Mitochondrial stress and glycoxidation increase with decreased kidney function.

Mitochondrial stress increases the production of fumarate, an intermediate of the Krebs cycle. Fumarate non-enzymatically reacts with the thiol group of cysteine, leading to the production of S-(2-succinyl)cysteine. Here, we quantified the concentration of fumarate, the free form of S-(2-succinyl)cysteine, and advanced glycation end-products, including N ε-(carboxymethyl)lysine and N δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine, in the serum of chronic kidney disease patients, using liquid chromatography-tandem mass spectrometry and an enzymatic assay. In a cross-sectional study, we evaluated the difference in metabolite concentration between healthy individuals (n = 22) and kidney transplant patients (n = 93). Additionally, we evaluated the metabolite concentration of end-stage renal disease patients (n = 17) before and 1, 3, 6, and 12 months after transplantation, in a longitudinal study. While the S-(2-succinyl)cysteine and AGEs levels were significantly increased in accordance with the rising chronic kidney disease severity, they were significantly decreased after transplantation. However, fumarate levels were only significantly different in end-stage renal disease patients. The S-(2-succinyl)cysteine levels correlated with the pre-existing kidney function marker. This study demonstrates that mitochondrial metabolic disorders contribute to impaired kidney function, and that measuring blood S-(2-succinyl)cysteine levels may be a minimally invasive way to evaluate the metabolic change in chronic kidney disease.

Changes in S-(2-succinyl)cysteine and advanced glycation end-products levels in mouse tissues associated with aging.

Cysteine is non-enzymatically modified by fumarate, which is an intermediate of the tricarboxylic acid cycle, leading to the formation of S-(2-succinyl)cysteine (2SC). Post-translational modification of physiological proteins by fumarate causes enzyme dysfunction. The aim of the study was to evaluate the changes in 2SC accumulation in physiological tissues associated with aging. Brain, liver, kidney, and serum samples were collected from 4-, 12-, and 96-week-old male C57BL/6J mice, and the level of 2SC was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after pretreatment, including delipidation, protein precipitation, and hydrolysis using hydrochloric acid. The 2SC level in the brain was higher than that in other tissues, and its accumulation significantly increased with age. Similarly, Nε-(carboxymethyl)lysine levels, an advanced glycation end-products (AGEs) that accumulates in tissues in an age-dependent manner, was found to be increased in the brain and kidneys of elderly mice. Accumulation of Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine increased significantly with age, but only in the kidneys. The fumarate content in the brain was similar to that in the liver and kidney at 4 and 12 weeks of age. Furthermore, fumarate contents increased in the liver and kidney at 96 weeks of age, whereas its level did not change in the brain. Our results demonstrated that the changes in 2SC and AGEs levels in tissues reflected differing metabolism and enhanced oxidative stress in each organ; in particular, the metabolism in the brain and kidneys is highly affected by aging.

Development of a conventional immunochemical detection system for determination of Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine in methylglyoxal-modified proteins.

Methylglyoxal (MGO) produced during glycolysis is known to react with arginine residues on proteins to generate advanced glycation end products, such as Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1). Since the production of MGO is increased during hyperglycemia or metabolic disorders in vivo, it is considered that the measurement of MG-H1 is useful for evaluating abnormalities in carbohydrate metabolism. Thus, we prepared a monoclonal antibody against MG-H1 to develop a conventional measurement system for MG-H1. Reactivity and specificity of the antibody to MGO-modified protein were confirmed by enzyme-linked immunosorbent assay and western blotting, respectively. The measurement of MG-H1 content by the antibody was positively correlated with that by electrospray ionization-liquid chromatography-tandem mass spectrometry and the ratio of modified arginine residues by amino acid analysis. Our results demonstrated that immunochemical methods could be useful for the estimation of MG-H1 content in modified proteins.

Antibody-based detection of advanced glycation end-products: promises vs. limitations.

Advanced glycation end-products (AGEs) of the Maillard reaction were originally measured according to their fluorescent and browning properties. A subsequent study with instrumental analyses such as high-performance liquid chromatography and gas chromatography mass spectrometry more clearly demonstrated the involvement of each AGE structure in pathological conditions. Furthermore, immunochemical methods have also been developed to clarify the localization of AGEs in tissues and measurement of AGEs in multiple clinical samples. Although the involvement of AGEs in age-related diseases has progressed due to immunochemical techniques, the relationship between AGE structure and diseases has not been clear because little was known about the epitope structure of each anti-AGE antibody. However, the development of epitope-identified antibodies against AGEs has made it possible to clarify AGE structures involved in diseases. This review discusses not only the usability of anti-AGE antibodies to evaluate AGEs and disease pathology and screen AGE inhibitors, but also describes their usage.

Soft-shelled turtle eggs inhibit the formation of AGEs in the serum and skin of diabetic rats.

Although soft-shelled turtle eggs (STE) have been used as a folk medicine for revitalization and the prevention of lifestyle-related diseases, the scientific evidence to support the use of STE in this manner is scarce. To clarify the physiological evidence, STE was administered to diabetic rats and the inhibitory effects on the formation of advanced glycation end-products (AGEs), which are known to increase with the progression of lifestyle-related diseases, were examined. STE and citric acid were administered to diabetic rats for 3 months, and serum N (ε)-(carboxymethyl)lysine (CML) contents were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the administration of STE did not affect the body weight, glycoalbumin or ketone body levels, it significantly reduced the serum level of CML. The accumulation of AGEs, which was measured by fluorescence intensity in the auricle skin and the lower gums, was also reduced by the administration of STE to a similar extent to that observed with citric acid. This report provides the first evidence that the oral administration of STE reduces the formation of AGEs, suggesting that one of the health effects of STE may be the inhibition of AGEs formation.

Mangosteen pericarp extract inhibits the formation of pentosidine and ameliorates skin elasticity.

The inhibition of advanced glycation end-products (AGEs) by daily meals is believed to become an effective prevention for lifestyle-related diseases. In the present study, the inhibitory effect of hot water extracts of mangosteen (Garcinia mangostana L.) pericarp (WEM) on the formation of pentosidine, one of AGEs, in vitro and in vivo and the remedial effect on skin conditions were measured. WEM significantly inhibited pentosidine formation during gelatin incubation with ribose. Several compounds purified from WEM, such as garcimangosone D and rhodanthenone B, were identified as inhibitors of pentosidine formation. Oral administration of WEM at 100 mg/day to volunteer subjects for 3 months reduced the serum pentosidine contents. Because obtaining skin biopsies from healthy volunteers is ethically difficult, AGE accumulation in the skin was estimated by a fluorescence detector. The oral administration of WEM significantly reduced the skin autofluorescence intensity, demonstrating that WEM also reduced AGE accumulation in the skin. Furthermore, the elasticity and moisture content of the skin was also improved by WEM. These results demonstrate that intakes of WEM reduces the glycation stress and results in the improvement of skin conditions.

Inhibition of AGEs formation by natural products.

Since advanced glycation end-products (AGEs) inhibitors such as benfotiamine, pyridoxamine and aminoguanidine significantly inhibit the development of retinopathy and neuropathy in streptozotocin-induced diabetic rats, treatment with AGEs inhibitors is believed to be a potential strategy for preventing lifestyle-related diseases such as diabetic complications and atherosclerosis. Furthermore, preventive medicine is the most important approach to preventing lifestyle-related diseases, and improving daily nutritional intake is thought to prevent the pathogenesis of such diseases. Therefore, AGEs inhibitors that can be obtained from daily meals are preferred to prescribed drugs. In this article, we describe a strategy for developing new AGEs inhibitors from natural products.

Detection of AGEs as markers for carbohydrate metabolism and protein denaturation.

Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the "Amadori product", is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases.

Citric acid inhibits development of cataracts, proteinuria and ketosis in streptozotocin (type 1) diabetic rats.

Although many fruits such as lemon and orange contain citric acid, little is known about beneficial effects of citric acid on health. Here we measured the effect of citric acid on the pathogenesis of diabetic complications in streptozotocin-induced diabetic rats. Although oral administration of citric acid to diabetic rats did not affect blood glucose concentration, it delayed the development of cataracts, inhibited accumulation of advanced glycation end-products (AGEs) such as N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) in lens proteins, and protected against albuminuria and ketosis. We also show that incubation of protein with acetol, a metabolite formed from acetone by acetone monooxygenase, generate CEL, suggesting that inhibition of ketosis by citric acid may lead to the decrease in CEL in lens proteins. These results demonstrate that the oral administration of citric acid ameliorates ketosis and protects against the development of diabetic complications in an animal model of type 1 diabetes.

Inhibition of human catechol-O-methyltransferase (COMT)-mediated O-methylation of catechol estrogens by major polyphenolic components present in coffee.

In the present study, we investigated the inhibitory effect of three catechol-containing coffee polyphenols, chlorogenic acid, caffeic acid and caffeic acid phenethyl ester (CAPE), on the O-methylation of 2- and 4-hydroxyestradiol (2-OH-E(2) and 4-OH-E(2), respectively) catalyzed by the cytosolic catechol-O-methyltransferase (COMT) isolated from human liver and placenta. When human liver COMT was used as the enzyme, chlorogenic acid and caffeic acid each inhibited the O-methylation of 2-OH-E(2) in a concentration-dependent manner, with IC(50) values of 1.3-1.4 and 6.3-12.5 microM, respectively, and they also inhibited the O-methylation of 4-OH-E(2), with IC(50) values of 0.7-0.8 and 1.3-3.1 microM, respectively. Similar inhibition pattern was seen with human placental COMT preparation. CAPE had a comparable effect as caffeic acid for inhibiting the O-methylation of 2-OH-E(2), but it exerted a weaker inhibition of the O-methylation of 4-OH-E(2). Enzyme kinetic analyses showed that chlorogenic acid and caffeic acid inhibited the human liver and placental COMT-mediated O-methylation of catechol estrogens with a mixed mechanism of inhibition (competitive plus noncompetitive). Computational molecular modeling analysis showed that chlorogenic acid and caffeic acid can bind to human soluble COMT at the active site in a similar manner as the catechol estrogen substrates. Moreover, the binding energy values of these two coffee polyphenols are lower than that of catechol estrogens, which means that coffee polyphenols have higher binding affinity for the enzyme than the natural substrates. This computational finding agreed perfectly with our biochemical data.

Nω -(Carboxymethyl)arginine Is One of the Dominant Advanced Glycation End Products in Glycated Collagens and Mouse Tissues.

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure Nω -(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of Nε -(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I-IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen.

Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products.

Immunohistochemical approaches have been widely used in the localization and quantification of advanced glycation end-products (AGEs). Traditional approaches for production of anti-AGE antibodies use cross-linkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to conjugate the AGE antigen to the carrier protein. However, these approaches often fail to produce antibodies that are specific to the particular AGE of interest. In the present study, Nepsilon-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was conjugated to human serum albumin (HSA) using various cross-linkers, including EDC, bis(sulfosuccinimidyl)suberate (BS3) and glutaraldehyde, to compare their efficiency for the production of epitope-specific antibodies. All of the cross-linkers tested were capable of conjugating CML to HSA, and each CML-conjugated HSA was recognized by previously characterized anti-CML antibody. However, only the use of glutaraldehyde as the cross-linker resulted in the production of a CML-specific monoclonal antibody, termed 2G11. 2G11 significantly recognized CML-modified HSA and peptide, whereas it did not recognize Nepsilon-(carboxyethyl)lysine (CEL)-modified HSA and peptide, indicating that 2G11 is highly specific to CML, and can distinguish the difference of a single methyl group between the two epitopes. To further demonstrate the use of glutaraldehyde, anti-AGE antibodies against CEL, S-(2-succinyl)cysteine and S-(carboxymethyl)cysteine were obtained by conjugation with glutaraldehyde. These studies demonstrate the efficacy of glutaraldehyde as a cross-linker for the production of antibodies against small molecules.

Comparison of pharmacokinetics between highly and mildly modified AGE proteins in mice.

We previously demonstrated that RAW 264.7 cells (murine macrophage cell line) recognize highly modified advanced glycation end products (AGE)-bovine serum albumin (BSA) (high-AGE-BSA), which was prepared by incubating BSA with 1600 mmol/L glucose for 40 weeks. In the present study, we prepared mildly modified AGE-BSA (mild-AGE-BSA) and conducted an endocytic uptake study using human monocyte-derived macrophages and Chinese hamster ovary cells which overexpressed such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1). Although high-AGE-BSA was significantly recognized by these cells, mild-AGE-BSA did not show any ligand activity to these cells. Furthermore, when 111 In-labeled mild- or high-AGE-BSA was injected into the tail vein of male ddY mice, 111 In-high-AGE-BSA was rapidly cleared from the circulation, with about 80% of the injected 111 In-high-AGE-BSA being eliminated within 5 min. In contrast, the clearance rate of 111 In-mild-AGE-BSA was very slow, similar to the 111 In-native BSA. Taken together, our results indicate that the ligand activity of AGE-BSA to scavenger receptors and those pharmacokinetic properties depend on their rate of modification by AGEs.

Usefulness of antibodies for evaluating the biological significance of AGEs.

Polyclonal and monoclonal antibodies have been widely applied to demonstrate the presence of advanced glycation end products (AGEs) in vivo. However, our previous study showed that monoclonal anti-AGE antibody (6D12) and polyclonal anti-N epsilon-(carboxymethyl)lysine (CML) antibody recognize not only CML but also N epsilon-(carboxyethyl)lysine (CEL), thus indicating that we should pay attention to the specificity of the antibodies. As a result, we prepared specific monoclonal antibodies against CML, CEL, N omega-(carboxymethyl)arginine (CMA), and S-(carboxymethyl)cysteine (CMC). Our immunochemical study using anti-CMA antibody demonstrated that the CMA content increased in a time-dependent manner when collagen was incubated with glucose, indicating that immunological quantification using the specific antibody is especially useful for measuring an acid-labile AGE structure, such as CMA. Monoclonal antibody is also applied to identify a novel biological marker in pathological lesions. We prepared antibody libraries against proteins modified with aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde (GA), and one antibody, GA5, which specifically reacts with the GA-modified protein that is recognized in human atherosclerotic lesions. Following successive high-performance liquid chromatography purification, the GA5-reactive compound was isolated and its chemical structure was found to be 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation, which was named GA-pyridine. Taken together, these results demonstrate that a specific antibody is a powerful tool for analyzing novel biomarkers, formation pathways, and the efficacy of AGE inhibitors.

Aphanothece sacrum (Sur.) Okada Prevents Cataractogenesis in Type 1 Diabetic Mice.

Aphanothece sacrum (Sur.) Okada is a species of cyanobacteria found in Japan. Although it has been used in local cuisine in Kyushu, Japan, for 250 y, little is known about its beneficial effect as food. The daily intake of health beneficial phytochemicals is believed to be useful for preventing lifestyle-related diseases, such as diabetic cataracts. In this study, the inhibitory effect of freeze-dried A. sacrum (Asa) on the formation of diabetic cataracts (DCs) was evaluated. Type 1 diabetes was induced in mice using streptozotocin (STZ). The mice were divided into two groups: one was fed a normal diet (DM-control group) and the other was fed a diet containing 1% Asa (DM-Asa group). During the study, changes in blood glucose levels and the amount of food and water consumed were measured. After 3 mo, the amount of Nε-(carboxymethyl)lysine (CML), an oxidative stress marker, in the lens was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the blood glucose levels (p=0.91) and food consumption did not significantly change in any group, the oral administration of Asa tended to suppress CML accumulation (p=0.15) and significantly inhibited the progression of cataractogenesis in the diabetic lens compared with that reported for the normal diet (p=0.009). These results suggested that the daily intake of A. sacrum prevents the pathogenesis of cataracts, and indicated that may reduce the number of DC patients.

Eucommia ulmoides extracts prevent the formation of advanced glycation end products.

Proteins non-enzymatically react with reducing sugars to form advanced glycation end-products (AGEs), resulting in the induction of protein denaturation. Because the levels of AGE increase with age and are elevated in age-related diseases, such as diabetes and atherosclerosis, the intake of compound(s) that inhibit the formation of AGEs by daily meal may represent a potential strategy for preventing age-related diseases. In this study, we measured the inhibitory effects of several Eucommia ulmoides extracts on the formation of AGEs, N(ε)-(carboxymethyl)lysine (CML) and N(ω)-(carboxymethyl)arginine (CMA). Although a crude extract obtained from E. ulmoides bark is widely used as herbal medicine, E. ulmoides leaf extract (ELE) inhibited CML and CMA formation more effectively during the incubation of gelatin with ribose. Therefore, the inhibitory effects of compounds present in ELE on CML and CMA formation were studied. As a result, isoquercetin showed the strongest inhibitory effect of all the tested ELE components. These results indicate that the oral intake of ELE may inhibit the formation of AGEs, thereby ameliorating age-related diseases.

Salacia chinensis L. extract ameliorates abnormal glucose metabolism and improves the bone strength and accumulation of AGEs in type 1 diabetic rats.

Although extracts of the roots and stems of Salacia chinensis have been used in folk medicines for chronic diseases such as rheumatism, irregular menstruation, asthma and diabetes mellitus, little is known about the mechanism by which Salacia chinensis extract (SCE) ameliorates these diseases. To clarify whether SCE ameliorates the progression of lifestyle-related diseases, the inhibitory effect of SCE on the formation of advanced glycation end products (AGEs) was analyzed in a rat model of streptozotocin-induced diabetes. Although the oral administration of SCE did not ameliorate the diabetes-induced decrease in body weight, it ameliorated the increase in glycoalbumin levels in diabetic rats. An analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that the levels of N(ε)-(carboxymethyl)lysine (CML) were highest in the femurs and that they increased by the induction of diabetes. The administration of SCE also ameliorated the decreased femur strength and the accumulation of CML. Furthermore, when all of the carbohydrates in the chow of diabetic rats were replaced with free glucose, the administration of SCE significantly ameliorated a diabetes-induced increase in glycoalbumin and decrease in serum creatinine level and body weight. This study provides evidence to support that SCE ameliorates diabetes-induced abnormalities by improving the uptake of glucose by various organs.

Strong inhibitory effects of common tea catechins and bioflavonoids on the O-methylation of catechol estrogens catalyzed by human liver cytosolic catechol-O-methyltransferase.

In the present investigation, we studied the inhibitory effects of three tea catechins [catechin, epicatechin, and (-)-epigallocatechin-3-O-gallate] and two bioflavonoids (quercetin and fisetin) on the O-methylation of 2- and 4-hydroxyestradiol (2-OH-E(2) and 4-OH-E(2), respectively) by human liver cytosolic catechol-O-methyltransferase (COMT). We found that catechin and epicatechin each inhibited the O-methylation of 2-OH-E(2) and 4-OH-E(2) in a concentration-dependent manner. The IC(50) values for inhibition of 2-OH-E(2) methylation by catechin and epicatechin were 14 to 17 microM and 44 to 65 microM, respectively, and their IC(50) values for inhibition of 4-OH-E(2) methylation were 5 to 7 microM and 10 to 18 microM, respectively. Our data showed that these two catechins had 2- to 6-fold higher inhibition potency for the O-methylation of 4-OH-E(2) than for the O-methylation of 2-OH-E(2). (-)-Epigallocatechin-3-O-gallate was found to have a distinctly high inhibition potency for the O-methylation of 2- and 4-OH-E(2) (IC(50) values of 0.04-0.07 microM and 0.2-0.5 microM, respectively). The crude extracts from green tea and black tea also showed very strong activity in inhibiting human liver COMT-mediated O-methylation of catechol estrogens. We also determined, for comparison, two common bioflavonoids (quercetin and fisetin) for their inhibitory effects on human liver COMT-mediated O-methylation of catechol estrogens. The IC(50) values for quercetin and fisetin were 0.9 to 1.5 microM and 3.3 to 4.5 microM, respectively, for inhibiting the O-methylation of 2-OH-E(2), and 0.5 to 1.2 microM and 2.6 to 4.2 microM, respectively, for inhibiting the O-methylation of 4-OH-E(2). Enzyme kinetic analyses showed that both tea catechins and bioflavonoids inhibited human liver COMT-mediated O-methylation of 4-OH-E(2) (a representative substrate) with a mixed mechanism of inhibition (competitive plus noncompetitive). In summary, the catechol-containing tea catechins and bioflavonoids are strong inhibitors of human liver COMT-mediated O-methylation of catechol estrogens. More studies are warranted to determine the extent of such inhibition in human subjects and the potential biological consequences.