Identification and quantification of phosphatidylethanolamine-derived glucosylamines and aminoketoses from human erythrocytes--influence of glycation products on lipid peroxidation.

While the Maillard reaction of amino acids and proteins as well as its consequences in vivo has been thoroughly investigated, little attention has so far been paid to the glycation of aminophospholipids such as phosphatidylethanolamine (PE) or phosphatidylserine (PS), which are essential for structure and functionality of biological membranes. PE-derived glucosylamines (Schiff-PEs) and aminoketoses (Amadori-PEs) have now for the first time been simultaneously identified and quantified in erythrocytes from diabetics and healthy individuals by liquid chromatography-electrospray mass spectrometry (LC-(ESI)MS). The amounts of glycated PE (gPE) were significantly higher in diabetics (0.18-34.2 mol% Schiff-PE and 0.047-0.375 mol% Amadori-PE) than in controls (0.12-3.99 mol% Schiff-PE and 0.018-0.055 mol% Amadori-PE). A positive correlation between fructosylated hemoglobin (HbA(1c)) and the gPE levels was established. No advanced glycation endproducts (AGEs) like 5-hydroxymethylpyrrole-2-carbaldehyde (pyrrole-PE), carboxymethyl (CM-PE), or carboxyethyl (CE-PE) derivatives were detected. To investigate the influence of gPE on lipid peroxidation of biological membranes, liposomes consisting of soy-PE and synthetically prepared Amadori-PE (16:0-16:0) were incubated for several days and the formation of oxidation products was monitored. It could be shown that Amadori-PE extensively promotes lipid peroxidation even in the absence of transition metal ions like Cu(2+) and Fe(3+). Oxidative damage to membrane lipids therefore is supposed to be at least partially caused by the glycation of aminophospholipids.

Identification and quantitative evaluation of the lysine-arginine crosslinks GODIC, MODIC, DODIC, and glucosepan in foods.

The presence of the various protein crosslinks GOLD 2, MOLD 3, GODIC 4, MODIC 5, DODIC 6, and glucosepan 7 in foods has been established for the first time by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). In compounds 2 and 3 two lysine moieties, in 4-7 a lysine and an arginine side chain are joined by the crosslink. Unequivocal identification of 2-7 was achieved with independently synthesized reference material. The quantitative results for the investigated foodstuffs show MODIC 5 to be the most important Maillard crosslink. The concentrations of 5 and GODIC 4 are 5-10 fold higher than those of the corresponding imidazolium compounds 3 and 2, establishing 5 and 4 as the major food protein crosslinks derived from methylglyoxal and glyoxal, respectively. The maximum value of 151 mg MODIC 5/kg protein (equivalent to 0.42 mmol/kg protein) was found in a butter biscuit sample which also shows the highest overall Maillard crosslink content with 0.71 mmol 47/kg protein. These first quantitative results suggest that compounds 4-7 can be jointly responsible for protein polymerization in the course of food processing.

Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A.

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor.

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.

Formation of a phospholipid-linked pyrrolecarbaldehyde from model reactions of D-glucose and 3-deoxyglucosone with phosphatidyl ethanolamine.

Phospholipid-linked 'advanced glycation end products' (AGEs) are supposed to play an important role for lipid oxidation in vivo. The identification of the pyrrolecarbaldehyde 1-[2-formyl-5-(hydroxymethyl)-1 H-pyrrol-1-yl]-4,10-dioxo-7-(tetradecanoyloxy)-3,5,9-trioxa- 4lambda5-phosphatricosan-4-olate (7) from model reactions of D-glucose or 3-deoxyglucosone (4, 3-DG) with phosphatidyl ethanolamine (PE) is described. A preparation method is given for 1-(2-hydrox¿ethyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (8). Independent syntheses as well as unequivocal structural characterization are reported for the substitution products of 8 1-(2-hydroxyethyl)-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (9a) and 5-(ethoxymethyl)-1-(2-hydroxyethyl)-1H-pyrrole-2-carbaldehyde (9b). For all these compounds, chromatographic and spectroscopic data were established by GLC-MS and HPLC with diode array detection (DAD). PE and D-glucose or 3-DG 4 were either incubated at pH 7.4, 100 degrees C for 3 h or at pH 7.4, 37 degrees C for 5 weeks in neat buffer or ethanol buffer mixtures. The phospholipid fraction was purified on a C18 solid-phase extraction column and cleaved with ethanolic potassium hydroxide. The carbaldehyde 8, released in this process, was identified bs GLC-MS and quantified by HPLC-DAD. Formation of 7 is favored in the ethanol buffer reactions relative to those in buffer solution only although the amounts determined from the 37 degrees C incubations generally are very low. It seems likely, therefore, that phospholipid-linked pyrrolecarbaldehydes, such as 7, are biomarkers rather than effectors of membrane damage in vivo.

Independent synthesis of aminophospholipid-linked maillard products.

Phospholipid-linked glycation products are supposed to play an important role in lipid oxidation in vivo. Independent syntheses and unequivocal structural characterization are reported for the phosphatidyl ethanolamine (PE)-derived Amadori compound 4-hydroxy-4-oxo-1-[(palmitoyloxy)methyl]-9-(2,3,4,5-tetrahydrox ytetrahydro-2H-pyran-2-yl)-3,5-dioxa-8-aza-4lambda5-ph osphanon-1-yl palmitate, pyrrolecarbaldehyde 2-[[[2-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]ethoxy](hydroxy)phosph oryl]oxy]-1-[(palmitoyloxy)methyl]ethyl palmitate, the carboxymethyl (CM) derivative 7-hydroxy-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa-3-aza-+ ++7lambda5-phosphaoctacosan-1-oic acid, and the carboxyethyl (CE) derivative 7-hydroxy-2-methyl-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa++ +-3-aza-7lambda5-phosphaoctacosan-l-oic acid. With these reference compounds, a liquid chromatography-mass spectrometry (LCMS) method for the determination of such PE-linked Maillard products has been developed.

Identification and quantification of aminophospholipid-linked Maillard compounds in model systems and egg yolk products.

While the Maillard reaction of free amino acids and proteins is a well-established process, no defined structures from the nonenzymatic browning of aminophospholipids in foodstuffs have been described so far. Phosphatidylethanolamine (PE)-linked glucosylamines (Schiff-PE), Amadori products (Amadori-PE), 5-hydroxymethylpyrrole-2-carbaldehydes (Pyrrole-PE), and carboxymethyl (CM-PE) as well as carboxyethyl (CE-PE) derivatives were detected and quantified by liquid chromatography- electrospray mass spectrometry (LC-(ESI)MS). Model incubations of soy-PE and D-glucose were employed to firmly establish the LC-(ESI)MS procedure. Analyses of spray-dried egg yolk powders and lecithin products derived therefrom show one-fourth of the native D-glucose content of egg yolk to be transformed to Amadori-PE, corresponding to a PE derivatization quota of 11-15.5 mol %. Schiff-PE and Pyrrole-PE were present only in low amounts, no CM-PE and CE-PE could be identified in any of the investigated samples. The high glycation rate of egg yolk PE will influence the emulsifying properties and perhaps even the oxidation resistance of the respective products.

Reactivity of lysine moieties toward an epoxyhydroxylinoleic acid derivative: aminolysis versus hydrolysis.

Epoxyols are generally accepted as crucial intermediates in lipid oxidation. The reactivity of tert-butyl (9R,10S,11E,13S)-9, 10-epoxy-13-hydroxy-11-octadecenoate (11a,b) toward lysine moieties is investigated, employing N(2)-acetyllysine 4-methylcoumar-7-ylamide (12) as a model for protein-bound lysine. The prefixes R and S denote the relative configuration at the respective stereogenic centers. Independent synthesis and unequivocal structural characterization are reported for 11a,b, its precursors, and tert-butyl (9R,10R,11E, 13S)-10-(¿5-(acetylamino)-6-[(4-methyl-2-oxo-2H-chromen-7-yl)amino ]-6 -oxohexyl¿amino)-9,13-dihydroxy-11-octadecenoate (13a-d). Reactions of 11a,b and 12 in 1-methyl-2-pyrrolidone (MP) and MP/water mixtures at pH 7.4 and 37 degrees C for 56 days show formation of the aminols 13a-d to be favored by an increased water content. The same trend is observed for hydrolytic cleavage of 11a,b to tert-butyl (E)-9,10, 13-trihydroxy-11-octadecenoate (14) and tert-butyl (E)-9,12, 13-trihydroxy-10-octadecenoate (15). Under the given conditions, aminolysis proceeds via an S(N)2 substitution, in contrast with the S(N)1 process for hydrolysis. In the MP/water (8:2) incubation, 15. 8% of 12 has been transformed to 13a-d and 10.5% of 11a,b hydrolyzed to the regioisomers 14 and 15 after 8 weeks, respectively. Aminolysis of alpha,beta-unsaturated epoxides by lysine moieties therefore is expected to be an important mode of interaction between proteins and lipid oxidation products.

Cross-linking of proteins by Maillard processes--characterization and detection of a lysine-arginine cross-link derived from D-glucose.

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. Investigations are reported on how the cross-linking unit 2-ammonio-6-[2-[(4-ammonio-5-oxido-5-oxopentyl)amino]-6,7-dihydrox y-4,5,6,7,8,8a-hexahydroimidazo[4,5-b]azepin-4-yl] hexanoate (7), designated as glucosepan, can be identified and quantified from D-glucose-bovine serum albumin (BSA) incubations. Independent synthesis and unequivocal structural characterization are given for glucosepan 7. A protocol was established for its determination by LC-MS with electrospray ionization (ESI). BSA and D-glucose were incubated at 37 degrees C, pH 7.4 for eight weeks and the time-dependent formation of 7 was observed. Since glucosepan 7 is unstable under acid proteolytic conditions, BSA was cleaved enzymatically. The maximum value obtained from a solution containing 50 g/L BSA and 100 mM D-glucose after eight weeks incubation time corresponds to an arginine derivatization rate of 1.38 +/- 0.07 mmol 7/mol Arg (equivalent to 31.7 +/- 1.6 mmol 7/mol BSA). From these results, it seems justified to expect 7 to play an important role in the cross-linking of proteins in vivo as well as in foodstuffs. The structural similarity of glucosepan 7 and pentosidine 1 made it obvious to also look for an eventual parallelism in the respective formation pathways.

Cross-linking of proteins by Maillard processes: characterization and detection of lysine-arginine cross-links derived from glyoxal and methylglyoxal.

Alpha-dicarbonyl compounds, such as glyoxal and methylglyoxal, are crucial intermediates in the browning and cross-linking of proteins by reducing sugars in the course of the Maillard reaction. The cross-linking units 2-ammonio-6-([2-[(4-ammonio-5-oxido-5-oxopentyl)amino]-4,5-dihydro - 1H-imidazol-5-ylidene]amino)hexanoate (9) and 2-ammonio-6-([2-[(4-ammonio-5-oxido-5-oxopentyl) amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene]amino)hexanoate (10), designated as GODIC and MODIC, are identified and quantified from glyoxal/methylglyoxal-bovine serum albumin (BSA) incubations. Independent syntheses and unequivocal structural characterization are given for 9 and 10. A protocol was established for their determination by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). BSA and the respective alpha-dicarbonyl compound were incubated at 37 degrees C, pH 7.4 for 1 week, and the time-dependent formation of 9 and 10 was observed. The maximum value obtained from a solution containing 50 g/L BSA and 2 mM glyoxal or methylglyoxal after a 7-day incubation period corresponds to an arginine derivatization quota of 13.0 +/- 0.32 mmol 9/mol Arg or 3.0 +/- 0.12 mmol 10/mol Arg. The cross-links 9 and 10 were also detected in a D-glucose-BSA incubation. From these results, it seems justified to assign an important role to 9 and 10 in the cross-linking of proteins in vivo as well as in foodstuffs. In an additional model study, formation of 9 and 10 was compared to that of the imidazolium cross-links GOLD 3 and MOLD 4.

Cross-linking of proteins by Maillard processes--model reactions of D-glucose or methylglyoxal with butylamine and guanidine derivatives.

Advanced Maillard reaction in proteins leads to formation of covalently cross-linked aggregates the chemical nature of which is largely unknown. From model reactions of methylglyoxal and butylamine with creatine or alpha-N-acetyl-L-arginine, one main product each was isolated. These two compounds were identified, on the basis of unequivocal spectroscopic evidence, as 2-[(5-butylimino-4-methyl-4,5-dihydro-1H-2-imidazolyl)(methyl)amin o]acetic acid and 2-acetylamino-5-[(5-butylimino-4-methyl-4,5-dihydro-1H-2-imidazoly l)amino]pentanoic acid, respectively. Using D-glucose instead of methylglyoxal, two main products each were obtained from reaction with the respective guanidine derivative. The spectroscopic data definitively establish the formation of the diastereoisomeric 2-[(4-butyl-6,7-dihydroxy-4,5,6,7,8,8a-hexahydroimidazo[4,5-b]a zepin-2-yl)(methyl)amino]acetic acid from the reaction with creatine, and of the diastereoisomeric 2-acetylamino-5-[(4-butyl-6,7-dihydroxy-4,5,6,7,8,8a-hexahydroimidazo [4,5-b]azepin-2-yl)amino]pentanoic acid from the reaction with alpha-N-acetyl-L-arginine. All products were isolated in fairly good yield and represent 1:1:1 adducts of the respective reaction partners. Formation of these compounds thus constitutes an efficient reaction pathway for linking primary amines to guanidine derivatives. It seems justified, therefore, to expect cross-linking of proteins by action of reducing carbohydrates to proceed analogously.