Effect of tocopherols in the antioxidative activity of oxidized lipid-amine reaction products

Phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysine (Lys), and mixtures of them were tested for antioxidative activity in a tocopherol-stripped olive oil (TSO) and the same oil after addition of 250 microg of alpha-tocopherol g of oil/(tocopherol-added olive oil, TAO) to evaluate the role of tocopherol in the antioxidant activity of oxidized lipid-amine products. Neither PE nor PC nor Lys protected TSO when tested alone, but both PE and Lys increased the induction period (IP) of TAO. On the contrary, PE/Lys and PC/Lys mixtures, but not PC/PE mixtures, protected both TSO and TAO. These results were a consequence of both the formation of oxidized lipid-amine products, which were determined by gas chromatography-mass spectrometry after their conversion into volatile derivatives, and a synergism between alpha-tocopherol and the produced compounds. These results were confirmed by analyzing the antioxidative activity of two of the produced carbonyl-amine products: 6-amino-2-(1H-pyrrol-1-yl)hexanoic acid (1) and 2,3-dipalmitoylpropyl 2-(1H-pyrrol-1-yl)ethyl phosphate (2). The hydrophilic compound 1 was more antioxidant than the analogous lipophilic compound 2, and this antioxidative activity was observed in TAO and not in TSO. All these results suggested that antioxidative activity of carbonyl-amine products may be greatly increased with the addition of tocopherols, and those products derived from Lys are more antioxidant in bulk oils than those derived from PE.     

Changes produced in the antioxidative activity of phospholipids as a consequence of their oxidation

The antioxidative activities of native and oxidized soybean phosphatidylcholine (PC), phosphatidylthanolamine (PE), and phosphatidylinositol (PI) in the protection of soybean oil heated in the dark under air at 60 degrees C were studied in an attempt to clarify the consequences that phospholipid oxidation has on antioxidative activities. The three native phospholipids protected the oil when assayed at 200 ppm, and phospholipid oxidation decreased the antioxidative activity of both PC and PI. However, slightly oxidized PE was more antioxidative than native PE, most likely as a consequence of the formation by amino-carbonyl reactions of pyrrolized phospholipids, which were determined and for which antioxidative properties are known. Nevertheless, further increases in PE oxidation produced a decrease in its antioxidative activity. These results suggest that two opposite reactions are competing in the antioxidative activity of amino phospholipids upon oxidation: fatty acid chain oxidation, which decreases phospholipid antioxidative activity, and amino-carbonyl reactions, which produce derivatives with antioxidant properties. This last property may be useful to increase the antioxidative activity of commercial lecithins containing amino phospholipids.     

Effect of the pyrrole polymerization mechanism on the antioxidative activity of nonenzymatic browning reactions

The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic browning reactions is changing at the same time that the browning (by the pyrrole polymerization mechanism) is being produced. The antioxidative activities of eight model pyrroles (pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole, pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde) as well as the browning reaction of 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and the dimer (DIM) produced during HMP browning were determined. The results obtained suggest that the AAs observed in nonenzymatic browning reactions are the result of the AAs of the different oxidized lipid/amino acid reaction products formed. Thus, the different pyrrole derivatives produced in these reactions had different AAs, and the highest AAs were observed for alkyl-substituted pyrroles without free alpha-positions. Because some of these pyrrole derivatives are implicated in nonenzymatic browning production and this browning production implies the loss of hydroxyl groups and the transformation of some pyrroles with one type of substitution into others, changes in AA during browning production were observed, and the resulting DIM derivative was more antioxidant than HMP or higher polymers. These results explain the AA observed in fatty acid/protein mixtures after slight oxidation and suggest that, when the pyrrole polymerization mechanism is predominant, slightly browned samples may be more antioxidant than samples in which nonenzymatic browning has been highly developed.     

Effect of pH and temperature on comparative antioxidant activity of nonenzymatically browned proteins produced by reaction with oxidized lipids and carbohydrates

The antioxidative activity of nonenzymatically browned bovine serum albumin (BSA) produced by reaction with ribose (RI), hydroperoxides of methyl linoleate oxidation (HP), and secondary products of methyl linoleate oxidation (SP), at different pHs (4, 7, and 10) and temperatures (25, 37, 50, 80, and 120 degrees C), was studied to compare the antioxidative effects of carbohydrate- and oxidized lipids-modified proteins. The modified proteins (RIBSA, HPBSA, and SPBSA) were tested for antioxidative activity (at 100 ppm) in soybean oil using the thiobarbituric acid-reactive substances (TBARS) assay. All of them decreased significantly (p < 0.05) the TBARS formation in the oil and exhibited different effectiveness as a function of the temperature and the pH of the medium. In addition, there was a good correlation between the antioxidative activity of the protein and the amino acid losses produced during the nonenzymatic browning. These results are in agreement with an analogous and complimentary contribution of both Maillard and oxidized lipid/protein reactions to the antioxidative activity produced in foods during processing and storage.     

Studies on radical intermediates in the early stage of the nonenzymatic browning reaction of carbohydrates and amino acids

Determination of the color intensity of heated mixtures of L-alanine and carbohydrate degradation products revealed furan-2-carboxaldehyde and glycolaldehyde as by far the most effective color precursors. EPR studies demonstrated that furan-2-carboxaldehyde generated colored compounds exclusively via ionic mechanisms, whereas glycolaldehyde led to color development accompanied by intense radical formation. In agreement with literature data, these radicals were also detected in heated mixtures of L-alanine and pentoses or hexoses, respectively, and were identified as 1,4-dialkylpyrazinium radical cations by EPR as well as LC/MS measurements. Studies on the mechanisms of radical formation revealed that under the reaction conditions applied, glyoxal is formed as an early product in hexose/L-alanine mixtures prior to radical formation. Reductones then initiate radical formation upon reduction of glyoxal and/or glyoxal imines, formed upon reaction with the amino acid, into glycolaldehyde, which was found as the most effective radical precursor. LC/MS measurements gave evidence that these pyrazinium radicals cations are not stable but are easily transformed into hydroxylated 1,4-dialkyl-1, 4-dihydropyrazines upon oxidation and hydrolysis of intermediate diquarternary pyrazinium ions. Besides other types of color precursors, these intermediates might be involved in the formation of colored compounds in the Maillard reaction.     

Maillard reaction products inhibit oxidation of human low-density lipoproteins in vitro

Dietary intake of antioxidants has been associated with a reduced risk of cardiovascular diseases, which is very likely caused by their capability of prevent oxidation of low-density lipoproteins (LDL). During food processing and storage, substances with antioxidative properties are formed by Maillard reactions. In this study, the activity of Maillard products to inhibit copper-induced oxidation of human LDL in vitro was investigated. d-Glucose was heated with an equimolar amount of glycine, l-lysine, or l-arginine, for 1 h under reflux. The increase of the antioxidative activity (AOA) of the Maillard mixtures was highly significant compared to the control mixtures. Additionally, two defined Maillard products with amino reductone structure were tested. 3-Hydroxy-4-(morpholino)-3-buten-2-one (1) and amino hexose reductone (2) showed a significant and dose dependent AOA. Compound 1 was about half as active as ascorbic acid, which served as positive control. Thus, it can be concluded that Maillard products, particularly those with amino reductone structure, have the strong potential to inhibit LDL oxidation.     

Conversion of phenylalanine into styrene by 2,4-decadienal in model systems

2,4-Decadienal was heated under an inert atmosphere and in the presence of phenylalanine to investigate whether this secondary lipid oxidation product is a final product of lipid oxidation or it reacts with the amino acid. The results obtained showed that, in the presence of the alkadienal, the amino acid was degraded to styrene. This reaction was favored in dry systems at pH approximately 6 and in the absence of oxygen. If oxygen was present, the alkadienal was oxidized and the Strecker degradation of the amino acid was produced. The activation energy for the formation of styrene from phenylalanine was 150.4 kJ/mol. The reaction mechanism is suggested to be produced either by an electronic rearrangement of the imine produced between the aldehyde and the amino acid with the formation of styrene, 2-pentylpyridine, carbon dioxide, and hydrogen, or by Michael addition of the amino compound to the alkadienal followed by beta-elimination to produce the same compounds. Both reaction schemes were supported on the results obtained by studying both the degradation of phenylethylamine and phenylalanine methyl ester produced by 2,4-decadienal, and the formation of ethylbenzene in decadienal/phenylalanine reaction mixtures heated in the presence of platinum oxide. All these results suggest that, analogously to carbohydrates, certain lipid oxidation products may degrade appropriate amino acids to their corresponding vinylogous derivatives.     

Effect of pH and temperature on comparative nonenzymatic browning of proteins produced by oxidized lipids and carbohydrates

Bovine serum albumin (BSA) was incubated for 24 h in the presence of 10 mM ribose (RI), methyl linoleate hydroperoxides, or the secondary products of methyl linoleate oxidation (SP), at five temperatures (25, 37, 50, 80, and 120 degrees C) and different pHs (4, 7, and 10), to study the influence of these variables in the browning, fluorescence, amino acid losses, and pyrrolization of the modified proteins. All treated proteins exhibited similar colors and fluorescence spectra, and the spectra of their Ehrlich adducts were also analogous. However, at 25-50 degrees C the proteins treated with oxidized lipids exhibited higher color changes, amino acid losses, and pyrrolization than the BSA treated with RI, and these effects were much higher in proteins treated with RI at 80-120 degrees C. The effect of pH was similar in proteins treated with RI or SP. These results suggested a similarity for browned proteins obtained from both carbohydrates and oxidized lipids. In addition, both reactions seem to be complementary, because melanoidin formation derived from oxidized lipids can be produced under conditions different from those carbohydrate/protein reactions.     

Interaction of Oxidation Products from Caffeic Acid with Fe(III) and Fe(II)

Abstract

Phenolic substances in the soil–plant system can be oxidized by metal ions, inorganic components, molecular oxygen as well as by phenoloxidases, giving rise to the formation of products of low or high molecular weight. Interactions of these products with iron, in both reduced and oxidized form, can affect the iron mobility in soil and rhizosphere, and thus its availability to plants. Here we report the results of a study on the complexing and reducing activity of the oxidation products from caffeic acid (CAF), obtained via electrochemical means, towards Fe(III) and Fe(II) in aqueous solution in the 3.0–6.0 pH range. The HPLC analysis of the filtered solutions after the CAF oxidation showed the formation of two main groups of products: (i) CAF oligomers formed through radicalic reactions which do not involve the double bond of the CAF lateral chain and (ii) products where this bond is involved. These oxidation products (COP) were found to interact with both Fe(III) and Fe(II) with formation of soluble and insoluble Fe(III)‐, and Fe(II)‐COP complexes. The COP were found to be able to reduce Fe(III) to Fe(II) mainly at pH < 4.0. A low redox activity was observed at pH ≥ 4.5 due to Fe(III) hydrolysis reactions as well as to the decrease in the redox potential of the Fe(III)/Fe(II) couple. Formation of hydroxy Fe(III)‐COP polymers occurs at pH > 3.5.     

A new insight into the formation of odor active carbonyls by thermally-induced degradation of phospholipids in self-assembly structures

The role of molecular organization in heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) was studied with respect to the formation of key odorants. Evidence was found for the crucial role of self-assembly structures adopted by phospholipid molecules on the quantitative composition of volatile constituents. The concentrations of seven aldehydes and one vinyl ketone were determined by isotope dilution assay in heated aqueous dispersions of PC and PE present in various ratios. Addition of PE to PC drastically decreased the amount of (E,E)-2,4-decadienal formed, which cannot be explained by the differences in the fatty acid composition of PC and PE. The free amino group in PE does not explain this phenomenon either, as replacing PE by phosphatidic acid distearylester also reduced the amounts of (E,E)-2,4-decadienal. We suggest that the type of self-assembly structure adopted by phospholipids in water significantly influences the reaction yields. However, the mechanisms leading to the preferred formation of phospholipid-derived odorants in a lamellar phase, as compared to the reversed hexagonal phase, remain unknown.     

Methyl linoleate oxidation in the presence of bovine serum albumin

The oxidation of methyl linoleate (LMe) in the presence of bovine serum albumin (BSA) was studied to analyze both the processes involved when lipid oxidation occurs in the presence of proteins and the relative progression of the several reactions implicated. The disappearance of LMe, the formation of primary and secondary lipid oxidation products, the loss of essential amino acids, and the production of oxidized lipid/amino acid reaction products (OLAARPs) were studied as a function of incubation time. During the first steps of lipid oxidation, LMe was converted quantitatively to methyl linoleate hydroperoxides, which were very rapidly degraded to either secondary products of lipid oxidation or OLAARPs. No significant differences were identified in the major lipid oxidation products formed in incubations with or without proteins, indicating that mechanisms for formation of these compounds are similar in both cases. In addition, no significant differences were observed between the time-courses of formation of secondary oxidation products and OLAARPs, suggesting that hydroperoxide decomposition and OLAARP formation occur simultaneously when the lipid oxidation process takes place in the presence of proteins. Furthermore, OLAARP formation seems to be an unavoidable process that should be considered as a last step in the lipid peroxidation process.     

Quantitative model studies on the efficiency of precursors in the formation of cooling-active 1-pyrrolidinyl-2-cyclopenten-1-ones and bitter-tasting cyclopenta-[b]azepin-8(1H)-ones

The yields of the cooling-active compounds 3-methyl-2-(1-pyrrolidinyl)-2-cyclopenten-1-one (1) and 5-methyl-2-(1-pyrrolidinyl)-2-cyclopenten-1-one (2) as well as the bitter tastants 7-methyl-2,3,6,7-tetrahydrocyclopenta-[b]azepin-8(1H)-one (3) and 7-methyl-2,3,4,5,6,7-hexahydrocyclopenta-[b]azepin-8(1H)-one (4) obtained by heating mixtures of possible Maillard-type precursors in model systems varying in temperature, pH value, or water content were determined quantitatively. The results showed that hexose-derived cyclotene is the common precursor for all four tastants and that the formation of each individual tastant is strongly determined by the structure of the nitrogen-containing precursor, e.g., reaction of cyclotene with pyrrolidine formed by thermal decarboxylation of L-proline produced the cooling compounds 1 and 2 only, whereas in the presence of 1-pyrroline formed upon Strecker reactions of L-proline, the bitter tasting azepinone 3 was produced exclusively. In contrast, the structure of the secondary amino acid L-proline enabled the formation of compound 4, whereas the pyrrolidine and 1-pyrroline, respectively, do not generate this tastant. In addition, a nonvolatile, tasteless intermediate, (S)-3-methyl-2-[(2'-carboxy)-1-pyrrolidinyl]-2-cyclopenten-1-one (5), was isolated from the cyclotene/L-proline reaction mixture and could be confirmed as an efficient precursor for the cooling compound 1. The data, obtained by these studies, are the scientific basis to tailor the desired overall flavor of foods by means of a more controlled Maillard-type technology.     

Comparative methyl linoleate and methyl linolenate oxidation in the presence of bovine serum albumin at several lipid/protein ratios

The oxidation of methyl linoleate (LMe) and methyl linolenate (LnMe) in the presence of bovine serum albumin (BSA) in the dark at 60 degrees C was studied to analyze the role of the type of fatty acid and the protein/lipid ratio on the relative progression of the processes involved when lipid oxidation occurs in the presence of proteins. The disappearance of the fatty acid, the formation of primary and secondary products of lipid peroxidation, the loss of amino acid residues, the production of oxidized lipid/amino acid reaction products, and the development of color and fluorescence were studied as a function of incubation time in protein/lipid samples at 10:1, 6:1, and 3:1 w/w ratios. The incubation of LMe and LnMe in the presence of BSA at 60 degrees C rapidly produced lipid peroxidation and protein damage. Although reaction rates were much faster for LnMe than for LMe, both fatty acids had similar behaviors, and LnMe seemed to be only slightly more reactive than LMe for BSA by producing a higher increase of protein pyrroles in the protein and the development of increased browning and fluorescence. The protein/lipid ratio also influenced the relative progress of the reactions implicated. Thus, a lower protein/lipid ratio increased sample oxidation and protein damage. This also produced an increased browning, in accordance with the mechanisms proposed for browning production by oxidized lipid/protein reactions. On the contrary, browning of extracted lipids increased at higher protein/lipid ratios. This opposite tendency allowed evaluation of the overall significance of the different browning processes implicated in the final colors observed, concluding that color changes observed in BSA/lipid samples were mostly a consequence of oxidized lipid/protein reactions.     

Heterocyclic volatiles formed by heating cysteine or hydrogen sulfide with 4-hydroxy-5-methyl-3(2H)-furanone at pH 6.5

The reaction of 4-hydroxy-5-methyl-3(2H)-furanone (HMF) with cysteine or hydrogen sulfide at pH 6.5 for 60 min at 140 degrees C produced complex mixtures of volatile compounds, the majority of these containing either sulfur or nitrogen. Of the 68 compounds detected, 63 were identified, some tentatively, by GC-MS. Among the identified compounds were thiophenes (10), thiophenones (6), thienothiophenes (5), thiazoles (5), trithiolanes (4), pyrazines (6), and oxazoles (4). More compounds were produced in the reaction of HMF with cysteine (63) than were formed in the reaction with hydrogen sulfide (33). In both systems, thiophenones were major reaction products, accounting for 25-36% of the total volatiles formed. Possible reasons for the differences in the composition of the two systems are discussed. The contributions of these reactions, and their products, to the flavor of heated foods are considered.     

Effects of phenolic propyl esters on the oxidative stability of refined sunflower oil

The oxidative stability of refined sunflower oil in the presence and in the absence of propyl caffeate (PC), propyl hydrocaffeate (PHC), propyl ferulate (PF), and propyl isoferulate (PI) has been evaluated according to the Rancimat method. The antioxidant activity of the phenolic derivatives was compared with that obtained with native [alpha-tocopherol (alpha-TOH)] and synthetic [propyl gallate (PG)] antioxidants. The results allow the establishment of a decreasing order of antioxidant power: PG > PHC > PC > alpha-TOH > PI > PF. The oxidative stability was improved neither by the addition of PF nor by a supplement of alpha-TOH. Moreover, a positive antioxidant effect was obtained for PC that was placed between those of alpha-TOH and PG. The antioxidant activity of PHC was higher than that of its analogue (PC). A dose-dependent effect was observed for PG, PHC, and PC. A chain-breaking mechanism was proposed for the antioxidant activity of propyl phenolic esters because the same ranking order of efficacy was obtained for their antiradical activities evaluated by using the 2,2-diphenyl-1-picrylhydrazyl radical method.     

Comparative study of virgin olive oil behavior under Rancimat accelerated oxidation conditions and long-term room temperature storage

Oxidative stability should be one of the most important quality markers of edible oils; nevertheless, it is not recognized as a legal parameter. The results reported in this study highlight the differences in the olive oil oxidation process under Rancimat accelerated conditions with respect to long-term storage at room temperature and clearly show the lack of correlation between shelf life and the Rancimat induction period. A better correlation, although not yet satisfactory, was found when the same oxidation end-point was used in both assays. The parameter K 270, a marker of secondary oxidation products, was the first index to reach the established upper legal limit under Rancimat conditions, whereas at 25 degrees C it was an index of primary oxidation products ( K 232). Furthermore, the ratio of oxidation rate at Rancimat conditions to oxidation rate at 25 degrees C was more than double for secondary oxidation products compared with primary ones. Notable differences were also observed in degradation rates of the different unsaturated fatty acids and in rates of formation of polar oxidation compounds. Moreover, under the Rancimat conditions antioxidants such as o-diphenols and alpha-tocopherol rapidly depleted, and when they had practically disappeared, there was a sharp increase in oxidation indices, such as peroxide value, and in oxidation products. At 25 degrees C, on the other hand, the depletion was much lower.     

Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction

Typical heterocyclic compounds substituted with various functional groups found in Maillard reaction products were examined for antioxidant activity. Pyrroles exhibited the greatest antioxidant activity among all heterocyclic compounds tested. All pyrroles inhibited hexanal oxidation by almost 100% at a concentration of 50 microg/mL over 40 days. Addition of formyl and acetyl groups to a pyrrole ring enhanced antioxidative activity remarkably. Pyrrole-2-carboxaldehyde, 2-acetylpyrrole, 1-methyl-2-pyrrolecarboxaldehyde, and 2-acetyl-1-methylpyrrole inhibited hexanal oxidation by >80% at 10 microg/mL. Unsubstituted furan exhibited the greatest antioxidant activity among furans tested. Addition of all functional groups used in this study to furan decreased antioxidative activity. The antioxidant activity of thiophene increased with the addition of methyl and ethyl groups, but the addition of formyl or acetyl groups to thiophene decreased antioxidant activity. Thiazoles and pyrazines were ineffective antioxidants at all concentrations tested. Reaction of all heterocyclic compounds with hydrogen peroxide resulted in the formation of various oxidized products.     

Monitoring of monooctanoylphosphatidylcholine synthesis by enzymatic acidolysis between soybean phosphatidylcholine and caprylic acid by thin-layer chromatography with a flame ionization detector

Thin-layer chromatography with a flame ionization detector (TLC-FID) was used for monitoring the production of structured phospholipids (ML type: L, long-chain fatty acids; M, medium-chain fatty acids) by enzyme-catalyzed acidolysis between soybean phosphatidylcholine (PC) and caprylic acid. It was found that the structured PC fractionated into two to three distinct bands on both plate thin-layer chromatography (TLC) and Chromarod TLC. These three bands represented PC of the LL type, ML type, and MM type, respectively. The TLC-FID method was applied in the present study to examine the influence of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio (caprylic acid/PC, mol/mol) on formation of ML-type PC in a batch reactor with Thermomyces lanuginosa lipase as the catalyst. The formation of ML-type PC was dependent on all parameters examined except for the substrate ratio. The ML-type PC content increased with increasing enzyme dosage, reaction temperature, solvent amount, and reaction time. The substrate ratio had no significant effect on the formation of ML-type PC within the tested range (3-15 mol/mol). The formation of MM-type PC was observed in some experiments, indicating that acyl migration is taking place during reaction since the lipase is claimed to be 1,3-specific. The TLC-FID method offers a simple and cheap technique for elucidation of product and byproduct formation during enzyme-catalyzed reactions for production of phospholipids containing mixtures of long- and medium-chain fatty acids.     

Model studies on the pattern of volatiles generated in mixtures of amino acids, lipid-oxidation-derived aldehydes, and glucose

The development of flavor and browning in thermally treated foods results mainly from the Maillard reaction and lipid degradation but also from the interactions between both reaction pathways. To study these interactions, we analyzed the volatile compounds resulting from model reactions of lysine or glycine with aldehydes originating from lipid oxidation [hexanal, (E)-2-hexenal, or (2E,4E)-decadienal] in the presence and absence of glucose. The main reaction products identified in these model mixtures were carbonyl compounds, resulting essentially from amino-acid-catalyzed aldol condensation reactions. Several 2-alkylfurans were detected as well. Only a few azaheterocyclic compounds were identified, in particular 5-butyl-2-propylpyridine from (E)-2-hexenal model systems and 2-pentylpyridine from (2E,4E)-decadienal model reactions. Although few reaction products were found resulting from the condensation of an amino acid with a lipid-derived aldehyde, the amino acid plays an important role in catalyzing the degradation and further reaction of these carbonyl compounds. These results suggest that amino-acid-induced degradations and further reactions of lipid oxidation products may be of considerable importance in thermally processed foods.     

Structural and functional characterization of pronyl-lysine,a novel protein modification in bread crust melanoidins showing in vitro antioxidative and phase I/II enzyme modulating activity

Application of an in vitro antioxidant assay to solvent fractions isolated from bread crust, bread crumb, and flour, respectively, revealed the highest antioxidative potential for the dark brown, ethanol solubles of the crust, whereas corresponding crumb and flour fractions showed only minor activities. To investigate whether these browning products may also act as antioxidants in biological systems, their modulating activity on detoxification enzymes was investigated as a functional parameter in intestinal Caco-2 cells. The bread crust and, in particular, the intensely brown, ethanolic crust fraction induced a significantly elevated glutathione S-transferase (GST) activity and a decreased phase I NADPH-cytochrome c reductase (CCR) activity compared to crumb-exposed cells. Antioxidant screening of Maillard-type model mixtures, followed by structure determination, revealed the pyrrolinone reductones 1 and 2 as the key antioxidants formed from the hexose-derived acetylformoin and N(alpha)-acetyl-L-lysine methyl ester or glycine methyl ester, chosen as model substances to mimic nonenzymatic browning reactions with the lysine side chain or the N terminus of proteins, respectively. Quantitation of protein-bound pyrrolinone reductonyl-lysine, abbreviated pronyl-lysine, revealed high amounts in the bread crust (62.2 mg/kg), low amounts in the crumb (8.0 mg/kg), and the absence of this compound in untreated flour. Exposing Caco-2 cells for 48 h to either synthetically pronylated albumin or purified pronyl-glycine (3) significantly increased phase II GST activity by 12 or 34%, respectively, thus demonstrating for the first time that "pronylated" proteins as part of bread crust melanoidins act as monofunctional inducers of GST, serving as a functional parameter of an antioxidant, chemopreventive activity in vitro.