Glycated Lysine Residues Research Articles

Comprehensive profiling and kinetic studies of glycated lysine residues in human serum albumin

Lysine residues of proteins slowly react with glucose forming Amadori products. In hyperglycemic conditions, such as diabetes mellitus, this non-enzymatic glycation becomes more pervasive causing severe medical complications. The structure and conformation of a protein predisposes lysine sites to differing reactivity influenced by their steric availability and amino acid microenvironment. The goal of our study was to identify these sites in albumin and measure glycation affinities of lysine residues. We applied a bottom-up approach utilizing a combination of three LC-MS instruments: timsTOF, Orbitrap, and QTRAP. To prove applicability to samples of varying glycemic status, we compared in vitro glycated and non-glycated HSA, as well as diabetic and non-diabetic individual samples. The analysis of lysine glycation affinities based on peptide intensities provide a semi-quantitative approach, as the results depend on the mass spectrometry platform used. We found that glycation levels based on multiple reaction monitoring (MRM) quantitation better reflect individual glycemic status and that the glycation percentage for each site is in linear relation to all other sites. To develop an approach which more accurately reflects glycation affinity, we developed a kinetics model which uses results from stable isotope dilution HPLC-MRM methodology. Through glycation of albumin at different glucose concentrations, we determine the rate constants of glycation for every lysine residue by simultaneous comparative analysis.

The role of non-enzymatic glycation on Tau-DNA interactions: Kinetic and mechanistic approaches

Despite the regulatory role of Tau protein in the stabilization and assembly of microtubules, this protein has an important function in the protection and stabilizing of DNA molecules in the cell nucleus. In the present study, it has been indicated that glycation of lysine residues (Lys-267, Lys-274, and Lys-280) in the microtubule-binding domain (MBD) can considerably decrease its binding affinity to DNA molecules. The structural analysis also confirmed that the decreased glycated tau-DNA complex's stability was due to structural modification of this protein after the glycation process. The study of hippocampal cells under hyperglycemic conditions showed that near to 70% of Tau proteins glycated in these cells, although the expression of Tau remained unaffected. The assessment of H3K9me2, as a marker for binding of Tau to pericentromeric heterochromatin (PCH), indicated that localization of Tau protein on PCH was remarkably decreased at high glucose conditions relative to the controls. It is suggested that increasing the structural stability of Tau protein limits the ability of this protein for DNA binding, while the molecular and physical barrier of glycated Lys residues should not be neglected.

Mechanical stretching changes crosslinking and glycation levels in the collagen of mouse tail tendon

Collagen I is a major tendon protein whose polypeptide chains are linked by covalent crosslinks. It is unknown how the crosslinking contributes to the mechanical properties of tendon or whether crosslinking changes in response to stretching or relaxation. Since their discovery, imine bonds within collagen have been recognized as being important in both crosslink formation and collagen structure. They are often described as acidic or thermally labile, but no evidence is available from direct measurements of crosslink levels whether these bonds contribute to the mechanical properties of collagen. Here, we used MS to analyze these imine bonds after reduction with sodium borohydride while under tension and found that their levels are altered in stretched tendon. We studied the changes in crosslink bonding in tail tendon from 11-week-old C57Bl/6 mice at 4% physical strain, at 10% strain, and at breaking point. The crosslinks hydroxy-lysino-norleucine (HLNL), dihydroxy-lysino-norleucine (DHLNL), and lysino-norleucine (LNL) in-creased or decreased depending on the specific crosslink and amount of mechanical strain. We also noted a decrease in glycated lysine residues in collagen, indicating that the imine formed between circulating glucose and lysine is also stress labile. We also carried out mechanical testing, including cyclic testing at 4% strain, stress relaxation tests, and stress-strain profiles taken at breaking point, both with and without sodium borohydride reduction. The results from both the MS studies and mechanical testing provide insights into the chemical changes during tendon stretching and directly link these chemical changes to functional collagen properties.

The inefficacy of donepezil on glycated-AChE inhibition: Binding affinity, complex stability and mechanism

Donepezil (DPZ) is a well-known drug for Alzheimer's disease that inhibits acetylcholinesterase activity (AChE). In the present study, the inhibitory effect of DPZ on non-enzymatic glycated-AChE (GLY-AChE) was studied by different experimental and simulation techniques. The initial investigation revealed that glycation process could reduce AChE activity approximately 60% in the pure enzyme and 38% in the extracted crude AChE from neural cells cultured in the presence of high glucose (HG) concentration. It is suggested that glycation of lysine residues on the structure of AChE could change the conformation of the active site (Trp-86 and His-447) in a way that the orientation of acetylcholine interrupted. The further studies indicated that DPZ is although a strong inhibitor for the native enzyme, it is not able to affect the GLY-AChE activity. The KD values of AChE-DPZ and GLY-AChE-DPZ complexes were estimated to be 1.88 × 10−9 and 2.10 × 10−6, respectively. The stability assessment showed that AChE-DPZ complex is more stable than the glycated complex. Our results indicate that, glycation process could impact on the conformation of the residues involved in the DPZ binding cavity on α-helix domain. Therefore, DPZ is not able to bind its specific cavity to induce its inhibitory effects on GLY-AChE.

The glycation site specificity of human serum transferrin is a determinant for transferrin's functional impairment under elevated glycaemic conditions

The mechanisms involving iron toxicity in diabetes mellitus are not completely understood. However, the spontaneous reaction of reducing sugars with protein amino groups, known as glycation, has been shown to compromise the action of Tf (transferrin), the systemic iron transporter. In order to understand the structural alterations that impair its function, Tf was glycated invitro and the modification sites were determined by MS. Iron binding to glycated Tf was assessed and a computational approach was conducted to study how glycation influences the iron-binding capacity of this protein. Glycated Tf samples were found to bind iron less avidly than non-modified Tf and MS results revealed 12 glycation sites, allowing the establishment of Lys534 and Lys206 as the most vulnerable residues to this modification. Their increased susceptibility to glycation was found to relate to their low side-chain pKa values. Lys534 and Lys206 participate in hydrogen bonding crucial for iron stabilization in the C- and N-lobes of the protein respectively, and their modification is bound to influence iron binding. Furthermore, the orientation of the glucose residues at these sites blocks the entrance to the iron-binding pocket. Molecular dynamics simulations also suggested that additional loss of iron binding capacity may result from the stereochemical effects induced by the glycation of lysine residues that prevent the conformational changes (from open to closed Tf forms) required for metal binding. Altogether, the results indicate that Tf is particularly vulnerable to glycation and that this modification targets spots that are particularly relevant to its function.

Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC-ESI-QqTOF-MS/MS

In this manuscript, we present the determination of glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.

Glycated lysine residues: a marker for non-enzymatic protein glycation in age-related diseases.

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

The structural feature surrounding glycated lysine residues in human hemoglobin

Complications derived from diabetes mellitus are caused by nonenzymatic protein glycation at the specific sites. LC/MS/MS was performed for the identification of the tryptic peptides of glycated hemoglobins using glyceraldehyde. After the identification of the glycation or non-glycation site, computer analysis of the structure surrounding the sites was carried out using PDB data (1BZ0). Five glycated lysine residues (Lys-16(α), -56(α), -8(β), -82(β), and -144(β)) and four non-glycated lysine residues (Lys-7(α), -40(α), -99(α), and -132(β)) were identified. The non-glycated lysine residues, Lys-7(α), -40(α), and -132(β), are most likely to form electrostatic interactions with the β carboxyl group of Asp-74(α), C-terminal His-146(β), and Glu-7(β) by virtue of their proximity, which is 2.67-2.91 Å (N-O). Additionally, there are histidine residues within 4.55-7.38 Å (N-N) around eight sites except for Lys-7(α). We conclude that the following factors seem to be necessary for glycation of lysine residues: (i) the apparent absence of aspartate or glutamate residues to inhibit the glycation reaction by forming an electrostatic interaction, (ii) the presence of histidine residues for acid-base catalysis of the Amadori rearrangement, and (iii) the presence of an amino acid residue capable of stabilizing a phosphate during proton transfer.

Preferential recognition of Amadori-rich lysine residues by serum antibodies in diabetes mellitus: Role of protein glycation in the disease process

This study analyzes effect of glycation on proteins rich in lysine residues as hyperglycemia induced protein glycation has been mainly reported in diabetes mellitus at the intrachain lysine residues leading to the formation of Amadori modified proteins. We have studied the effect of glucose on poly- l-lysine (PLL), a homopolymer of lysine residues. Levels of Amadori products in the glycated PLL were evaluated by fructosamine assay and the presence of 5-hydroxymethylfurfural (HMF) in the glycated PLL was analyzed by thiobarbituric acid assay. Fluorescence and FT-IR spectroscopy were applied to characterize the modified PLL. Binding characteristics of experimentally induced antibodies against glycated PLL and the presence of antibodies against glycated PLL in the sera of diabetes patients was evaluated by solid phase enzyme immunoassays. The fructosamine assay showed significantly high yield of early glycation (Amadori) products in the glycated PLL, which was confirmed by increased yield of HMF from Amadori products of glycated PLL. Loss in fluorescence intensity and appearance of a new band corresponding to Amadori products were observed in FT-IR spectrum of the glycated PLL. Glycated PLL was found to be highly immunogenic in rabbits as compared to the native form. Serum antibodies from diabetes patients showed appreciably high recognition of the glycated PLL. The results conclusively show the glycation induced damage to the lysine molecules and specific recognition of Amadori-lysine residues by serum antibodies from diabetes patients. The glycated lysine residues may serve as a diagnostic biomarker for early glycation process in diabetes mellitus.

Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease.

In healthy individuals fibrinogen occurs in more than one million non-identical forms because of the many possible combinations of biosynthetically or postbiosynthetically modified or genetically polymorphic sites. The various forms may show considerable differences in their functional properties. Normal variant sites are due to alternative splicing, modification of certain amino acid residues, and proteolysis. Both the A alpha and the gamma chain occur in two splice forms, and it is known that only the shorter gamma chain can interact with platelets, but the longer may bind thrombin and factor XIII. Many types of posttranslationally modified amino acid residues are present in fibrinogen. The A alpha chain is partially phosphorylated at two sites, possibly leading to protection against proteolysis. The B beta chain is N-glycosylated and partially proline hydroxylated, each at one site. The gamma chain is N-glycosylated at one site and the longer splice form doubly tyrosine-sulfated. The glycosylations are believed to protect against polymerization and proteolysis. All three chains are partially oxidized at methionine residues and deamidated at asparagine and glutamine residues. The A alpha and gamma chain are partially carboxy-terminally degraded by proteolysis, the shorter forms causing a decrease in polymerization, crosslinking, and clot stability. Abnormal variants occur in patients with diabetes mellitus, in the form of glycated lysine residues; in patients with certain types of cancer, in the form of crosslinked degradation products; in patients with certain types of autoimmune disease, in the form of complexes with antibodies; in cigarette smokers; and in individuals treated with acetylsalicylic acid, in the form of acetylated lysine residues.

Reactivity of lecithin-cholesterol acyl transferase (LCAT) towards glycated high-density lipoproteins (HDL)

Hyperglycaemia in diabetic patients results in non-enzymatic glycation of plasma proteins, including lipoproteins such as high-density lipoproteins (HDL). We studied the effects of in vitro HDL glycation on the activity of lecithin-cholesterol acyl transferase (LCAT), a key enzyme in HDL plasma metabolism. LCAT was prepared from non-diabetic subjects and HDL by sequential density ultracentrifugation (in the density range of 1.063–1.21 g/ml) from both diabetic and non-diabetic patients. HDL from non-diabetic patients were glycated in vitro by incubating lipoproteins with 100 mmol/l glucose for various times at 37°C with sodium cyanoborohydride as reducing agent. Glycation of HDL protein was quantified by measuring the percentage of derived amino acid residues using the TNBS assay. Kinetic parameters of LCAT were first determined using native HDL from non-diabetic patients and in vitro glycated HDL. With native HDL, K m and V max were 51.1 ± 4.2 μmol/l ( n = 8) and 12.9 ± 2.4 nmol/ml/h ( n = 8), respectively. Enzyme reactivity, calculated as the V max/ K m ratio, was 0.25 ± 0.04 h −1 ( n = 8). In the case of moderate glycation (derived residues < 30%; n = 19) a significant increase in both K m (18.2 ± 3.4%; mean ± S.D.) and V max (9.3 ± 2.4%) was observed. In contrast, with a high level of glycation (derived residues > 30%; n = 8), both parameters fell ( K m, 25 ± 6.3%; V max, 34.1 ± 3.3%). In addition, whatever the level of glycation, enzyme reactivity was lower in the presence of in vitro glycated HDL. This decrease in LCAT reactivity was not due to a peroxidative process nor to an alteration of the protein and lipid composition of in vitro glycated HDL. It could, however, be explained by glycation of lysine residues in apolipoprotein A-I, which is the most potent activator of LCAT. In a second series of experiments, native diabetic HDL preparations were used as LCAT substrate. No alteration in K m values was observed, but there was a significant decrease in both V max (28%) and enzyme reactivity (32%). This difference in K m and V max alterations between native diabetic HDL and in vitro glycated HDL with low levels of glycation might be explained by the impact of physiological modifications, other than glycation, which could differently affect the chemicophysical properties of HDL in diabetic patients. In conclusion, the decrease in LCAT reactivity observed with both native diabetic HDL and in vitro glycated HDL could affect the reverse cholesterol transport of HDL and thereby contribute to the atherosclerotic process in diabetic patients.

Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Pathophysiological concentrations of glucose promote oxidative modification of low density lipoprotein by a superoxide-dependent pathway.

Oxidized lipoproteins may be important in the pathogenesis of atherosclerosis. Because diabetic subjects are particularly prone to vascular disease, and glucose autoxidation and protein glycation generate reactive oxygen species, we explored the role of glucose in lipoprotein oxidation. Glucose enhanced low density lipoprotein (LDL) oxidation at concentrations seen in the diabetic state. Conjugated dienes, thiobarbituric acid reactive substances, electrophoretic mobility, and degradation by macrophages were increased when LDL was modified in the presence of glucose. In contrast, free lysine groups and fibroblast degradation were reduced. Although loss of reactive lysine groups could be due to either oxidative modification or nonenzymatic glycation of apolipoprotein B-100, inhibition of lipid peroxidation by the metal chelator, diethylenetriamine pentaacetic acid, blocked the changes in free lysines. Thus, glycation of lysine residues is unlikely to account for the alterations in macrophage and fibroblast uptake of LDL modified in the presence of glucose. Glucose-mediated enhancement of LDL oxidation was partially blocked by superoxide dismutase and nearly completely inhibited by butylated hydroxytoluene. These findings indicate that glucose enhances LDL lipid peroxidation by an oxidative pathway involving superoxide and raise the possibility that the chronic hyperglycemia of diabetes accelerates lipoprotein oxidation, thereby promoting diabetic vascular disease.

Mechanism of fructosamine assay: evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction

We studied the chemistry of the fructosamine assay for glycated serum proteins by using the model Amadori compound N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Free lysine was formed at approximately 70% yield during a standard 20-min incubation of fFL with alkaline nitroblue tetrazolium (NBT) at 37 degrees C. Although superoxide dismutase (SOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) decreased the yield of the product, monoformazan dye (MF+), the yield of MF+ was slightly greater under anaerobic than aerobic conditions, excluding a role for superoxide as an intermediate in the reduction of NBT during the fructosamine assay. SOD added to diabetic patients' sera at physiological concentrations also caused a significant (approximately 50%) inhibition of MF+ formation. This inhibition was reduced by addition of nonionic detergents, which contain organic peroxide inhibitors of SOD, to the fructosamine reagent. Overall, these data indicate that the Amadori compound is the direct reductant of NBT in the fructosamine assay and that superoxide is not an intermediate in the reaction. The inhibitory effects of SOD and catalase are most likely the result of oxygen regeneration in the assay mixture.

Studies on the solubilization of the water-insoluble fraction from human lens and cataract

Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and + 1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 m m sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agent, however, disulfide-linked and lysine modified crystallins were best solubilized with urea.

Oxidative degradation of glucose adducts to protein. Formation of 3-(N epsilon-lysino)-lactic acid from model compounds and glycated proteins.

The chemistry of Maillard or browning reactions of glycated proteins is being studied in model systems in vitro in order to characterize potential reaction pathways and products in biological systems. In previous work with the Amadori rearrangement product N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in proteins, we showed that fFL was oxidatively cleaved between C-2 and C-3 of the carbohydrate chain to yield N epsilon-carboxymethyllysine (CML) and D-erythronic acid. We then detected CML in proteins glycated in vitro, as well as in human lens proteins and collagen in vivo (Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894). This work provided an explanation for the origin of CML in human urine and evidence for non-browning pathways of the Maillard reaction in vivo. In this report we describe the identification of a second set of products resulting from oxidative cleavage of fFL between C-3 and C-4 of the sugar chain, i.e. 3-(N epsilon-lysino)-lactic acid (LL) and D-glyceric acid. The formation of LL from fFL was increased at slightly acid pH, representing about 30% of the yield of CML at pH 6.4, compared with 4% at pH 7.4 in phosphate buffer. By gas chromatography-mass spectroscopy, LL was detected in proteins glycated in vitro and then identified as a natural product in human lens proteins and urine. Our results indicate that oxidative degradation of Amadori adducts to proteins occurs in vivo, leading to formation and excretion of CML and LL. These non-browning pathways for reaction of Amadori compounds may be physiologically relevant mechanisms for averting potentially damaging consequences of the Maillard reaction.

Accelerated glycation of the aorta in diabetic rats

Glycation of the aorta in rats with streptozotocin-induced diabetes was estimated by determining the early-stage product and the advanced product of the Maillard reaction. The early-stage product of the Maillard reaction was determined using furosine, which is derived from glycated lysine residues by acid hydrolysis. The advanced product was determined by fluorescence high-performance liquid chromatography. The levels of both early-stage and advanced products in diabetic rats were significantly higher than those in non-diabetic rats at the age of both 20 and 50 weeks. The levels of both early-stage and advanced products at 50 weeks in rats tended to be higher than those at 20 weeks. However, the level of glycated hemoglobin in both non-diabetic and diabetic rats showed no significant change between 20 and 50 weeks of age. These results suggest that tissue glycation may be involved in the development of diabetic complications and may be related to the aging mechanism.

Age-related acceleration of glycation of tissue proteins in rats.

The Maillard reaction on proteins is a nonenzymatic glycosylation that is now termed glycation and produces a ketoamine linkage on the protein. The extent of this glycation was estimated by determining furosine (epsilon-N-(2-furoylmethyl)-L-lysine), which is derived from glycated lysine residues. Glycation of rat sciatic nerve and aorta increased with advancing age for up to 50 weeks and the high degree of glycation was maintained thereafter. The level of hemoglobin did not change significantly after 14 weeks. These results suggest that tissue glycation may increase with aging and may be involved in the mechanism of aging.

Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein.

The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.