Carboxymethyllysine Content Research Articles

Glycolaldehyde Induces Oxidative Stress in the Heart: A Clue to Diabetic Cardiomyopathy?

Cardiovascular complications account for 80% of the mortality related to diabetes mellitus. Hyperglycemia is believed to be the major culprit of angiopathy and cardiomyopathy. High glucose levels and oxidative stress cause elevation of Advanced Glycation End-products that are known to contribute to diabetic complications and correlate with many diseases. However, there are few reports describing the effects of glycating agents other than glucose. Here, we aimed to evaluate the effects of glycolaldehyde (GA) on oxidative stress parameters in the heart of Wistar rats. Male Wistar rats received a single injection of GA (10, 50 or 100mg/Kg) and were sacrificed 6, 12 or 24h after injection. As indexes of oxidative stress, we quantified protein carbonylation, lipid peroxidation and total reduced thiols. The activities of superoxide dismutase, catalase and glyoxalase I were assayed. Also, the content of N (ɛ)-(carboxymethyl)lysine (CML) was quantified. Glycolaldehyde induced an imbalance in the redox status, with increased protein carbonylation and lipoperoxidation. Catalase and glyoxalase I had a decrease in their activities. Despite the oxidative stress, we observed no increase in CML content. These results suggest that short-chain aldehydes such as GA might have a significant role in the development of diabetic cardiomyopathy.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy.

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy.

Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats

Advanced glycation end products (AGEs) play a key role in the pathogenesis of diabetes and its complications, including the diabetic nephropathy. The renoprotective effects of exercise are well known; however, the mechanisms remain elusive. Here we examined whether a regular moderate exercise in obese Zucker rats (OZR), a model of diabetes- and obesity-associated nephropathy, will affect the development of early renal injury in OZR possibly via alteration of AGEs formation. The OZR were left without exercise (sedentary) or subjected to 10 weeks intermittent treadmill running of moderate intensity. Compared with sedentary OZR, kidneys of running OZR had significantly less glomerular mesangial expansion and tubulointerstitial fibrosis. Running OZR had significantly lower plasma AGEs-associated fluorescence and N ɛ -carboxymethyllysine. Correspondingly, renal AGEs and N ɛ -carboxymethyllysine content were lower in running OZR. Systemically, exercise increased aerobic metabolism, as apparent from urinary metabolite profiling. No differences in plasma glucose, insulin, or lipid profile were found between the 2 groups. Apart from lower advanced oxidation protein products (a marker of myeloperoxidase activity), no other marker of inflammation was altered by exercise, either systemically or locally in kidneys. No indication of changed oxidative status was revealed between the groups. Exercise in OZR decreased advanced glycation. This might represent the early event of exercise-induced renoprotection in diabetic nephropathy in OZR. If confirmed in clinical studies, regular moderate exercise could represent an easy and effective nonpharmacologic approach to reduce advanced glycation.

Plasma Concentration and Urinary Excretion of Nɛ‐(Carboxymethyl)lysine in Breast Milk– and Formula‐fed Infants

Industrial processing of infant formulas (IFs) induces the formation of Maillard products, namely N epsilon-(carboxymethyl)lysine (CML). CML content is expected to be several times higher in IFs than in fresh human breast milk. To elucidate whether CML is absorbed from IFs into the bloodstream, CML concentration in the plasma and urine were analyzed in 6-month-old infants (34 breast fed and 25 fed exclusively with IFs) and in 56 samples of human breast milk and 16 commercial IFs. We found that IFs contain higher amounts of CML compared to mother's milk (median: 70-fold; range: 28- to 389-fold), and CML content was higher in hydrolyzed IFs than in nonhydrolyzed IFs (P < 0.03). Plasma CML levels were 46% higher (P < 0.01) and urinary excretion of CML was 60-fold higher (P < 0.001) in the formula-fed infants than in the breast-fed group. Infants fed with hydrolyzed IFs displayed significantly higher plasma CML levels than those on nonhydrolyzed formulations. We conclude that CML from IFs is absorbed into the circulatory system and is rapidly excreted in the urine.

Advanced glycation endproducts: A biomarker for age as an outcome predictor after cardiac surgery?

Objective. A decline in the function of all organs can be detected during ageing. Although the trend appears to be stable, deviation within the elderly population is much greater in comparison to young controls. The aim of the study was to identify a marker of senescence which correlates to heart function. Advanced glycation endproducts (AGEs) accumulate with age and are associated with degenerative diseases. Methods. Carboxymethyllysine (CML) concentrations in the pericardial fluid (as a measure of AGEs) were analysed with ELISA technique in 75 patients undergoing cardiac surgery and correlated with clinical parameters and outcome of these patients. Results. CML content of pericardial fluid increases significantly with age. AGEs show an inverse correlation to left ventricular ejection fraction. High CML levels correlate with poor outcome of patients as shown by adverse cardiac events, prolonged ventilation time and prolonged stay within the Intensive Care Unit. Within all parameters, AGE concentration of the pericardial fluid fits better with the outcome of the patients in comparison to age alone. Interestingly, medical treatment with nitrates correlates with increased CML content. Conclusion. AGEs, in addition to being a marker of senescence, appear to represent a prognostic factor in cardiac surgery, which can be used as a predictor of patient outcome.

Effect of Glucose Concentration on Formation of AGEs in Erythrocytes in Vitro

Posttranslational modifications, such as advanced glycoxidation and lipoxidation end products (AGE/ALEs), are implicated in the pathogenesis of diabetic complications and atherosclerosis. Recent studies have demonstrated that AGE/ALEs are generated not only in extracellular matrix proteins, but also in intracellular proteins from metabolic intermediates. In this study we investigate the effect of glucose concentration on the formation of the AGE/ALEs, Nepsilon-(carboxymethyl)lysine (CML), Nepsilon-(carboxyethyl)lysine (CEL), S-(carboxymethyl)cysteine (CMC), and S-(2-succinyl)cysteine (2SC) in erythrocytes as a function of glucose concentration. Human erythrocytes (10% hematocrit) were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 5 mM or 30 mM glucose for 5 days at 37 degrees C. Globin was recovered by precipitation with 0.25 M HCl in acetone. Following acid hydrolysis, amino acids were converted to their trifluoroacetyl methyl ester derivatives and analyzed by GC/MS/MS. The CML and CEL content of globin increased in a time- and glucose-dependent manner and also increased 1.3- and 1.8-fold, respectively, in incubations containing 30 mM glucose; whereas CMC and 2SC content did not change during the five-day incubations. Furthermore, CEL content of globin in erythrocytes incubated with 30 mM was the highest in the other AGEs, indicating that methylglyoxal may play a major role in AGE formation in erythrocytes. The erythrocyte system should be useful for cellular screening of the efficacy of inhibitors of AGE/ALE formation.

Conventional Antibody against N�-(Carboxymethyl)Lysine (CML) Shows Cross-Reaction to N�-(Carboxyethyl)Lysine (CEL): Immunochemical Quantification of CML with a Specific Antibody

Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.

Reversal by aminoguanidine of the age-related increase in glycoxidation and lipoxidation in the cardiovascular system of Fischer 344 rats

Non-enzymatic glycoxydation and lipoxidation of proteins continues to stimulate great interest in gerontology as both markers and promoters of aging. The first aim of the study was to determine the age-related changes in levels of N ɛ-(carboxymethyl)lysine (CML) and 4-hydroxy-2-nonenal (HNE) present on proteins of the cardiovascular system of Fischer 344 rats and identify the particular polypeptides being modified. The second objective was to evaluate whether pharmacological administration of aminoguanidine (1 g/L in the drinking water) could reverse protein glycoxidation and lipoxidation. CML content in serum, aorta, and heart proteins from 28-month-old rats was double of that found in 4-month-old animals. AG administration to old rats for 3 months from the age of 25 months lowered CML content by 15 ( P = .2275), 44 ( P < .0001), and 28% ( P = .0072) in serum, aorta, and heart, respectively. Serum albumin, transferrin and immunoglobulins were most prominently adducted by both CML and HNE. While the extent of albumin and transferrin modification was comparable between age groups, CML and HNE bound to immunoglobulins increased in the sera of old rats as a result of the accumulation of immunoglobulin heavy and light chains. AG treatment prevented immunoglobulin accumulation in serum, suggesting a beneficial action on renal filtration. Lipoxidation of heart mitochondrial proteins was prevalent over glycoxidation, either as CML or pentosidine. Although AG prevented HNE-induced inactivation of the α-ketoglutarate dehydrogenase complex in vitro, it had no effect in rat hearts, suggesting AG could not reach the mitochondrial matrix.

O0102 PROTEIN HYDROLYSIS OF INFANT FORMULAS STRONGLY ACTIVATES THE MAILLARD REACTION

Introduction: The particular composition of infant formulas favours the development of the Maillard reaction during processing, inducing nutritional and health hazard effects such as loss of available lysine and promotion of the inflammatory response. Lactose, the main substrate of the reaction in milk, is present at high concentrations. Ascorbate and polyunsaturated fatty acids are oxidable nutrients which give rise upon heat treatment to many other reactive dicarbonyle substrates. These substrates attack the free amino groups of proteins, mainly the e-NH2 group of lysine in milk proteins. Hydrolysis of the proteins induces a tremendous increase of the reactive amino groups, via the terminal a-NH2 of peptides or free amino-acids. The aim of this study is to evaluate the effect of protein hydrolysis on the Maillard reaction in the formula. Four indicators were selected to estimate the nutritional quality and safety of infant formulas. Methods: Hydrolyzed and non hydrolyzed formulas processed as powders or sterilized liquids were purchased from the market (Germany and France). Furosine and free hydroxymethylfurfural were quantified by HPLC-UV; carboxymethyllysine (CML) was estimated by ELISA and fluorescent Maillard products (FMP) were measured directly on the powders or liquid samples by front-face fluorescence. These indicators were also measured on samples stored at 60°C for 4–7 days, to mimic long storage. Results: Hydrolysis of the proteins strongly (2 to 3 times) increased the furosine and CML contents of the formulas. Similarly, FMP were significantly higher indicating that protein hydrolysis is an important parameter for the rate of development of the Maillard reaction. The type of process also had a significant effect, as powders were less altered than sterilized liquid formulas. However, accelerated aging had a much stronger effect on powders, especially when the water content was higher than 6 %. Conclusion: Protein hydrolysis is proposed to decrease the allergenicity to milk proteins. However, the liberation of free reactive amino-groups strongly increases the Maillard reaction with possible nutritional and safety consequences. Milder heat treatments for drying and sterilizing should limit the extent of the Mail-lard reaction.

Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose.

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.

Acute hyperglycemia causes intracellular formation of CML and activation of ras, p42/44 MAPK, and nuclear factor kappaB in PBMCs.

Twenty-three nondiabetic volunteers were divided into three groups. In group A (n = 9), the glucose infusion was adjusted to maintain blood glucose at 5 mmol/l (euglycemic clamp). In group B (n = 9), the glucose infusion was adjusted to maintain blood glucose at 10 mmol/l (hyperglycemic clamp) over 2 h. Group C consisted of five volunteers who were studied as the control group. Peripheral blood mononuclear cells (PBMCs) were isolated before and at the end of a 2-h clamp. In group C, PBMCs were isolated before and after 2 h without performing a clamp. The euglycemic clamp as well as "no clamp" had no effects on all parameters studied. In contrast, a significant increase in carboxymethyllysine (CML) content and p21(ras) and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation was observed at the end of a 2-h hyperglycemic clamp. The nuclear factor (NF)-kappaB (but not Oct-1) binding activity increased significantly in the hyperglycemic clamp. Western blots confirmed NF-kappaB-p65-antigen translocation into the nucleus. IkappaBalpha did not change significantly in both groups. Hyperglycemia-mediated NF-kappaB activation and increase of CML content, p21(ras), and p42/44 MAPK phosphorylation was also seen in ex vivo-isolated PBMCs stimulated with 5 or 10 mmol/l glucose. Addition of insulin did not influence the results. Inhibition of activation of ras, MAPK, or protein kinase C blocked hyperglycemia-mediated NF-kappaB activation in ex vivo-isolated PBMCs stimulated with 10 mmol/l glucose. Similar data were obtained using an NF-kappaB-luciferase reporter plasmid. Therefore, we can conclude that an acute hyperglycemia-mediated mononuclear cell activation is dependent on activation of ras, p42/p44 MAPK phosphorylation, and subsequent NF-kappaB activation and results in transcriptional activity in PBMCs.

Advanced Glycation End Products and Nutrition.

Advanced glycation end products (AGEs) may play an important adverse role in process of atherosclerosis, diabetes, aging and chronic renal failure. Levels of Ne-carboxymethyllysine and fluorescent AGE values were estimated in two nutritional population groups - alternative group (vegetarians - plant food, milk products, eggs) and traditional group (omnivorous subjects). Vegetarians have a significantly higher carboxymethyllysine content in plasma and fluorescent AGE values. Intake of proteins, lysine and monosaccharides as well as culinary treatment, consumption of food AGEs (mainly from technologically processed products) and the routes of Maillard reaction in organism are the substantial sources of plasma AGEs. Vegetarians consume less proteins and saccharides. Lysine intake is significantly reduced (low content in plant proteins). Subjects on alternative nutrition do not use high temperature for culinary treatment and consume low amount of technologically processed food. Fructation induced AGE fluorescence is greater as compared with that induced by glucose. It is due to higher participation of a more reactive acyclic form of fructose. Intake of vegetables and fruit with predominance of fructose is significantly higher in vegetarians. Comparison of nutrition and plasma AGEs in vegetarian and omnivorous groups shows that the higher intake of fructose in alternative nutrition of healthy subjects may cause an increase of AGE levels.

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.

Carboxymethyllysine in Dermal Tissues of Diabetic and Nondiabetic Patients with Chronic Renal Failure: Relevance to Glycoxidation Damage

Carboxymethyllysine (CML) is currently recognized as a major advanced glycation end product and a marker for glycoxidation. Plasma CML levels are increased in patients with chronic renal failure (CRF). However, significance and mechanism of CML accumulation in these patients are poorly understood. The objective of the present study was to analyze CML in soluble and collagen-binding fractions of the dermis to investigate CML deposition and formation and collagen damage related to CML accumulation in patients with CRF. Skin samples (among them autopsy samples) were obtained from 33 subjects: 8 nondiabetic CRF patients, 7 diabetic predialysis patients with CRF (CRF-DM), 7 hemodialysis patients, and 11 control subjects without either CRF or DM. The dermal samples were extracted sequentially by phosphate-buffered normal saline, pepsin, and collagenase. The extracts were referred to as the soluble fraction and the proteinase-extracted fraction (including pepsin-extracted and collagenase-extracted fractions). Our ELISA assay for CML in dermal collagen from predialysis patients with CRF (CRF and CRF-DM groups) demonstrated that the levels of CML in both the soluble fraction (containing soluble CML which was mainly determined by serum clearance) and the structural collagen-binding proteinase-extracted fraction (in which high CML levels could be a strong indication of in situ formation) were increased and could not be completely reduced after hemodialysis in CRF-DM and CRF groups. These results suggest that accumulation of CML may be due to both a low serum clearance and/or increased in situ CML formation in CRF. CML contents in the proteinase extracted fraction inversely correlated with the susceptibility of collagen to extraction by proteinases (n = 33, r = –0.59, p < 0.001). Our results provide the first biochemical evidence that CML level is increased in both the soluble and collagen-binding fractions and that increased CML level resulted in increased fractions of proteinase-resistant collagen in dermal extracts of patients with CRF.

Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor.

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs) that are recognized by AGE receptors. Glyoxal, glycolaldehyde (GA), and methylglyoxal are potential intermediates for the formation of AGE structures such as Nomega-(carboxymethyl)lysine (CML). We evaluated the contribution of these aldehydes to the formation of AGE structure(s), particularly the structure important for the receptor-mediated endocytic uptake of AGE proteins by macrophages. GA-modified bovine serum albumin (BSA), methylglyoxal-modified BSA (MG-BSA), and glyoxal-modified BSA (GO-BSA) were prepared, and their physicochemical, immunological, and biologic properties were compared with those of glucose-derived AGE-BSA. CML contents were high in GO-BSA and low in GA-modified BSA (GA-BSA) but did not exist in MG-BSA. The fluorescence patterns of GA-BSA and MG-BSA were similar to those of glucose-derived AGE-BSA but were weak in GO-BSA. Immunochemically, the antibody against non-CML structures of glucose-derived AGE-BSA reacted strongly with GA-BSA and weakly with GO-BSA but did not react with MG-BSA. The negative charge of these ligands increased to a similar extent. However, GA-BSA, but not MG-BSA or GO-BSA, underwent receptor-mediated endocytosis by the macrophage-derived cell line RAW 264.7, which was effectively inhibited by glucose-derived AGE-BSA, acetylated LDL, and oxidized LDL, which are well-known ligands for the macrophage type I and type II class A scavenger receptors (MSR-A). The endocytic uptake of GA-BSA by mouse peritoneal macrophages was also significant, but that by peritoneal macrophages from MSR-A-deficient mice was markedly reduced. Our results suggest that GA serves as an important intermediate for the generation of AGE structure(s) responsible for recognition by MSR-A.

Glycoxidation in aortic collagen from STZ-induced diabetic rats and its relevance to vascular damage

Glycoxidation reactions lead to the formation of permanent, irreversible chemical modifications and cross-links in protein, such as the glycoxidation products carboxymethyllysine (CML) and pentosidine. It has been implicated that CML as well as Amadori products play a role in the formation of superoxidative products, such as H 2O 2 and advanced glycosylation endproducts in trapping LDL. Therefore, a possible relationship between glycoxidation and lipoperoxidation might exist because oxidized lipoprotein, which has been directly linked to atheroma formation, could be produced by the superoxidative products released from the pathway of CML formation. Using a CML-specific monoclonal antibody (6D12) and a specific antiserum against hexitol-lysine (HL), an Amadori product, we studied the relationship between glycoxidation and lipoperoxidation by determining the aortic CML contents with ELISA and the fluorescence levels of lipoperoxidation side products, malondialdehyde (MDA) and hydroxynonenal (HNE) from STZ-induced diabetic rats and age-matched control rats. The immunohistochemical and ultrastructural changes relevant to glycoxidation and lipoperoxidation were also studied. The CML content measured by ELISA in DM rats was significantly higher than that in the control rats at 28 weeks ( n=11, P<0.01). The levels of MDA-linked and HNE-linked fluorescence in the DM rats increased in a similar way and were significantly higher than the levels in control rats at 28 weeks ( n=11, both P<0.01 at 28 weeks). The CML contents correlated with the fluorescence levels of both MDA-linked ( n=19, r=0.638, P<0.01) and HNE-linked fluorescence ( n=19, r=0.629, P<0.01) only in the DM rats, but not in the control rats. Our immunohistochemical study thus demonstrated that CML was initially formed in the aortic media of diabetic rats in the 16th week of diabetes, localized primarily in the extracellular matrix surrounding the aortic smooth muscle cells after HL occurred early in the 2nd week of diabetes. Consequently, a significant increase in the extracellular matrix and decrease in the area of the SMCs were observed in the aortic media in the DM rats by a morphometrical study. The in vivo results of this study provided the first evidence that CML correlated with fluorescence levels of MDA and HNE, and thus suggested the existence of a close relationship between glycoxidation and lipoperoxidation in vivo. This information is thus considered to shed some new light on the etiology of atherogenesis in diabetes.

Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging.

N(epsilon)-(Carboxymethyl)lysine (CML), a major product of oxidative modification of glycated proteins, has been suggested to represent a general marker of oxidative stress and long-term damage to proteins in aging, atherosclerosis, and diabetes. To investigate the occurrence and distribution of CML in humans an antiserum specifically recognizing protein-bound CML was generated. The oxidative formation of CML from glycated proteins was reduced by lipoic acid, aminoguanidine, superoxide dismutase, catalase, and particularly vitamin E and desferrioxamine. Immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries provided evidence for an age-dependent increase in CML accumulation in distinct locations, and acceleration of this process in diabetes. Intense staining of the arterial wall and particularly the elastic membrane was found. High levels of CML modification were observed within atherosclerotic plaques and in foam cells. The preferential location of CML immunoreactivity in lesions may indicate the contribution of glycoxidation to the processes occurring in diabetes and aging. Additionally, we found increased CML content in serum proteins in diabetic patients. The strong dependence of CML formation on oxidative conditions together with the increased occurrence of CML in diabetic serum and tissue proteins suggest a role for CML as endogenous biomarker for oxidative damage.

The Advanced Glycation End Product, N∊-(Carboxymethyl)lysine, Is a Product of both Lipid Peroxidation and Glycoxidation Reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

N (epsilon)-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction.

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGE). Recent immunological studies have suggested the potential role of AGE in atherosclerosis, aging, and diabetic complications. We previously prepared a monoclonal (6D12) as well as a polyclonal anti-AGE antibody and proposed the presence of a common AGE structure(s) that may act as a major immunochemical epitope [Horiuchi, S., Araki, N., & Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332]. The purpose of the present study was to determine the major epitope. Amino acid analysis of AGE-proteins indicated that N(epsilon)-(carboxymethyl)lysine (CML) was a major modified lysine residue. Immunologic studies demonstrated the positive reaction of 6D12 not only to all CML-modified proteins tested, but also to BSA modified with several aldehydes known to generate a CML-protein adduct, and a linear correlation between the CML contents of CML-BSA and their immunoreactivity to 6D12 up to approximately 8 mol/mol of protein. Further experiments with CML analogs revealed that the epitope of 6D12 is a CML-protein adduct with an important carbonyl group. In contrast to 6D12, our polyclonal anti-AGE antibody showed a significant but much weaker immunoreactivity to CML-BSA, suggesting that the polyclonal antibody contains two populations, one reactive to CML (CML-PA) and the other unreactive to CML (Non-CML-PA). Non-CML-PA separated from CML-PA by CML-BSA affinity chromatography did not react with all CML-modified preparations, but retained its property to react commonly with AGE preparations obtained from proteins, lysine derivatives, and monoaminocarboxylic acids. Therefore, it is clear that a CML-protein adduct is a major immunological epitope in AGE structures, but there still exist other major epitope(s) expressed commonly in AGE-proteins.

Determination of N ε -carboxymethyllysine by a reversed-phase high-performance liquid chromatography method

To determine Nε -carboxymethyllysine (CML) in foods a RP-HPLC method after derivatisation with o-phthaldialdehyde was developed. To prevent an overestimation of the CML values by the formation of CML from Amadori products during hydrolysis a borohydride reduction precedes the hydrolysis. A comparison of the determination with and without reduction shows that during hydrolysis 2–12 times more CML than originally present can be formed. With the analytical conditions described in this paper it is possible to obtain measurable amounts of this trace substance in spite of the much higher values for other amino acids. The CML contents in selected processed food items varied between 11 mg in a preparation from mixed cereals for infants to 408 mg/kg protein in a processed malt product. CML is suitable as indicator of heat damage in processed or stored foods, being more stable than the Amadori compounds determined, e.g. in form of furosine.