Advanced Glycation End-products Structures Research Articles

Analysis of Crude, Diverse, and Multiple Advanced Glycation End-Product Patterns May Be Important and Beneficial.

Lifestyle-related diseases (LSRDs), such as diabetes mellitus, cardiovascular disease, and nonalcoholic steatohepatitis, are a global crisis. Advanced glycation end-products (AGEs) have been extensively researched because they trigger or promote LSRDs. Recently, techniques such as fluorimetry, immunostaining, Western blotting, slot blotting, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, matrix-assisted laser desorption-mass spectrometry (MALDI-MS), and electrospray ionization-mass spectrometry (ESI-MS) have helped prove the existence of intra/extracellular AGEs and revealed novel AGE structures and their modifications against peptide sequences. Therefore, we propose modifications to the existing categorization of AGEs, which was based on the original compounds identified by researchers in the 20th century. In this investigation, we introduce the (i) crude, (ii) diverse, and (iii) multiple AGE patterns. The crude AGE pattern is based on the fact that one type of saccharide or its metabolites or derivatives can generate various AGEs. Diverse and multiple AGE patterns were introduced based on the possibility of combining various AGE structures and proteins and were proven through mass analysis technologies such as MALDI-MS and ESI-MS. Kampo medicines are typically used to treat LSRDs. Because various compounds are contained in Kampo medicines and metabolized to exert effects on various organs or tissues, they may be suitable against various AGEs.

Toxic advanced glycation end-products (TAGE) are major structures of cytotoxic AGEs derived from glyceraldehyde

The habitual overconsumption of sugars has been implicated in the onset and/or development of lifestyle-related diseases (LSRD). Advanced glycation end-products (AGEs) have recently been proposed as one of the factors contributing to LSRD. Therefore, strategies that reduce AGEs may be used to effectively prevent and treat LSRD. AGE structures vary according to the types of reducing sugars/carbonyl compounds with which they react; therefore, the AGE structure primarily responsible for LSRD remains unknown. Among the various AGEs produced in the human body, glyceraldehyde (GA)-derived AGEs (GA-AGEs) exert strong cytotoxic effects that contribute to the onset/development of LSRD. The dysregulated metabolism of glucose/fructose associated with modern diets promotes the binding of excessive GA generated in cells to intracellular proteins, which results in the production and accumulation of GA-AGEs and, thus, many types of cellular damage. The toxic AGEs (TAGE) theory originated from our findings. The hypothesis proposed herein is that strongly cytotoxic TAGE structures are GA-derived 1,4-dihydropyrazine compounds, which differ from the known structures of GA-AGEs (i.e., GA-derived pyridinium compound, trihydroxy-triosidine, and pyrrolopyridinium lysine dimer derived from GA).

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.

Multistep Ultrahigh Performance Liquid Chromatography/Tandem Mass Spectrometry Analysis for Untargeted Quantification of Glycating Activity and Identification of Most Relevant Glycation Products

The use of advanced glycation end-products (AGEs) as biomarkers for diagnosis and clinical studies is still hampered by insufficient knowledge on clinically relevant structures formed from precursors associated with defined disease states. The present study conducted untargeted analysis of the glycating activity of AGE-precursors by ultrahigh performance liquid chromatography/tandem mass spectrometry multiple reaction monitoring (UHPLC/MSMS-MRM), monitoring the loss of a nonapeptide as the glycation target. Thus, the glycating activities of seven important AGE-precursors were determined (glucose 13% and the reactive carbonyl compounds glucosone 39%, 3-deoxyglucosone 15%, 3-deoxygalactosone 26%, 3,4-dideoxyglucosone-3-ene 79%, methylglyoxal 94%, and glyoxal 97% peptide loss; 12 h/37 °C). Furthermore, UHPLC/MSMS with simultaneous precursor ion scan and information-dependent acquisition of enhanced resolution spectra and subsequent product ion scan was applied for untargeted analysis of the major AGE-structures derived from various AGE-precursors. The 20 most important modifications could be assigned to 8 AGE-structures previously reported in the literature. Seven loosely bound AGEs not yet covered by conventional methods were detected and assigned to hemiaminals. Five AGE structures did not match any known products. The method can be applied to analyze glycating activity and AGE-structures formed from various other precursors under defined reaction conditions, supporting the selection and evaluation of diagnostic AGE-markers for clinical studies.

Heat Treatment Increases the Level of AGEs in Human Blood

92 Levels of plasma pentosidine, which is one of the advanced glycation end-products (AGEs) of the Maillard reaction, are elevated by the pathogenesis of diabetes 1) and end-stage renal failure 2). A recent study suggested that because plasma pentosidine levels in patients with mild renal dysfunction increase before plasma creatinine levels increase, the measurement of plasma pentosidine level can be a useful clinical marker for the early diagnosis of beginning renal failure 3). Several medical laboratories in Japan have measured the levels of blood AGEs, such as Ne-(carboxymethyl)lysine (CML) and pentosidine, by a competitive enzyme-linked immunosorbent assay (ELISA). According to the authors’ protocol, plasma samples are incubated with proteases for anti-AGE antibodies to assess the intramolecular AGE structures, and are incubated again at 100°C for 15 min 3) to inactivate the protease. However, our previous study demonstrated that CML, a major antigenic AGE structure, is generated from the Amadori products by short-term heating in vitro, demonstrating that CML generated from Amadori products are an artifact of immunochemical detection by heating process 4). Furthermore, Miyata et al. 5) reported that substance(s) with a molecular weight of less than 5,000 Da that are abundantly present in uremic plasma exhibit enhanced pentosidine formation during in vitro incubation of uremic plasma at 37°C, thus strongly demonstrating that pentosidine is also generated by incubation in vitro. To confirm this notion, we conducted additional experiments to clarify whether the pentosidine content measured by high-performance liquid chromatography (HPLC) 6) in blood increases using the same procedure as that used by medical laboratories. As a result, the levels of pentosidine in patients with nondiabetic hemodialysis increased 1.2to 3-fold by heating at 100°C for 15 min compared to unheated samples. Moreover, the increased rate of pentosidine levels generated by the heating process was found to differ between patients. Therefore, the heating process as a pretreatment for pentosidine measurement by competitive ELISA appears to compromise the accuracy of the pentosidine concentration present in plasma. Taken together, previous reports 4,5) and the present study indicate that the level of pentosidine in clinical samples is overestimated by using a heating process. Heating enhances both the oxidative cleavage of Amadori products and the production of α-oxoaldehydes such as glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, which were indicated to be important precursors for AGE formation 4). We next measured the inhibitory effects of reduction of Amadori products on CML formation. Because CML is generated from the Amadori compounds by oxidative cleavage, whereas the Amadori compounds reduced into hexitol lysine and glucitol lysine are stable and do not easily generate CML, sodium cyanoborohydride (NaBH3CN) is used for the blood CML analysis using a competitive ELISA 7) to prevent CML formation during the heating process. However, the present study demonstrated that preincubation of glycated bovine serum albumin (glycated BSA), a model Amadori proteins, in the presence of 100 mM sodium borohydride (NaBH4), a stronger reducing agent than NaBH3CN, for 1 h did not inhibit CML formation induced by heating at 100°C for 15 min. From these data, it is likely that the conventional protocol used by medical laboratories which measures plasma pentosidine and CML after pretreatment may be in fact quantifying artifacts of heat treatment. Therefore, the establishment of a more reliable method for the quantification of AGEs in patient blood samples will clarify the physiological significance as well as the clinical usefulness of AGEs. Masako Nakano, Ryoji Nagai

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, Nɛ-(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 Nɛ-(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.

Advanced glycation endproducts in peripheral nerve in type 2 diabetes with neuropathy

Advanced glycation endproducts (AGE) accumulate over proteins as a consequence of diabetic hyperglycemia, and thus contribute to the pathogenesis of diabetic complications. To improve the understanding of the pathology of diabetic neuropathy, AGE accumulation was analyzed in sural and/or femoral nerves obtained under spinal anesthesia from 8 type 2 diabetic patients with both distal symmetrical polyneuropathy and proximal neuropathy. Pronounced AGE immunoreactivity was detected on axons and myelin sheaths in 90% of diabetic peripheral nerves but not in the control specimen. The intensity of axonal AGE immunopositivity significantly correlated with the severity of morphological alterations (p<0.005). AGE localization, demonstrated by immunohistochemical methods, was also present in the endoneurium, perineurium and microvessels. Morphometric analysis of the diabetic peripheral nerve showed perineurial thickening (diabetic vs. control, 15.5+/-4.9 vs. 6.6+/-2.1 microm, p<0.001), narrowing of the microvessel lumina (66.6+/-50.5 vs. 579.5+/-38.4 x10(3) microm(2), p<0.001) and significant reduction in the number of preserved axons (3.6+/-3 vs. 8.9+/-2.3 per 10(5) microm(2) per area, p<0.037). The sera of diabetic patients contained epitope(s) of AGE structure and soluble immune complexes containing AGE moiety. In conclusion, to the best of our knowledge, this is the first study providing evidence for excessive AGE formation on peripheral nerve components, primarily axons, and a significantly higher level of circulating AGE-immune complexes in patients with both distal diabetic polyneuropathy and proximal neuropathy. Humoral immune mechanisms, including the production of anti-AGE autoantibody, may potentially be involved in the development of structural abnormalities described in this report.

In vitro evidence for immune activating effect of specific AGE structures retained in uremia

Advanced glycation end-products (AGEs) have been identified to be accumulated in blood and tissues of patients with end-stage renal disease (ESRD). AGEs have been shown to modulate immune competent cell activities and in this way they may contribute to the progression of atherosclerosis. All studies in this context have been performed, however, with generated mix of glycation compounds, and not with structures similar to those encountered in uremia. In the present study, the immunologic effect of specific AGE compounds, known to be retained in uremia, has been evaluated. Four albumin preparations, modified chemically at lysine or arginine residues, respectively, to contain N-epsilon-carboxymethyllysine (CML albumin), N-epsilon-carboxyethyllysine (CEL albumin), glyoxal-induced imidazolinones (Arg I albumin) or methylglyoxal-induced imidazolinones (Arg II albumin) were applied. Their effect on chemiluminescence production, CD14 expression, and the DNA synthesis of calcitriol-differentiated HL-60 (monocyte/macrophage phenotype) was studied. The phorbol 12-myristate 13-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of CEL albumin (44.1 +/- 18.5 vs. 64.7 +/- 28.1 counts 10(3)/30 min) (P < 0.05), Arg I albumin (46.4 +/- 18.8 vs. 66.1 +/- 32.6 counts 10(3)/30 min) (P < 0.05) and CML albumin (41.9 +/- 25.5 vs. 60.9 +/- 5.5 counts 10(3)/30 min) (P= 0.0625) pointing to an increase in free radical production. The latter AGE compounds also significantly increased the calcitriol-induced CD14 expression on HL-60 cells (1675 +/- 796 vs. 2075 +/- 1044; 768 +/- 143 vs. 890 +/- 150; 647 +/- 63 vs. 716 +/- 69 mean fluorescence intensity) (P < 0.05, respectively) pointing to an increase in expression of the lipopolysaccharide (LPS) receptor. Finally, the DNA synthesis of the calcitriol-differentiated HL-60 cells was enhanced in the presence of Arg I albumin [34.5 +/- 4.6 vs. 27.7 +/- 9.7% 5-bromo-2'-deoxyuridine (BrdU)-positive cells] (P < 0.05) resulting in an increased cell proliferation. Genuine AGE compounds, as they are encountered in the uremic condition, activate leukocyte response, and hence could play a role in uremia related atherogenesis.

Involvement of Advanced Glycation End-products (AGEs) in Alzheimers Disease

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients. It has recently become clear that AGEs also influence physiological aging and neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Recently we have provided direct immunochemical evidence for the existence of six distinct AGE structures within the AGE-modified proteins and peptides that circulate in the serum of diabetic patients on hemodialysis (DM-HD). We showed a direct toxic effect of the synthetic AGE-2 (glyceraldehyde-derived AGEs) on cortical neuronal cells and provided evidence for a toxic effect of AGE-2 present in DM-HD serum. These results indicate that of the various types of AGE structures that can form in vivo, the AGE-2 structure is likely to play an important role in the pathophysiological processes associated with AGE formation. In AD brains, AGE-2 epitope was mainly present in the cytosol of neurons in the hippocampus and para-hippocampal gyrus. Protein cross-linking by AGE structures results in the formation of protease-resistant aggregates. Such protein aggregates may interfere with both axonal transport and intracellular protein traffic in neuron. In this review, we provide an outline of AGEs formation in vivo and propose that the novel structural epitope AGE-2 is an important toxic moiety for neuronal cells in AD.

TAGE (toxic AGEs) hypothesis in various chronic diseases

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients, in aging, and in neurodegenerative diseases. However, it is still not clear which AGEs subtypes play a pathogenetic role and which of several AGEs receptor mediate AGEs effects on cells. We have provided direct immunochemical evidence for the existence of six distinct AGEs structures (AGEs-1 to -6) within the AGEs-modified proteins and peptides that circulate in the serum of diabetic patients. Recently we demonstrated for the first time that glyceraldehyde-derived AGEs (AGEs-2) and glycolaldehyde-derived AGEs (AGE-3) have diverse biological activities on vascular wall cells, mesangial cells, Schwann cells, malignant melanoma cells and cortical neurons. We also demonstrated for the first time that acetaldehyde (AA)-derived AGEs (AA-AGE) have cytotoxic activity on cortical neurons and the AA-AGE epitope was detected in human brain of alcoholics. These results indicate that of the various types of AGEs structures that can form in vivo, the toxic AGEs (TAGE) structures (AGEs 2, 3, and AA-AGE), but not non-toxic AGEs ( N-carboxymethyllysine, pentosidine, pyrraline etc.) are likely to play an important role in the pathophysiological processes associated with AGEs formation.

Alternative routes for the formation of glyceraldehyde-derived AGEs (TAGE) in vivo

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients, and in Alzheimer's disease (AD). Recently we have provided direct immunochemical evidence for the existence of six distinct AGEs structures, designated AGEs-1 to -6, within the AGEs-modified proteins and peptides that circulate in the serum of diabetic patients. We found for the first time that glyceraldehyde-derived AGEs (AGE-2), which comprise main structure of TAGE (toxic AGEs), in the serum of diabetic patients have diverse biological activities on vascular wall cells and cortical neurons. These results suggest a causal role for AGE-2 in the pathogenesis of diabetic complications and AD in vivo. In AD brains, AGE-2 epitope was mainly present in the cytosol of neurons in the hippocampus and para-hipocampal gyrus. We propose three pathways for the in vivo formation of AGE-2 precursor, glyceraldehyde, by: (i) glycolytic pathway, (ii) polyol pathway, and (iii) fructose metabolic pathway. Glyceraldehyde can be transported or can leak passively across the plasma membrane. It can react non-enzymatically with proteins to lead to accelerated formation of AGE-2 at both intracellular and extracellular region.

Application of monoclonal antibody libraries for the measurement of glycation adducts.

Immunological approaches have been used to demonstrate the presence of advanced glycation end-products (AGEs) in several human and experimental animal tissues. We previously prepared polyclonal and monoclonal anti-AGE antibodies by immunizing AGE-modified proteins such as BSA and RNase. Although these antibodies contributed to demonstrate the presence of AGE-modified protein in vivo, the epitope structure of these antibodies had not been identified. We subsequently prepared several antibodies against AGE structures such as pentosidine, pyrraline, 3-deoxyglucosone imidazolone and N epsilon-(carboxyethyl)lysine by immunizing single AGE structures. These structure-specific antibodies have greatly helped broaden our understanding of AGE structures in aging and age-enhanced disease process. Monoclonal anti-AGE antibody is also used for the identification of major AGE structures in some pathological tissues, such as human atherosclerosis lesions. Based on the strategy, we successfully identified a novel AGE structure named glycolaldehyde-pyridine, which is the major antigenic AGE derived from glycolaldehyde. Therefore a monoclonal antibody library for AGE structures has served an important role in the elucidation of the biological significance of AGE.

Preparation of specific antibody against CML, one of major AGE structures

Nε-(Carboxymethyl)lysine (CML) is one of the major structures of advanced glycation endproducts (AGE). Immunological studies using monoclonal (6D12) and polyclonal anti-CML antibody demonstrated CML-accumulation in several pathological tissues such as kidneys of patients with diabetic nephropathy and atherosclerotic lesions of arterial walls. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly by glycolysis and polyol pathways, reacts with proteins to form MG-derived AGE structures such as Nε-(carboxyethyl)lysine (CEL), imidazolone and imidazolium salt cross-link like methylglyoxal-lysine dimmer (MOLD). Our preliminary studies demonstrated that 6D12 and polyclonal anti-CML antibodies significantly reacted not only with CML but also with CEL. In the present study, we determined the possibility that antibody could recognize a difference between CML and CEL. To prepare polyclonal CML-specific antibody (PCMS), rabbits were immunized with CML-keyhole limpet hemocyanin (CML-KLH) and CEL-reactive antibody was removed by a CEL-conjugated affinity chromatography. Similar to PCMS, polyclonal CEL-specific antibody (PCES) was purified by separating CML-reactive antibody from antiserum obtained by immunization with CEL-KLH. Next, the monoclonal antibody specific for CML (CMS-10) was obtained by immunization of CML-KLH, followed by successive screening by CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both PCMS and CMS-10 showed a significant reactivity to CML-proteins but not to CEL-proteins. In sharp contrast, PCES showed a high reactivity toward CEL-protein, but not to CML-protein. It is likely therefore that these antibodies were able to recognize the difference in one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with glucose-modified BSA (AGE-BSA) as well as BSA modified by several aldehydes such as glyoxal and 3-deoxyglucosone, potential intermediates for AGE formation. These results indicate that CMS-10 would help detect CML-modified proteins in vitro in a more specific way.

Conversion of Amadori product of the Maillard reaction to Nε-(carboxymethyl) lysine by short-term heating process

The accumulation of advanced glycation endproducts (AGE) of the Maillard reaction is increased by aging process and pathogenesis of age-enhanced disease such as atherosclerosis and diabetic complications. Especially, immunohistochemical analyses have demonstrated accumulation of AGE in vivo such as diabetic nephropathy, atherosclerosis and Alzheimer's disease. In some issues of these reports, localization of AGE has been determined using formalin-fixed paraffin-embedded tissue sections after heat-induced epitope retrieval treatment. However, we speculated that AGE might be artificially formed from Amadori product, a crucial intermediate for AGE formation, by the heating process. To evaluate this possibility, we compared the reactivity of monoclonal antibody against Nε-(carboxymethyl) lysine (CML), a major AGE structure, to heat-treated and untreated tissue sections. Normal human skin, rat skin and liver samples were divided into two groups, one was rapidly frozen and the other was formalin-fixed paraffin-embedded, followed by heat-induced epitope retrieval treatment. In the heat-treated sections, the nuclei of the epidermis in the skin obtained from human and rat, and cytoplasm of rat liver were strongly stained by anti-CML antibody (6D12) while in the frozen sections, the staining of the same site was negligible. In order to analyze the mechanism, we conducted heat-treatment to glycated-human serum albumin (glycated-HSA), a model Amadori product, and generation of CML was determined by 6D12 and high performance liquid chromatography (HPLC). CML was generated from glycated-HSA by heat-treatment at over 80 °C, and it was increased in time dependent manner. In contrast, the generation of CML from glycated-HSA was significantly inhibited in the presence of NaBH4 (132 mM), diethylenetriamine pentaacetic acid (DTPA) (1 mM) or aminoguanidine (90 mM). Furthermore, reactive intermediates such as glucosone, 3-deoxyglucosone, methylglyoxal and glyoxal were detected by HPLC after heat treatment of glycated-HSA at 100 °C, indicating that these aldehydes generated from Amadori product by oxidative cleavage can contribute to further AGE formation. These results strongly demonstrated that Amadori product in tissues was able to convert into CML by the heating process.

In Vitro Formation of Nϵ-(Carboxymethyl)lysine and Imidazolones under Conditions Similar to Continuous Ambulatory Peritoneal Dialysis

Conventional peritoneal dialysis fluids (PDFs) lead to formation of advanced glycation end-products (AGE) in the peritoneal membrane. In this study, we investigated in vitro the dependence of AGE formation on regular changes of PDFs, as performed during continuous ambulatory peritoneal dialysis (CAPD), and on the contribution of high glucose concentration versus glucose degradation products (GDPs). Under conditions similar to CAPD, protein glycating activity of a conventional single chamber bag PDF (CAPD 4.25%), two double chamber bag PDFs (CAPD Balance 4.25% and CAPD Bicarbonate 4.25%) and a sterile filtered control was measured in vitro by Nϵ-(carboxymethyl)lysine (CML) and imidazolones, two well characterized, physiologically relevant AGE structures. Regular changes of PDFs increased AGE formation (CML 3.3-fold and imidazolone 2.6-fold) compared to incubation without changes. AGE formation by CAPD 4.25% was increased compared to control (imidazolones 7.9-fold and CML 3.3-fold) and the use of double chamber bag PDFs led to a decrease of imidazolones by 79% (CAPD Bicarbonate 4.25%) and by 66% (CAPD Balance 4.25%) and to CML contents similar to the control. These results indicate that a major part of AGEs were formed by GDPs in PDFs, whereas only a minor part was due to high glucose concentration. The use of double chamber bag fluids can reduce AGE formation considerably.

Detection of autoantibodies against advanced glycation endproducts and AGE-immune complexes in serum of patients with diabetes mellitus

Advanced glycation of protein causes their immunogenicity. The evidence that advanced glycation endproducts (AGEs) have antigenic properties has led to a hypothesis that the AGE structure found in vivo may exert an autoimmune response. In the present study, we showed the sera of diabetic patients as well as of nondiabetic individuals to contain autoantibodies to epitopes of AGE structures. Contrary to what might be expected, we observed lower AGE antibody titers in diabetic subjects, and postulated that the antibodies against AGEs form immune complexes in vivo, hampering their determination. The existence of immune complexes containing AGE moiety was established by two independent criteria: (a) serum AGE-immune complexes (AGE-IC) were detected by enzyme-linked immunosorbent assay (ELISA) using an immunochemical bridge; and (b) soluble AGE-IC were precipitated from serum by polyethylene glycol and analyzed. We demonstrated the presence of circulating AGE-IC in sera, predominantly in the sera of diabetic subjects. We also found an inverse correlation between serum AGE level and AGE-IC ( r=−0.8, P<0.000), indicating the serum level of AGEs to decline with an increasing presence of AGE-IC. The content of AGE in soluble immune complexes was significantly higher in diabetic patients than in control subjects (3.51±1.9 vs. 1.89±1.0 μgEq/ml ( P<0.00004), and correlated inversely with free antibodies ( r=−0.26, P<0.01). Interactions of AGE autoantibodies with AGE as a continuously produced antigen result in the formation of AGE-immune complexes that may play a role in the atherogenic processes.

Neurotoxicity of Advanced Glycation End-Products for Cultured Cortical Neurons

The Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in aging. AGEs are believed also to contribute to the pathology of Alzheimer disease (AD) and other neurodegenerative processes. Incubation of cortical neurons with 5 immunochemically distinct AGEs, designated AGEs-1 to -5, produced a dose-dependent increase in neuronal cell-death, as assessed by MTT assay, Trypan blue and Hoechst 33258 staining. The structural epitope designated AGE-2 was found to have the greatest cytopathic effect and the neurotoxicity of AGE-2 was neutralized by the addition of an anti-AGE-2-specific antibody, but not by other types of anti-AGE antibodies. Distinct classes of AGE structures also have been established to circulate in the blood of individuals with diabetes mellitus and end-stage renal disease treated by hemodialysis (DM-HD). We fractionated serum from normal control and DM-HD patients by gel filtration and identified 2 fractions that contained AGE epitopes-1 to -5 and as well as the defined AGE structure carboxymethyllysine (CML). The addition of these 2 fractions led to the death of cultured neuronal cells and this cytotoxic effect was completely prevented by the addition of the anti-AGE-2-specific antibody. We propose that the structural epitope AGE-2 is an important toxic moiety for neuronal cells.

Advanced glycation endproducts are deposited in neuronal hyaline inclusions: a study on familial amyotrophic lateral sclerosis with superoxide dismutase-1 mutation.

To determine the role of advanced glycation endproducts (AGE) in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) with superoxide dismutase-1 (SOD1) mutation, we investigated the immunohistochemical localization of N(epsilon)-carboxymethyl-lysine (CML), one of the major AGE structures, in spinal cords from three familial ALS patients with a heterozygous Ala to Val substitution at codon 4 in the gene for SOD1. Neuronal hyaline inclusions (NHIs), the abnormal structures seen in some of the remaining lower motor neurons of familial ALS patients with SOD1 mutation, were intensely stained by a monoclonal antibody specific for CML in contrast to the only weakly stained cytoplasm. Immunoelectron microscopy depicted the CML determinants restricted to the granule-associated thick linear structures that mainly compose the NHIs. The NHIs were also recognized by antibodies to SOD1, phosphorylated neurofilament protein and ubiquitin. No focal collection of either CML or SOD1 was found in neurons of the control individuals. Our results indicate that CML is a component of the NHIs of familial ALS patients with SOD1 mutation, and suggest that the CML formation may be mediated by protein glycoxidation or lipid peroxidation in the presence of oxidative stress from mutant SOD1, in association with motor neuron degeneration.

Localization of identified advanced glycation end-product structures, N epsilon(carboxymethyl)lysine and pentosidine, in age-related inclusions in human brains.

The recent identification of age-related accumulation of advanced glycation end-products (AGE) of the Maillard reaction in neurons and vessels of the human brain suggests the involvement of AGE in the aging process. A variety of inclusions such as lipofuscin granules, corpora amylacea, Hirano bodies, granulovacuolar degenerations and ubiquitin-positive granular structures are found in the aged human brain. These age-related inclusions contain insoluble and non-degradable proteins. Advanced glycation end-product-modified proteins are also known to be insoluble and protease resistant. The similarity between proteins in such inclusions and AGE-modified proteins suggests the presence of AGE in inclusions. To investigate this possibility, the presence of two known AGE structures, N epsilon(carboxymethyl)lysine (CML) and pentosidine, was examined in age-related inclusions. Immunohistochemical examination of the medial temporal area of brain tissues obtained at autopsy from seven non-demented elderly individuals demonstrated positive reactions in lipofuscin granules and corpora amylacea but not in other inclusions for anti-CML and anti-pentosidine antibodies. As CML and pentosidine are glycoxidation products among AGE, the results suggest that glycation and/or oxidation may be involved in the formation of lipofuscin granules and corpora amylacea.