Relative quantification of N(epsilon)-(Carboxymethyl)lysine,imidazolone A,and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry

The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, has received increasing recognition from nutritional science and medical research. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform relative and simultaneous quantification of the Amadori product, which is an early glycation product, and of N(epsilon)-(carboxymethyl)lysine and imidazolone A, two important advanced glycation end products. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1, 4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 degrees C. After enzymatic digestion with endoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE) and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were used for relative quantification of the three Maillard products. Amadori product, N(epsilon)-(carboxymethyl)lysine, and imidazolone A were the main glycation products formed under these conditions. Their formation was dependent on glucose concentration and reaction time. The kinetics were similar to those obtained by competitive ELISA, an established method for quantification of N(epsilon)-(carboxymethyl)lysine and imidazolone A. Inhibition experiments showed that coincubation with N(alpha)-acetylargine suppressed formation of imidazolone A but not of the Amadori product or N(epsilon)-(carboxymethyl)lysine. The presence of N(alpha)-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations of imidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completely inhibited the formation of N(epsilon)-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved to be a new analytical tool for the simultaneous, relative quantification of specific products of the Maillard reaction. For the first time, kinetic data of defined products on specific sites of glycated protein could be measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillard reaction in the course of food processing.     

Reactivities of D-glucose and D-fructose during glycation of bovine serum albumin.

Glycation of bovine serum albumin by D-glucose and D-fructose under dry-heating conditions was studied. The reactivities of D-glucose and D-fructose, with respect to their ability to utilize primary amino groups of proteins, to cross-link proteins, to develop Maillard fluorescence, and to reduce protein solubility in the presence and absence of air (molecular oxygen) were investigated. D-Glucose showed a higher initial rate of utilization of primary amino groups than D-fructose, both in the presence and in the absence of oxygen. Subsequent reactions of the Amadori and Heyns rearrangement products, cross-linking, development of Maillard fluorescence, oxidation, and fragmentation, indicated that the alpha-hydroxy carbonyl group of Amadori products is more reactive than the aldehydo group of Heyns products. D-Fructose showed a greater sensitivity than D-glucose toward the presence of oxygen at the initial stages of the Maillard reaction. The presence or absence of oxygen in the glycation mixture did not seem to have an influence on the nature of products generated in the glycation mixtures during the advanced stages of the Maillard reaction.     

Degradation of oligosaccharides in nonenzymatic browning by formation of alpha-dicarbonyl compounds via a "peeling off" mechanism

The formation of alpha-dicarbonyl-containing substances and Amadori rearrangement products was studied in the glycine-catalyzed (Maillard reaction) and uncatalyzed thermal degradation of glucose, maltose, and maltotriose using o-phenylenediamine as trapping agent. Various degradation products, especially alpha-dicarbonyl compounds, are formed from carbohydrates with differing degrees of polymerization during nonenzymatic browning. The different Amadori rearrangement products, isomerization products, and alpha-dicarbonyls produced by the used carbohydrates were quantified throughout the observed reaction time, and the relevance of the different degradation pathways is discussed. In the Maillard reaction (MR) the amino-catalyzed rearrangement with subsequent elimination of water predominated, giving rise to hexosuloses with alpha-dicarbonyl structure, whereas under caramelization conditions more sugar fragments with an alpha-dicarbonyl moiety were formed. For the MR of oligosaccharides a mechanism is proposed in which 1,4-dideoxyosone is formed as the predominating alpha-dicarbonyl in the quasi-water-free thermolysis of di- and trisaccharides in the presence of glycine.     

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.     

High-pressure effects on Maillard reaction between glucose and lysine

Glucose-lysine model systems prepared over a range of pH values (5-10) in unbuffered and buffered media were incubated at 60 degrees C either under atmospheric pressure or at 400 MPa. The results obtained showed that high pressure affected in different ways the different stages of the Maillard reaction and that such effects were strongly influenced by pressure-induced changes in the pH of the systems. In unbuffered media, at an initial pH < or =8.0, the formation of Amadori rearrangement products (ARP) was not considerably affected by pressure, whereas the intermediate and advanced stages of the Maillard reaction were suppressed, suggesting a retardation of the degradation of the ARP. In buffered media, at pH values < or =8.0, pressure slowed the Maillard reaction from the initial stages. These effects are attributed to the pH drop caused by the pressure-induced dissociation of the acid groups. In unbuffered and buffered media at initial pH = 10.2, high pressure accelerated the formation and subsequent degradation of ARP, leading to increased levels of intermediate and advanced reaction products.     

Monitoring carbonyl-amine reaction and enolization of 1-hydroxy-2-propanone (Acetol) by FTIR spectroscopy

Infrared absorption bands characteristic of the aldehydo, keto, and enediol forms of 1-hydroxy-2-propanone (acetol) were identified and used to study the effect of solvent on the absorption frequencies and the effect of temperature and acid/base catalysis on the enolization reactions. The data indicated that, in addition to water, acids and bases can catalyze the enolization of 1-hydroxy-2-propanone and that the temperature inversely effects the rate of enolization under basic conditions. However, under acidic conditions, increasing the temperature favors the enolization process. In addition, the reaction of 1-hydroxy-2-propanone with a primary and a secondary amine was also monitored by Fourier transform infrared spectroscopy. The data indicated that at room temperature the rate of amine reaction was faster than the rate of its catalysis of enolization; however, below room temperature, the rate of base-catalyzed enolization became comparable with the rate of carbonyl-amine reaction forming both Heyns and Amadori adducts.     

3-deoxypentosulose: an alpha-dicarbonyl compound predominating in nonenzymatic browning of oligosaccharides in aqueous solution

The thermal degradation of D-glucose, maltose, and maltotriose in aqueous solution was investigated under caramelization (no glycine) and Maillard (with glycine) conditions. Degradation of the sugar and alpha-dicarbonyls product was monitored. Under both caramelization and Maillard reaction conditions, 3-deoxypentosulose was the predominating alpha-dicarbonyl compound formed from maltose and maltotriose. In the absence of an amino compound, however, 3-deoxypentosulose is formed in much lower concentration. It was concluded that 3-deoxypentosulose is formed by a pathway specific for oligo- and polysaccharides since this alpha-dicarbonyl is formed from the alpha-1-->4 glucans such as maltose and maltotriose but not from glucose. For its formation, a retro Claisen reaction of an enolization product of 1-amino-1,4-dideoxyhexosulose is proposed as the route to its formation. 1-Amino-1,4-dideoxyhexosulose could be formed by vinylogous alpha-elimination from the 2,3-enediol structure after Amadori rearrangement, favored by planar alignment of the bonds between C1 and C4. Subsequent rearrangement by keto-enoltautomerization leads to a 1-imino-3-keto structure. In this structure, attack of a hydroxyl anion, provided by water at neutral pH, could cause a splitting off of the C1. This reaction gives rise to formic acid or formamide and a pentose derivative, which reacts further to give 3-deoxypentosulose.     

Reactions of monosaccharides during heating of sugar-casein systems: building of a reaction network model

The Maillard reaction is important during the heating and processing of foods for its contribution to food quality. To control a reaction as complex as the Maillard reaction, it is necessary to study the reactions of interest quantitatively. In this paper the main reaction products in monosaccharide-casein systems, which were heated at 120 degrees C and pH 6.7, were identified and quantified, and the reaction pathways were established. The main reaction routes were (i) sugar isomerization, (ii) degradation of the sugar into carboxylic acids, and (iii) the Maillard reaction itself, in which not only the sugar itself but also its reaction products react with the epsilon-amino group of lysine residues of the protein. Significant differences in reaction mechanism between aldose and ketose sugars were observed. Ketoses seemed to be more reactive in the sugar degradation reactions than their aldose isomers, and whereas the Amadori product was detected as a Maillard reaction intermediate in the aldose-casein system, no such intermediate could be found in the ketose-casein system. The reaction pathways found were put together into a model, which will be evaluated by kinetic modeling in a subsequent paper.     

Formation of Maillard reaction products during heat treatment of carrots

As indicators of the early stage of the Maillard reaction in carrots, N-(furoylmethyl) amino acids (FMAAs) formed during acid hydrolysis of the corresponding Amadori products were analyzed using RP-HPLC with UV detection. N(ε)-FM-Lys (furosine), FM-Gly, FM-Ala, FM-Val, FM-Ile, FM-Leu, and FM-GABA were identified using synthesized standard material by means of mass spectrometry. Furthermore, N(ε)-carboxymethyllysine (CML) and pyrraline were analyzed as indicators for advanced stages of glycation. For commercial samples with high water content, the formation of Amadori compounds predominates, whereas the advanced stage of Maillard reaction plays only a minor part. Carrot juices, baby food, and tinned carrots showed quite low rates of amino acid modification up to 5%. For dehydrated carrots, significantly higher values for Amadori products were measured, corresponding to a lysine derivatization of up to 58% and nearly 100% derivatization of GABA. Drying experiments revealed great differences in reactivity between the amino acids studied. Whereas furosine reached constant values quite quickly, some FMAAs showed a continuous increase with heating time, indicating that selected FMAAs can be used as a hallmark for the early Maillard reaction to control processing conditions.     

Strecker-type degradation produced by the lipid oxidation products 4,5-epoxy-2-alkenals

Strecker degradation is one of the most important reactions leading to final aroma compounds in the Maillard reaction. In an attempt to clarify whether lipid oxidation products may be contributing to the Strecker degradation of amino acids, this study analyzes the reaction of 4,5-epoxy-2-alkenals with phenylalanine. In addition to N-substituted 2-(1-hydroxyalkyl)pyrroles and N-substituted pyrroles, which are major products of the reaction, the formation of both the Strecker aldehyde phenylacetaldehyde and 2-alkylpyridines was also observed. The aldehyde, which was produced at 37 degrees C-as could be determined by forming its corresponding thiazolidine with cysteamine-and pH 6-7, was not produced when the amino acid was esterified. This aldehyde is suggested to be produced through imine formation, which is then decarboxylated and hydrolyzed. This reaction also produces a hydroxyl amino derivative, which is the origin of the 2-alkylpyridines identified. All these data indicate that Strecker-type degradation of amino acids is produced at 37 degrees C by some lipid oxidation products. This is a new proof of the interrelations between lipid oxidation and Maillard reaction, which are able to produce common products by analogue mechanisms.     

Thermal generation of 3-amino-4,5-dimethylfuran-2(5H)-one, the postulated precursor of sotolone, from amino acid model systems containing glyoxylic and pyruvic acids

4,5-Dimethyl-3-hydroxy-2(5H)-furanone (sotolone), a naturally occurring flavor impact compound, can be isolated from various sources, especially fenugreek seeds. It can also be thermally produced from intermediates generated from the Maillard reaction such as pyruvic and ketoglutaric acids, glyoxal, and 2,3-butanedione. A naturally occurring precursor of sotolone, 3-amino-4,5-dimethyl-2(5H)-furanone, was thermally generated for the first time from pyruvic acid and glycine or from glyoxylic acid and alanine model systems. Isotope labeling studies have implicated 4,5-dimethylfuran-2,3-dione as an intermediate that can be converted into 3-amino-4,5-dimethyl-2(5H)-furanone through Strecker-like interaction with any amino acid. Furthermore, these studies have also indicated the presence of two pathways for the formation of 4,5-dimethylfuran-2,3-dione, one requiring pyruvic acid and a formaldehyde source and the other requiring glyoxylic acid and acetaldehyde. Self-aldol condensation of pyruvic acid followed by lactonization and further aldol reaction with formaldehyde can generate the same intermediate as the self-aldol addition product of acetaldehyde with glyoxylic acid followed by lactonization. The pyruvic acid pathway was found to be a more efficient route than the glyoxylic acid pathway. Furthermore, the pyruvic acid/glycine model system was able to generate sotolone in the presence of moisture, and in the presence of ammonia, commercial sotolone was converted back into 3-amino-4,5-dimethyl-2(5H)-furanone.     

Nonenzymatic browning of lactose and caseinate during dry heating at different relative humidities

Dry mixtures of lactose and caseinate were heated at 60 degrees C for up to 96 h at different relative humidities (RHs) ranging from 29 to 95%. The resulting nonenzymatic browning was studied by determining lactulosyl lysine formation in the caseinate (as measured by the conversion to furosine), amount of reacted lactose, loss of lysine, color formation, and fluorescent intensity. For each measurement, the maximum reaction occurred at intermediate RHs. While there is general agreement between the results obtained by different methods, discrepancies are understandable given the complex nature of nonenzymatic browning. It was shown that the degradation of the Amadori product, lactulosyl lysine, increased with RH. Moreover, the Maillard reaction, as opposed to caramelization of lactose, was the major pathway at all RHs. Visible browning occurred when the destruction of Amadori product became dominant, and interactions between sugar fragments and caseinate were not the rate-limiting steps in the nonenzymatic browning.     

Why asparagine needs carbohydrates to generate acrylamide

Structural considerations dictate that asparagine alone may be converted thermally into acrylamide through decarboxylation and deamination reactions. However, the main product of the thermal decomposition of asparagine was maleimide, mainly due to the fast intramolecular cyclization reaction that prevents the formation of acrylamide. On the other hand, asparagine, in the presence of reducing sugars, was able to generate acrylamide in addition to maleimide. Model reactions were performed using FTIR analysis, and labeling studies were carried out using pyrolysis-GC/MS as an integrated reaction, separation, and identification system to investigate the role of reducing sugars. The data have indicated that a decarboxylated Amadori product of asparagine with reducing sugars is the key precursor of acrylamide. Furthermore, the decarboxylated Amadori product can be formed under mild conditions through the intramolecular cyclization of the initial Schiff base and formation of oxazolidin-5-one. The low-energy decarboxylation of this intermediate makes it possible to bypass the cyclization reaction, which is in competition with thermally induced decarboxylation, and hence promote the formation of acrylamide in carbohydrate/asparagine mixtures. Although the decarboxylated Amadori compound can be formed under mild conditions, it requires elevated temperatures to cleave the carbon-nitrogen covalent bond and produce acrylamide.     

Monitoring glycation of lysozyme by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to study the glycation of lysozyme by D-glucose (LZM-G) and by D-fructose (LZM-F) under dry heating conditions in the presence and in the absence of oxygen. ESI-MS proved to be a precise method for monitoring protein glycation with respect to following the extent of glycation and changes in the glycoconjugate profile with time. The ESI-MS spectrum of glycated LZM revealed a heterogeneous distribution of glycoforms of LZM at different reaction stages. D-Glucose showed a higher level of reactivity with the amino groups of LZM than D-fructose, both in the presence and in the absence of oxygen. The presence of oxygen in the reaction system induced oxidative side reactions, which competed with and slowed the initial rate of formation of Amadori or Heyns products. The more reactive glycoxidation products formed during the initial stages of incubation in the presence of oxygen accelerated the rate of glycation during the later stages of incubation and increased the involvement of arginine residues of LZM in the glycation reaction. The interaction between the initial glycoxidation product(s) of the reducing sugars and intact lysozyme during the later stages of incubation was observed by the appearance of a different cluster of glycoconjugates in the mass spectrum during the latter stages of incubation. The molecular weight differences between the molecular ions of the new cluster of glycoconjugates are consistent with the formation of D-glucosone from the autoxidation of D-glucose or from the oxidative cleavage of the glucose-lysozyme imine adduct in the lysozyme-glucose system. The effect of oxygen-induced glycoxidation on the glycation reaction was also more pronounced in the LZM-G system compared with that in the LZM-F system.     

Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids

To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.     

Characterization and functional properties of lactosyl caseinomacropeptide conjugates

Ovine caseinomacropeptide (CMP) was modified with lactose through Maillard reaction under 44% relative humidity and 40 degrees C for various periods (0-11 days). Different lactosylated CMP forms were separated by capillary electrophoresis and reversed phase high-performance liquid chromatography (RP-HPLC) and identified by RP-HPLC coupled with electrospray ionization mass spectrometry (ESI-MS). Around 55-60% of CMP was lactosylated under the conditions assayed, with the monolactosylated form being the most abundant one, followed by the di-, tri-, and tetralactosylated species. During the first days of incubation amino acid analyses showed a decrease in lysine and NH(2)-terminal methionine, which coincided with an increase in the furosine content. However, from the ninth day of incubation, further degradation of Amadori compounds prevailed over their formation. Solubility, heat stability, and emulsifying capacity of the native and modified CMP were investigated. Lactosylation improved the emulsifying activity, but it did not modify the great solubility and heat stability of native CMP.     

Rye Flour Extraction Rate Affects Maillard Reaction Development,Antioxidant Activity,and Acrylamide Formation in Bread Crisps

This report shows the effect of rye flour extraction rate on Maillard reaction, antioxidant activity, and acrylamide formation during toasting of rye bread crisps. Four rye flours with extraction rates of 70, 85, 95, and 100% were tested. Maillard reaction development was studied by measuring browning development, hydroxymethylfurfural (HMF), and glucosilisomaltol (GIM) formation, as well as antioxidant activity. Results showed that HMF and GIM concentrations in toasted bread crisps were higher as the flour extraction rate increases. Antioxidant activity increased during toasting as a consequence of antioxidant Maillard reaction product formation. Acrylamide concentration was clearly affected by free asparagine content of flour, while no effect of dietary fiber and natural antioxidant content of flours had an effect on acrylamide formation. Overall data suggest that the rate of Maillard reaction is higher in whole flours because of their higher free amino acid and protein content.     

Formation of early and advanced Maillard reaction products correlates to the ripening of cheese

The present study deals with the characterization of the ripening of cheese. A traditional German acid curd cheese was ripened under defined conditions at elevated temperature, and protein and amino acid modifications were investigated. Degree of proteolysis and analysis of early [Amadori compound furosine (6)] and advanced [N(ε)-carboxymethyllysine (4), N(ε)-carboxyethyllysine (5)] Maillard reaction products confirmed the maturation to proceed from the rind to the core of the cheese. Whereas 6 was decreased, 4 and 5 increased over time. Deeper insight into the Maillard reaction during the ripening of cheese was achieved by the determination of selected α-dicarbonyl compounds. Especially methylglyoxal (2) showed a characteristic behavior during storage of the acid curd cheese. Decrease of this reactive structure was directly correlated to the formation of 5. To extend the results of experimental ripening to commercial cheeses, different aged Gouda types were investigated. Maturation times of the samples ranged from 6 to 8 weeks (young) to more than 1 year (aged). Again, increase of 5 and decrease of 2 were able to describe the ripening of this rennet coagulated cheese. Therefore, both chemical parameters are potent markers to characterize the degree of maturation, independent of coagulation.     

Further insight into thermally and pH-induced generation of acrylamide from glucose/asparagine model systems

On the basis of numerous studies on the mechanism of formation of acrylamide (AA) from asparagine and reducing sugars, the decarboxylated Schiff base [ N-( d-glucos-1-yl)-3'-aminopropionamide] and its corresponding Amadori product [ N-(1-deoxy- d-fructos-1-yl)-3'-aminopropionamide) are considered to be possible direct precursors in addition to 3-aminopropionamide (AP). Furthermore, the mechanism of decarboxylation of the initially formed N-( d-glucos-1-yl)asparagine to generate the above-mentioned precursors also remains to be confirmed. To identify the relative importance of AA precursors, the decarboxylated Amadori product (AP ARP) and the corresponding Schiff base were synthesized and their relative abilities to generate AA under dry and wet heating conditions were studied. Under both conditions, the N-( d-glucos-1-yl)-3'-aminopropionamide had the highest intrinsic ability to be converted into AA. In the dry model system, the increase was almost 4-fold higher than the corresponding AP ARP or AP; however, in the wet system, the increase was 2-fold higher relative to AP ARP but >20-fold higher relative to AP. In addition, to gain further insight into the decarboxylation step, the amino acid/sugar reactions were analyzed by FTIR to monitor the formation of the previously proposed 5-oxazolidinone intermediate known to exhibit a peak in the range of 1770-1810 cm (-1). Spectroscopic studies clearly indicated the formation of an intense peak in the indicated range, the precise wavelength being dependent on the amino acid and the sugar used. The identity of the peak was verified by observing a 40 cm (-1) shift when [(13)C-1]-labeled amino acid was used.     

Formation of pyrazines and a novel pyrrole in Maillard model systems of 1,3-dihydroxyacetone and 2-oxopropanal

Alkylpyrazines are a very important class of Maillard flavor compounds, but their mechanism of formation is complex and consists of different pathways. The model reaction of 20 different amino acids with 1,3-dihydroxyacetone, as a precursor of 2-oxopropanal, was studied by means of SPME-GC-MS to investigate the involvement of the amino acid side chain in the substitution pattern of the resulting pyrazines. 2,5-Dimethylpyrazine was quantitatively the most important pyrazine formed from all of the amino acids. The amino acid side chain is not involved in its formation. The substituents of other less abundant pyrazines resulted mainly from the incorporation of the Strecker aldehyde or aldol condensation products in the intermediate dihydropyrazine. The importance of different reaction mechanisms was evaluated, taking into account the pattern of pyrazines identified. In the solvent extracts of aqueous model reactions of 2-oxopropanal with amino acids, the main reaction product was not a pyrazine but a novel pyrrole. This pyrrole was identified as 2,5-diacetyl-3-methyl-1 H-pyrrole by means of spectral analysis, secured by chemical synthesis. A reaction mechanism for its formation was proposed and evaluated. The influence of various reaction conditions on the formation of 2,5-diacetyl-3-methyl-1 H-pyrrole and 2,5-dimethylpyrazine in the model reaction of alanine with 2-oxopropanal was studied. These results underscore the importance of the ratio of the different reagents and the presence of water in the resulting flavor formation in the Maillard reaction.