Mitsugu Akagawa

Production of hydrogen peroxide by polyphenols and polyphenol-rich beverages under Quasi-physiological conditions

To investigate the ability of the production of H2O2 by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37°C), and then measured the formation of H2O2 by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H2O2. We also examined the effects of a metal chelator, pH, and O2 on the H2O2-generating property, and the generation of H2O2 by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H2O2-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H2O2 in beverages. From the results, the possible significances of the H2O2-generating property of polyphenols for biological systems are discussed.

Mechanism of formation of elastin crosslinks.

We examined the formation of quaternary pyridinium crosslinks of elastin formed by condensation of lysine and allysine residues using the model compounds propanal (allysine) and n-butylamine (lysine) under quasi-physiological conditions. The resulting pyridinium compounds were characterized and the structure compared with the known pyridinium crosslinks. Three pyridinium compounds were identified and the structures were identical with the skeleton of the crosslinking amino acids, desmosine (DES), isodesmosine (IDE), and pentasine. We concluded that a non-enzymatic pathway is available for the spontaneous generation of pyridinium crosslinks. To elucidate the intermediates and the mechanism of the formation of DES and IDE, we synthesized model intermediates from propanal and n-butylamine, and they were allowed to react in three kinds of solvents. Then, the products were analyzed by an ion-pair reverse-phase HPLC. The results of this model system indicated that DES and IDE can be formed by condensation of dehydromerodesmosine with dehydrolysinonorleucine and by condensation of allysine with dehydrolysinonorleucine, respectively. We also describe the mechanism of DES and IDE crosslinking.

Formation of α-Aminoadipic and γ-Glutamic Semialdehydes in Proteins by the Maillard Reaction

Abstract: Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. α‐Aminoadipic semialdehyde (AAS) and γ‐glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high‐performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p‐aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA‐derivatized protein, ABA‐AAS and ABA‐GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 μM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.

Oxidative Deamination by Hydrogen Peroxide in the Presence of Metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.

LYSYL OXIDASE COUPLED WITH CATALASE IN EGG SHELL MEMBRANE

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).

Oxidative deamination of benzylamine by glycoxidation.

In the present study, model reactions for the oxidative deamination by glycoxidation using benzylamine were undertaken to elucidate the detail of the reaction. Glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) oxidatively deaminated benzylamine to benzaldehyde in the presence of Cu(2+) at a physiological pH and temperature but not glyoxal. 3-DG and MG were more effective oxidants than glucose. We have determined the effects of metal ions, pH, oxygen, and radical scavengers on the oxidative deamination. The formation of benzaldehyde was greatest with Cu(2+), and was accelerated at a higher pH and in the presence of oxygen. EDTA, catalase, and dimethyl sulfoxide significantly inhibited the oxidation, suggesting the participation of reactive oxygen species. From these results, we propose a mechanism for the oxidative deamination by the Strecker-type reaction and the reactive oxygen species-mediated oxidation during glycoxidation.

Oxidative deamination of lysine residue in plasma protein from diabetic rat: α-dicarbonyl-mediated mechanism

Abstract The lysine residue of bovine serum albumin was deaminated to allysine (α-aminoadipic-δ-semialdehyde) during the incubation with glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) in the presence of Cu2+ at a physiological pH and temperature but not with glyoxal. Further, glucose, 3-DG, and MG oxidatively deaminated benzylamine to benzaldehyde in the presence of Cu2+. The formation of benzaldehyde was greatest with Cu2+, and was accelerated in the presence of oxygen. EDTA, catalase, and dimethyl sulfoxide (DMSO) significantly inhibited the oxidation. Analysis of plasma proteins revealed significantly higher levels of allysine in streptozotocin (STZ)-induced diabetic rats compared with normal controls. From these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes via the Maillard reaction.