Veronika Faist

Metabolic Transit and in vivo Effects of Melanoidins and Precursor Compounds Deriving from the Maillard Reaction

Metabolic transit data on food-borne advanced MRPs (Maillard reaction products) termed melanoidins are yet not completely elucidated and it is still an open question whether isolated melanoidin structures undergo metabolic biotransformation and subsequently cause physiological effects in vivo. Advanced MRPs, acting as premelanoidins, and melanoidins are formed under severe heat treatment of foods and are ingested with the habitual diet at considerable amounts. Metabolic transit data are known for Amadori compounds classified as early MRPs, like, e.g., fructose-lysine. For rats and humans, the percentages of ingested free versus protein-bound fructose-lysine excreted in the urine were found within ranges of 60–80% and 3–10%, respectively. Balance studies on free advanced MRPs are still lacking, but protein-bound low-molecular-weight premelanoidins and high-molecular-weight melanoidins have already been investigated in animal experiments using 14C-tracer isotopes. The amount of ingested radioactivity absorbed and excreted in the urine was found at levels ranging from 16 to 30% and from 1 to 5% for premelanoidins and melanoidins, respectively. These different metabolic transit data of premelanoidins and melanoidins can be explained by the following mechanisms involved: (i) intestinal degradation by digestive and microbial enzymes; (ii) absorption of these compounds or their degradates, and (iii) tissue retention. Structure specific in vivo effects have been identified for protein-bound premelanoidins on intestinal microbial activity, xenobiotic biotransformation enzymes and further glycation reactions. The latter are hypothesized to be involved in the aging process and in the course of different diseases. Further investigations are needed to clarify synergistic in vivo effects of dietary ingested melanoidins and endogenously formed glycation products.

Determination of Nϵ-carboxymethyllysine in milk products by a modified reversed-phase HPLC method

Abstract A modified reversed-phase-HPLC method with o-phthalaldehyde pre-column-derivatisation for determination of N ϵ -carboxymethyllysine in food samples is presented. It is shown, that the method has to be modified if applied to milk products, including specific modifications in sample preparation and chromatographic separation conditions. The increased selectivity of a double endcapped RP 18 phase is necessary for reliable separation of N ϵ -carboxymethyllysine in hydrolysates of complex products like cheese. With a detection limit of 0.5 pMol the method shows high sensitivity and a very good reproducibility ( s =2.81%). In total, several different milk products ( n =50) as well as fresh, processed and ripened cheese samples ( n =50) were analysed. The highest amounts of N ϵ -carboxymethyllysine were found in a whey cheese (1016 mg/kg protein), evaporated milk (1691 mg CML/kg protein), coffee cream (613 mg CML/kg protein) and cocoa milk (413 mg CML/kg protein). N ϵ -carboxymethyllysine could not be detected in UHT milk, fresh, processed and ripened cheese. The results show that N ϵ -carboxymethyllysine can give valuable information on lysine damage in severely heat-treated milk products and in products, with added sugar, pre-damaged constituents or stabilising agents. ©

Metabolic transit of Amadori products

In several studies, the absorption and urinary excretion of free and protein bound Amadori products were measured in rats and humans. Both, in vitro tests with everted intestinal sac preparations and in vivo experiments, showed that there is no active intestinal transport of these compounds but an absorption by diffusion. Trials with tissue slices have shown that there was an uptake into the cells of the liver, kidneys and muscles. Metabolism of Amadori products, if it exists in animals, tends to be very low. Micoorganisms in the large intestines decompose the Amadori products almost completely. The profile of urinary excretion of Amadori products after the ingestion of test meals showed a rapid elimination of the absorbed part, while the fecal output, although low because of the hind gut fermentation, persisted up to 3 days. Only 1-3% of the ingested amounts of protein bound Amadori products were recovered in the urine, which suggests a low absorption rate.

RAGE-mediated MAPK activation by food-derived AGE and non-AGE products

Investigating the cellular effects of food compounds formed by heat treatment during processing, we recently demonstrated the expression of the receptor for advanced glycation endproducts (RAGE) and the p44/42 MAP kinase activation by casein-N(epsilon )-(carboxymethyl)lysine (casein-CML), a food-derived AGE, in the intestinal cell line Caco-2. In this work, we report a Caco-2 p44/42 MAP kinase activation by bread crust and coffee extract. After identification, quantification, and synthesis of two key compounds formed in association with the process-induced heat impact applied to bread dough and coffee beans, those compounds, namely the AGE pronyl-glycine and the non-AGE N-methylpyridinium, were also demonstrated for the first time to activate the p44/42 MAP kinase through binding to RAGE in Caco-2 cells. Blocking of RAGE by an antagonistic antibody and expression of C-terminally truncated RAGE resulted in a reduced Caco-2- and HEK-293-MAP kinase activation. These findings unequivocally point to a RAGE-mediated activating effect of chemically defined food-derived, thermally generated products, both, AGEs and non-AGEs, on cellular signal transduction pathways involved in inflammatory response and cellular proliferation.

Effect of Heat-Treated Proteins on Selected Parameters of the Biotransformation System in the Rat

The intake of heat-damaged proteins from food causes various effects, like the loss of essential amino acids and a reduced protein digestibility. There is also an influence on gastrointestinal microorganisms and different digestion enzymes. Until now, very little is known about the influence of heat-treated proteins on the enzymes of the biotransformation system. In the present study, the influence of protein-bound L-lysino-D,L-alanine, NΕ-fructoselysine, and NΕ-carboxymethyllysine (CML) on selected enzymes of the biotransformation in liver, kidney, and intestinal mucosa of male Wistar rats was examined. The contents of cytochrome P-450 and cytochrome b5 and the activity of NADPH-cytochrome c reductase served as indicators of phase I biotransformation. The influence on phase II biotransformation was shown by the content of glutathione and the glutathione S-transferase activity. The results showed that treatment with heat-damaged proteins mainly affected phase II biotransformation enzymes with CML, yielding the strongest effect. The activity of glutathione S-transferase in the kidney was 86% higher in animals treated with diets containing 4,930 mg·kg–1 protein-bound CML than in animals of the control group which received a diet without any detectable CML. In addition, a higher level of glutathione was found in the kidneys of animals fed on diets containing CML. The glutathione S-transferase activity was 64% higher in the intestinal mucosa of animals fed on protein-bound NΕ-fructoselysine (2,700 mg·kg–1). The glutathione S-transferase activity was higher (p >0.05) in the intestinal mucosa of animals fed on protein-bound L-lysino-D,L-alanine (2,582 and 12,474 mg·kg–1). In conclusion, ingestion of heat-treated proteins led to an activation of the enzymes of phase II biotransformation. Whether or not the released pure compounds or the degradation products of the test proteins are responsible for the altered enzyme activities remains to be evaluated.

Selective fortification of lysinoalanine, fructoselysine and Nε-carboxymethyllysine in casein model systems

In the present study, a promising strategy to study nutritional effects of selected chemical reaction products formed in heat treated protein containing foods is addressed. In due course, a selective fortification of different marker compounds for lysine damage in casein-sugar mixtures was performed to provide model systems being applicable to investigate biological effects of the cross-link lysinoalanine (LAL), the MRPs fructoselysine (FL) and N epsilon-carboxymethyllysine (CML) in a casein-linked preparation. The three different model proteins, casein-LAL, casein-FL and casein-CML were prepared by heating casein either in strong alkaline conditions at 105 degrees C for 1 h, in the presence of glucose at 65 degrees C for 68 h, or in the presence of glyoxylic acid at 37 degrees C for 19 h. Finally, the degree of lysine modification achieved was 39%, 75% and 55% for the casein-LAL, casein-FL and casein-CML, respectively. The calculation of lysine recovery and the respective analysis of each single modified casein (LAL-, FL- and CML-MP) for the selected fortified compound and each other compound vice versa proved that the individual procedure provides a specific fortification for LAL, FL and CML, respectively. The modified proteins are suitable as reference model proteins to be investigated for specific biological and toxicological effects of casein-linked LAL, FL and CML.

Effects of dietary Nε-carboxymethyllysine on expression of the biotransformation enzyme, glutathione-S-transferase, in the rat

Abstract N e -carboxymethyllysine (CML) is an advanced-glycation endproduct found in both heat-treated foods and in living organisms. In the experiments described below, we have evaluated the effects of CML on the expression of the detoxification enzyme, glutathione- S -transferase, in the rat and in intestinal cells in cell culture. In the first animal experiment, casein-linked CML was administered to Wistar rats at two pharmacological doses (110 and 300 mg CML/kg body weight/day) for 10 days. Phase II glutathione- S -transferase (GST) enzyme activity was analysed in the kidneys. In a second study, CML was administered in a diet supplemented with bread crust (25% w/w) such that rats received a moderate dose of 11 mg CML/kg body weight/day. The induction of GST isoenzymes was analysed in the kidneys. In vitro experiments were also performed to study the effects of casein-CML and bread crust on GST induction in the intestinal Caco-2 cell line. Both animal experiments revealed an inductive effect of casein-linked CML and of bread crust on GST activity in the kidneys. The results obtained from the cell culture experiments by enzyme activity analysis and Western blotting confirmed these inductive effects on the GST. In conclusion, dietary-advanced glycation products containing CML were shown to enhance the expression of GST in both animal models and in cell culture.

Molecular weight distribution of non-enzymatic browning products in Japanese soy sauce and studies on their effects on NADPH–cytochrome c–reductase and glutathione-S-transferase in intestinal cells

Low molecular weight browning products (<1 kDa) isolated from Japanese soy sauce by ultrafiltration inhibited Phase-I CCR and increased Phase-II GST activity at low concentrations, thus showing evidence for a chemopreventive effect in intestinal cells in vitro.