Teruyuki Usui

Identification and Determination of α-Dicarbonyl Compounds Formed in the Degradation of Sugars

The α-dicarbonyl compounds formed in the degradation of glucose and fructose were analyzed by HPLC using 2,3-diaminonaphthalene as derivatizing reagent, and identified as glucosone (GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), triosone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and methylglyoxal (MGO). The results suggest that α-dicarbonyl compounds were formed from glucose via non-oxidative 3-deoxyglucosone formation and oxidative glucosone formation in glucose degradation. In addition, TRIO, GO, and MGO were also formed from glyceraldehyde as intermediate. The α-dicarbonyl compounds might be formed from glucose via these pathways in diabetes.

Cytotoxicity and Oxidative Stress Induced by the Glyceraldehyde-related Maillard Reaction Products for HL-60 Cells

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nα-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress. We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nα-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930–932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production. This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.

Isolation and identification of the 3-hydroxy-5-hydroxymethyl-pyridinium compound as a novel advanced glycation end product on glyceraldehyde-related Maillard reaction.

Glyceraldehyde (200 mM) and Nα-acetyllysine (100 mM) were incubated in 0.2 M sodium phosphate buffer (pH 7.4) at 37°C for a week. A major compound, glyceraldehyde-related Maillard reaction product, was purified from the reaction mixture using reverse phase (ODS)-HPLC. It was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named as GLAP (Glyceraldehyde derived Pyridinium compound), using NMR and MS analyses. It was suggested that GLAP as a novel advanced glycation end product (AGE) is one of the key compounds in the glyceraldehyde-related Maillard reaction.

Detection and determination of glyceraldehyde-derived pyridinium-type advanced glycation end product in streptozotocin-induced diabetic rats.

GLAP, glyceraldehyde-derived pyridinium-type advanced glycation end product (AGE), formed by glyceraldehyde-related glycation, was identified in the plasma protein and the tail tendon collagen of streptozotocin (STZ)-induced diabetic rats. It was detected in the plasma protein and the collagen in diabetic rats by LC-MS and LC-MS/MS analysis, but was not detected in normal rats. In addition, GLAP was formed from glyceraldehyde-3-phosphate (GA3P) with lysine as well as glyceraldehyde (GLA) with lysine in vitro. Accordingly, it is suggested that an increase in the GLAP level reflects an increase in the GLA level and the GA3P level. GLAP might be a biomarker for reduced activity of the glyceraldehyde-related enzymes in the metabolic diseases such as diabetic complications.

Formation Mechanisms of Melanoidins and Fluorescent Pyridinium Compounds as Advanced Glycation End Products

The formation mechanisms of melanoidins as advanced glycation end products (AGEs) have not been resolved. Blue and red pigments generated in the D‐xylose–glycine reaction system are postulated to be intermediate oligomers in the generation of melanoidins. A novel blue pigment, designated blue‐M5, was identified as a similar structure to blue‐M1 except for the side chain of two dihydroxypropyl groups. Blue pigments were also generated in the D‐glucose–glycine and D‐xylose–β‐alanine reaction systems as well as in the D‐xylose–glycine reaction system. Blue pigments by the Maillard reaction might be formed by the decarboxylation of two molecules of pyrrolopyrrole‐2‐carbaldehydes (PPA). PPA, composed of a side chain of a dihydroxypropyl group, was identified as a precursor of blue pigments. In fact, blue‐M5 was generated by the incubation of PPA alone. Blue pigments, which involved pyrrolopyrrole structures, were readily changed to brown polymers. Glyceraldehyde‐derived pyridinium (GLAP) compound, a glyceraldehyde‐derived fluorescent AGE, and lysyl‐pyrropyridine, a 3‐deoxyglucosone‐derived fluorescent AGE, were detected at higher levels in the plasma proteins and the tail tendon collagen of streptozotocin‐induced diabetic rats compared to normal rats. GLAP and lysyl‐pyrropyridine, therefore, might be related to the progression of diabetic complications.

Chemistry and biological effects of melanoidins and glyceraldehyde-derived pyridinium as advanced glycation end products.

Abstract: Blue pigments (blue‐M1 and blue‐M2) and red pigments (red‐M1 and red‐M2) were generated in a xylose‐glycine reaction system. Blue‐M2 was identified as an addition compound of di‐xylulose‐glycine to blue‐M1 that involved two pyrrolopyrrole structures. We identified red pigments as isomers of addition compounds of xylulose‐glycine to the condensed compound between pyrrolopyrrole‐2‐carbaldehyde and pyrrole‐2‐carbaldehyde. These pigments have polymerizing activity, suggesting that they are important Maillard reaction intermediates through the formation of melanoidins. Melanoidins induced IFN‐γ and IL‐12 expression in spleen cells exposed to allergen and in macrophages, respectively. These findings suggest that melanoidins have a suppressive effect on allergic reaction as a novel physiological effect. On the other hand, we identified a glyceraldehyde‐derived advanced glycation end product (AGE) formed from glyceraldehyde and N‐acetylarginine as well as glyceraldehyde‐derived pyridinium (GLAP) in physiological conditions. The AGE was identified as 5‐methylimidazoline‐4‐one (MG‐H1), which has been reported to be formed from arginine and methylglyoxal. GLAP, which induced reactive oxygen species (ROS) production in HL‐60 cells, is supposed to be a toxic AGE, while MG‐H1 is a nontoxic AGE.

Protective Effect of Blue-M1 against Oxidative Stress on COS-1 Cells

Blue-M1 is a blue pigment formed from xylose and glycine in the Maillard reaction. Previous work revealed that Blue-M1 scavenged hydroxyl radicals, and prevented the autoxidation of linoleic acid in vitro. We investigated the protective effect of Blue-M1 for 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH)-induced toxicity in COS-1 cells. COS-1 cells were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 24 h. Blue-M1 decreased the AAPH-induced toxicity in COS-1 cells, and this effect was dose-dependent. Furthermore, COS-1 cells were treated with diphenyl-1-pyrenylphosphine (DPPP), as a reagent for the detection of lipid peroxide, and then were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 6 h. Blue-M1 prevented the AAPH-induced peroxidation of cell membrane on COS-1 cells, and this effect was also dose-dependent. These results suggest that Blue-M1 prevents the oxidative cell injury. Therefore, Blue-M1 will be an antioxidant, which protect against the oxidative stress in living systems.

Determination of Glyceraldehyde Formed in Glucose Degradation and Glycation

Glyceraldehyde (GLA) was determined in glucose degradation and glycation. GLA was detected as a decahydroacridine-1,8-dione derivative on reversed phase HPLC using cyclohexane-1,3-dione derivatizing reagent. The glucose-derived GLA level was higher than the glycation-derived GLA level, because GLA was converted to intermediates and advanced glycation end products (AGE) in glycation. GLA was also generated from 3-deoxyglucosone and glucosone as intermediates of glucose degradation and glycation. This study suggests that glyceraldehyde is generated by hyperglycemia in diabetes, and that it is also formed in medicines such as peritoneal dialysis solution.

Isolation and Identification of 5-Methyl-imidazolin-4-one Derivative as Glyceraldehyde-Derived Advanced Glycation End Product

We isolated and identified the glyceraldehyde-derived advanced glycation product (AGE) formed from glyceraldehyde and N α-acetylarginine. A major product was identified as N α-acetyl-N δ-(5-methyl-imidazolin-4-one-2-yl)-ornithine. The compound has been reported as methylglyoxal-derived AGE, MG-H1. This study suggests that MG-H1 is formed through both glyceraldehyde-related and methylglyoxal-related pathways. There is a possibility that MG-H1 becomes an index of injury to glyceraldehyde and methylglyoxal-related enzymes.

The Formation of Argpyrimidine in Glyceraldehyde-Related Glycation

Three major glyceraldehyde-related advanced glycation end products (AGEs) were formed from a mixture of Nα-acetyllysine, Nα-acetylarginine, and glyceraldehyde. Two of the compounds were MG-H1 and GLAP, as previously reported, and the other compound was identified as Nα-acetyl-Nδ-(5-hydroxy-4,6-dimethyl-pyrimidin-2-yl)-ornithine, argpyrimidine (APN). APN is a modification product of arginine residue, but it did not form from glyceraldehyde with arginine residue. The coexistence of lysine residue was necessary to APN formation.