylxanthine analogues theobromine, paraxanthine and theophylline, respectively. Isolable amounts of the imidazole ring operated 6-amino-5-(N-formylmethyl-amino)-1,3-dimethyl-uracil (1,3,7-DAU) derivative were also detected, which was characterised by 1H NMR and mass spectroscopy. The identified products indicate that the pertinent chemical reactions, i.e. C-8 hydroxylation, demethylations, and C8-N9 bond scission, are comparable to the primary metabolic pathways of caffeine in humans. The influence of pH, transition metals, hydrogen peroxide, free radical scavengers and metal chelators on caffeine oxidation was studied. This report illustrates that natural food-borne reactants can aid in identifying specific chemical markers of free radical induced damage. Furthermore, potentially anti-and pro-oxidative reactions can be elucidated which may be important in assessing the impact of nutrient additives and supplements on the shelf life and stability of foods and beverages.

Evaluating the Extent of Protein Damage in Dairy Products

An isotope dilution liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed to determine lysine (Lys), Nε‐fructosyllysine (FL), Nε‐carboxymethyllysine (CML), and pyrraline (Pyr) in dairy products. The presented approach entails protein cleavage via enzymatic digestion to liberate the aforementioned compounds, which were then quantified using a stable isotope dilution assay. LC‐MS/MS analysis was performed by positive electrospray ionization recording two transition reactions per analyte in selected reaction monitoring mode. The CML and Lys values obtained with enzymatic digestion were compared to those acquired with acid hydrolysis HCl (6 mol/L), and the two proteolysis methods yielded comparable quantifications. Allowing for the fact that the investigated compounds are formed during different stages of the glycation process, the method is able to reveal the progress of protein glycation in dairy products.