Characterization of coloured compounds obtained by enzymatic extraction of bakery products.

Melanoidins, the brown-colored polymers formed through Maillard type reaction in several heat-treated foods, represent a significant part of our diet, with an average intake of grams per day. Most of the studies on the physiological effects of these compounds have been performed using the water soluble melanoidin fractions. But dietary melanoidins formed on the surface of bakery products are poorly soluble in water as well as in organic solvents. In this work, an enzymatic solubilization procedure was developed on a gluten-glucose model system and it was applied to bread and biscuits. The soluble material obtained was tested for its antioxidant activity, for its effect on phase-I and phase-II xenobiotic enzymes and for potential cytotoxic effects. Soluble melanoidins from model system and biscuits exhibit a strong antioxidant activity and do not show any cytotoxicity on Caco-2 cells. Melanoidins extracted from biscuits was able to inhibit the activity of Phase I (NADPH-cytochrome-c reductase) and Phase II (Glutathione-S-transferase) enzymes, whereas the low molecular weight melanoidins isolated from gluten-glucose model system inhibit the activity of NADPH-cytochrome-c reductase.

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, N(epsilon)-fructoselysine, and N(epsilon)-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 b(5) 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 N(epsilon)-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.

Influence of molecular weight fractions isolated from roasted malt on the enzyme activities of NADPH-cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells.

In the present study, water-soluble nonenzymatic browning products (melanoidins) formed in roasted malt were separated, quantified, and investigated for their effects on detoxifying mechanisms in intestinal Caco-2 cells. The melanoidins were prepared from roasted malt by hot water extraction, and the water-soluble compounds were separated into different molecular weight (MW) fractions by gel filtration chromatography. By monitoring the effluent at 300 nm, seven molecular fractions I-VII were consecutively collected, revealing that approximately 2.3% of the water-soluble compounds had mean MWs between 10000 and 30000 Da. Thus, the bulk of water-soluble malt melanoidins consisted of MW > 30000 Da, among which approximately 58% showed mean MWs between 60000 Da and 100000 Da, whereas approximately 32% exhibited mean MWs of 200000 Da. Biotransformation enzyme activities of NADPH-cytochrome c-reductase (CCR) and glutathione-S-transferase (GST) were analyzed in Caco-2 Cells after 48 h of exposure to the different MW fractions. The low MW fraction of 10000 Da was most effective in activating the CCR and the GST activities (+122 and +33% vs control, respectively). The majority of the mid molecular weight compounds tested showed an activating effect on CCR activity and an inhibitory effect on GST activity. These effects were most pronounced for compounds of up to 70000 Da and >200000 Da but less distinct for fractions of an average molecular weight of 100000 Da.

RAGE expression and AGE-induced MAP kinase activation in Caco-2 cells.

RAGE (receptor for advanced glycation end products) is a multiligand cell surface molecule of the immunoglobulin superfamily. It was originally described as a receptor for protein adducts formed by glycoxidation (AGEs) that accumulate in diseases such as diabetes and renal failure. Performing RT-PCR and Western blot analysis we intended to determine RAGE expression in the human colon adenocarcinoma cell line Caco-2. Moreover, Caco-2 cells were incubated in the presence of AGEs. Since RAGE ligation triggers the p21(ras) signal transduction pathway the activation state of p44/42 (ERK1/2) MAP kinases was determined. Here we demonstrate for the first time that Caco-2 cells express RAGE and that administration of the food-derived casein-linked AGE N(epsilon)-(carboxymethyl)lysine (Cas-CML) results in Caco-2 p44/42 (ERK1/2) MAP kinase activation.

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.

Determination of the molecular weight distribution of non-enzymatic browning products formed by roasting of glucose and glycine and studies on their effects on NADPH-cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells.

After thermal treatment of a mixture of glucose and glycine for 2 h at 125 degrees C, about 60% of the starting material was converted into non-soluble, black pigments, whereas 40% of the mixture was still water-soluble. Dialysis of the latter fraction revealed 30.4% of low molecular weight compounds (LMWs; MW < 10,000 Da) and 10.0% high-molecular weight products (HMWs; MW > or = 10,000 Da). The water-soluble Maillard reaction products (MRPs) were separated by gel permeation chromatography and ultrafiltration, revealing that 60% of the water-soluble products of the total carbohydrate/amino acid mixture had MWs < 1,000 Da and consisted mainly of non-coloured reaction products. MRPs with MWs between 1,000 and 30,000 Da were found in comparatively low yields (about 1.3%). In contrast, about 31.1% of the MRPs exhibited MWs > 30,000 Da, amongst which 14.5% showed MWs > 100,000 Da, thus indicating an oligomerisation of LMWs to melanoidins under roasting conditions. To investigate the physiological effects of these MRPs, xenobiotic enzyme activities were analysed in intestinal Caco-2 cells. For Phase-I NADPH-cytochrome c-reductase, the activity in the presence of the LMW and HMW fraction was decreased by 13% and 22%, respectively. Phase-II glutathione-S-transferase activity decreased by 15% and 18%, respectively, after incubation with the LMW and the HMW fractions. Considering the different yields, 30% and 10%, respectively, of the LMW and the HMW fractions, the total amount of the LMW fraction present in the glucose-glycine mixture is more active in modulating these enzyme activities than that of the HMW fraction.

Selective fortification of lysinoalanine, fructoselysine and N epsilon-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.

Decreased mitochondrial oxygen consumption and antioxidant enzyme activities in skeletal muscle of dystrophic mice after low-intensity exercise.

To evaluate low-intensity exercise training induced changes of mitochondrial metabolism in dystrophic skeletal muscle, oxygen consumption, reactive oxygen species (ROS) scavengers and antioxidant enzymes were measured in control (C57BL/10) and dystrophic (mdx) mice at 10 (young) and 22 (adult) weeks of age. Dystrophic and control mice were either kept sedentary or daily exercised on a treadmill (480 m/day, exercise training was initiated at 4 and 16 weeks of age for 6 weeks' duration). Mitochondrial oxygen consumption was significantly lower in skeletal muscle from exercised young compared to sedentary young dystrophics. Whereas oxygen consumption was unchanged in exercised adult dystrophics, exercised adult controls exhibit a significant increase vs. sedentary adult controls. Contents of TBARS and lipofuscin were increased (+48%, +24%), while alpha-tocopherol concentration tended to decrease (p > 0.05) in exercised vs. sedentary young dystrophics. Compared to sedentary groups, glutathione peroxidase activity was decreased in exercised young dystrophic muscle (-12%), but increased in exercised controls (young controls +60%, adult controls +47%). In conclusion, adaptation to exercise-induced formation of ROS was limited in young dystrophic skeletal muscle but regained in that of adults.

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 (14)C-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.

Plasma levels of advanced glycation end products in healthy, long-term vegetarians and subjects on a western mixed diet.

BACKGROUND: Evidence indicates that food-derived Maillard's reaction products are absorbed and yet can be detected in the circulation. AIM OF THE STUDY: We postulated that consumption of the heat-treated food by omnivores could be reflected by higher plasma levels of advanced glycation end products (AGEs) in comparison with vegetarians, who in cooking (by keeping away from meat) use lower temperatures and less time for heating. METHODS: Plasma fluorescent AGEs (350/450 nm) and N(epsilon)-(carboxymethyl)lysine (CML, competitive ELISA) levels were investigated in 3 groups of healthy vegetarians (9 vegans-V, 19 lactoovo-vegetarians--VLO and 14 semi-vegetarians--VS) and compared with those of age-matched omnivores (O, n=19). Mean duration of vegetarian diet was V: 7.2 +/- 1.0,VLO: 8.2 +/- 0.8 and VS: 7.9 +/- 1.1 years. RESULTS: Both fluorescent AGE (O: 9.9 +/- 0.5; V: 10.8 +/- 0.7, LO: 13.1 +/- 0.8* and SV: 11.6 +/- 1.2 x 10(3) AU), and CML levels (O: 427.1 +/- 15.0,V: 514.8 +/- 24.6*, LO: 525.7 +/- 29.5**, SV: 492.6 +/- 18.0* ng/ml) were significantly lower in omnivores than in vegetarians. Plasma glucose, parameters of renal function (plasma concentration of creatinine and cystatin C, calculated glomerular filtration rate--GFR) as well as C-reactive protein levels were within the normal range and did not differ significantly between the groups. Thus, neither decline of kidney function nor inflammatory processes contributed to the rise in plasma AGEs. CONCLUSION: Enhanced plasma AGE levels in vegetarians in comparison to omnivores are herein presented for the first time. Mechanisms of AGE elevation and potential pathophysiological relevance of this finding are to be elucidated in prospective studies.

Mitochondrial oxygen consumption, lipid peroxidation and antioxidant enzyme systems in skeletal muscle of senile dystrophic mice.

To evaluate age-dependent abnormalities in mitochondrial redox metabolism specifically in dystrophic skeletal muscle, oxygen consumption, thiobarbituric acid reactivity (TBARS), free-radical scavengers and oxidative marker enzymes were measured in the skeletal muscle from adult and senile control (C57BL/10) and dystrophic (mdx/+) mice. Mitochondrial oxygen consumption in state 3 was significantly lowered with age in the senile dystrophic (-52%) and less markedly in the senile control (-30%) skeletal muscle. Compared with adult muscle, mitochondrial concentration of TBARS and cellular concentration of lipofuscin were significantly increased in senile control and dystrophic muscle. Enzymatic activity of glutathione peroxidase (GSH-Px) and concentration of alpha-tocopherol were significantly increased in the senile control (GSH-Px 43+/-5.7 vs 53+/-8.7 U/g protein, alpha-tocopherol 0.19+/-0. 09 vs 0.29+/-0.14 micromol/g total lipids), but significantly decreased in the senile dystrophic (GSH-Px 80+/-8.0 vs 53+/-12 U/g protein, alpha-tocopherol 0.45+/-0.13 vs 0.19+/-0.03 micromol/g total lipids) muscle. Selenium content was significantly decreased only in senile dystrophic muscle (1.37+/-0.42 vs 0.78+/-0.21 nmol/g wet muscle). In conclusion, the enzymatic adaptation to reactive oxygen species was limited in the dystrophic skeletal muscle, suggesting a higher need for antioxidants, especially alpha-tocopherol.