Oxidative Glycation and Free Radical Production: A Causal Mechanism of Diabetic Complications

Glucose may oxidise under physiological conditions and lead to the production of protein reactive ketoaldehydes, hydrogen peroxide and highly reactive oxidants. Glucose is thus able to modify proteins by the attachment of its oxidation derived aldehydes, leading to the development of novel protein fluoro-phores, as well as fragment protein via free radical mechanisms.

The fragmentation of protein by glucose is inhibitable by metal chelators such as diethylenetriamine pentaacetic acid (DETAPAC) and free radical scavengers such as benzoic acid, and sorbitol. The enzymic antioxidant, catalase, also inhibits protein fragmentation.

Protein glycation and protein oxidation are inextricably linked. Indeed, using boronate affinity chromatography to separate glycated from non-glycated material, we demonstrate that proteins which arc glycated exhibit an enhanced tryptophan oxidation. Our observation that both glycation and oxidation occur simultaneously further supports the hypothesis that tissue damage associated with diabetes and ageing has an oxidative origin.     

The assessment of protein glycation in human atherosclerotic plaques by affinity chromatography

SUMMARY

Human atherosclerotic plaques are characterized by a massive deposition of lipid within arterial walls. The lipids accumulated are partly oxidized, as assessed by gas chromatography of lipids and their oxidation products. Both advancing age and diabetes mellitus are associated with an increased prevalence and severity of atherosclerosis.

In diabetes mellitus the development of secondary complications appear to be increased by poor glucose control. Indeed, the post-translational modification of protein by non-enzymatic glycation may provide the link between abnormal glucose control and diabetic complications. For atherosclerosis however, the relationship between glucose control and disease is unclear, with evidence available to support and discount such a link. To study protein glycation in a condition associated with a significant level of lipid oxidation products poses several methodological problems, most of which are associated with interference by lipid-derived aldehydes. Many chemical assays of protein glycation monitor aldehydic products common to the chemistry of both protein glycation and lipid oxidation. Studies of protein glycation in human atheroma, obtained at necropsy, are presented which make use of a commercially available boronic acid affinity-based chromatographic assay of glycated protein. The commercially available affinity-based chromatographic assay of glycated protein appears to be free from such interference and may well prove useful in the study of other conditions in which the non-enzymatic glycation of protein is suspected.     

Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes

Non-enzymatic glycation of reactive amino groups in model proteins increased the rate of free radical production at physiologic pH by nearly fifty-fold over non-glycated protein. Superoxide generation was confirmed by electron paramagnetic resonance measurements with the spin-trap phenyl-t-butyl-nitrone. Both Schiff base and Amadori glycation products were found to generate free radicals in a ratio of 1:1.5. Free radicals generated by glycated protein increased peroxidation of membranes of linoleic/arachidonic acid vesicles nearly 2-fold over control, suggesting that the increased glycation of proteins in diabetes may accelerate vascular wall lipid oxidative modification.     

Oxidative stress in Alzheimer disease

Alzheimer disease (AD) is a progressive dementia affecting a large proportion of the aging population. The histopathological changes in AD include neuronal cell death, formation of amyloid plaques and neurofibrillary tangles. There is also evidence that brain tissue in patients with AD is exposed to oxidative stress (e.g., protein oxidation, lipid oxidation, DNA oxidation and glycoxidation) during the course of the disease. Advanced glycation endproducts (AGEs) are present in amyloid plaques in AD, and its extracellular accumulation may be caused by an accelerated oxidation of glycated proteins. AGEs participate in neuronal death causing direct (chemical) and indirect (cellular) free radical production and consequently increase oxidative stress. The development of drugs for the treatment of AD that breaks the vicious cycles of oxidative stress and neurodegeneration offer new opportunities. These approaches include AGE-inhibitors, antioxidants and anti-inflammatory substances, which prevent free radical production.Key words: ageing, advanced glycation endproducts, Alzheimer disease, amyloid, oxidative stress     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Inhibitory effect of some acetyl esters and acetamides on glycation of the histone H1

Non-enzymatic glycosylation (glycation) is a spontaneous set of reactions between reducing sugars and free amino groups in proteins or other biomolecules leading to the formation of fluorescent and coloured compounds known as advanced glycation end products (AGEs). AGEs cause structural changes of key proteins in humans, and therefore they are related with a number of physiological processes and diseases such as aging, atherosclerosis, cataract, arthritis, Alzheimer's disease. Two main strategies have been employed to prevent the formation of AGEs: a) low carbohydrate diet and b) pharmacological intervention. The latter includes treatment with reactive compounds which might be either sugar competitors (type A), carbonyl traps (type B) or free radical trapping antioxidants (type C). Acetylsalicylic acid (ASA, aspirin) is a good example of sugar competitor capable of inhibiting glycation by acetylating epsilon-amino groups of lysine residues in proteins. Taking into consideration the inhibiting effect of ASA on glycation we designed to study the antiglycation activity of other acetyl group-containing compounds (acetamides and acetyl esters) using the lysine-rich protein histone H1 as a model. The glycation of the histone H1 was carried out by either fructose or a complex mixture of glycating agents obtained from E. coli and monitored by fluorescent spectroscopy, SDS-PAGE and measurement of the content of reactive carbonyl groups in the target protein. Our results showed that the inhibitory effect of phenyl acetate, acetanilide, 4-acetamidophenylacetic acid and isopropenyl acetate was comparable to that of ASA. Based on the obtained results we conclude that these compounds act as free radical scavengers protecting proteins from the damaging effect of reactive oxygen species produced during the formation of AGEs.     

The role of glycation and autoxidation on crystallin aggregation

SUMMARY

Incubation of α-crystallin with glucose and CuSO₄ resulted in crystallin changes similar to those observed in cataracts. Examination of the reaction mixtures by polyacrylamide slab gel electrophoresis showed progressive crystallin aggregation through non-disulfide covalent bonds and parallel increases in ultraviolet absorbance and non-tryptophan fluorescence. Both glucose and copper were required; iron was less effective. The reaction can be accelerated by increasing glucose concentration or by utilizing ribose which has a higher percentage of free aldehyde groups than glucose. These observations are consistent with a mechanism involving crystallin glycation. The reaction is mediated by hydrogen peroxide and transition metals since it is inhibited by catalase and by chelating agents. These results, in turn, are consistent with copper-catalyzed autoxidation of glucose and of glycated crystallin. This reaction generates superoxide free radical which dismutates to yield hydrogen peroxide. The latter, in turn, generates hydroxyl radicals in presence of transition metal ions (Fenton reaction). Hydroxyl radical attack leads to cross-linking which is enhanced in glycated proteins. Under hyperglycemic conditions, such as in diabetes mellitus, high levels of glucose occur in insulin-independent tissues such as the lens. Elevated cupremia and oxidative stress are also known to occur in diabetic patients. There-fore, our findings are consistent with crystallin glycation and superimposed oxyradical generation during diabetic cataractogenesis.     

Oxidative stress in Alzheimer disease

Alzheimer disease (AD) is a progressive dementia affecting a large proportion of the aging population. The histopathological changes in AD include neuronal cell death, formation of amyloid plaques and neurofibrillary tangles. There is also evidence that brain tissue in patients with AD is exposed to oxidative stress (e.g., protein oxidation, lipid oxidation, DNA oxidation and glycoxidation) during the course of the disease. Advanced glycation endproducts (AGEs) are present in amyloid plaques in AD, and its extracellular accumulation may be caused by an accelerated oxidation of glycated proteins. AGEs participate in neuronal death causing direct (chemical) and indirect (cellular) free radical production and consequently increase oxidative stress. The development of drugs for the treatment of AD that breaks the vicious cycles of oxidative stress and neurodegeneration offer new opportunities. These approaches include AGE-inhibitors, antioxidants and anti-inflammatory substances, which prevent free radical production.     

Glycated proteins can enhance photooxidative stress in aged and diabetic lenses

This study intends to clarify the ability of different carbonyl-containing lens metabolites to form advanced glycation end products, which possess photosensitizer activity and to investigate whether these modified proteins could be implicated in lens photodamage. Calf lens protein was experimentally glycated with either methylglyoxal, glyoxal, ascorbic acid, or fructose to obtain models of aged and diabetic cataractous lenses. Being exposed to 200 J/cm 2 UVA radiation the model glycated proteins produced 2-3-fold more singlet oxygen compared to the unmodified protein and the superoxide radical formation was 30-80% higher than by the native protein. Ascorbylated proteins demonstrated the highest photosensitizer activity. Biological responses of glycation-related photosensitizers were studied on cultured lens epithelial cells irradiated with 40 J/cm 2 UVA. Tissue culture studies revealed a significant increase in thiobarbituric acid reactive substances in the culture medium of lens epithelial cells after irradiation and treatment with glycated proteins. Lens proteins had a protective effect against UVA induced cytotoxicity, however, this protective effect decreased with the increasing photosensitizer activity of experimentally glycated proteins. The documented glycation-related photosensitization could explain the accelerated pathogenic changes in human lens at advanced age and under diabetic conditions.     

In Vitro Nonenzymatic Glycation Enhances the Role of Myoglobin as a Source of Oxidative Stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO² and GMbO², respectively. H²O²-induced iron release was significantly higher from GMbO² than that from MbO². This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H²O² was studied. GMbO² degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO². Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H²O² with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO². Compared to MbO², GMbO² was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO². GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H²O². These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Glycated Proteins Can Enhance Photooxidative Stress in Aged and Diabetic Lenses

This study intends to clarify the ability of different carbonyl-containing lens metabolites to form advanced glycation end products, which possess photosensitizer activity and to investigate whether these modified proteins could be implicated in lens photodamage. Calf lens protein was experimentally glycated with either methylglyoxal, glyoxal, ascorbic acid, or fructose to obtain models of aged and diabetic cataractous lenses. Being exposed to 200 J/cm 2 UVA radiation the model glycated proteins produced 2-3-fold more singlet oxygen compared to the unmodified protein and the superoxide radical formation was 30-80% higher than by the native protein. Ascorbylated proteins demonstrated the highest photosensitizer activity. Biological responses of glycation-related photosensitizers were studied on cultured lens epithelial cells irradiated with 40 J/cm 2 UVA. Tissue culture studies revealed a significant increase in thiobarbituric acid reactive substances in the culture medium of lens epithelial cells after irradiation and treatment with glycated proteins. Lens proteins had a protective effect against UVA induced cytotoxicity, however, this protective effect decreased with the increasing photosensitizer activity of experimentally glycated proteins. The documented glycation-related photosensitization could explain the accelerated pathogenic changes in human lens at advanced age and under diabetic conditions.     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species.

Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction.     

Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.     

Methylglyoxal induces glycation and oxidative stress in Saccharomyces cerevisiae

Purpose

Hyperglycemia causes abnormal accumulation of methylglyoxal (MGO) and concomitant DNA, protein glycation. These pathophysiological changes further leads to diabetic complications. Yeast Saccharomyces cerevisiae is one of the best model to study MGO-induced glycation modifications. The aim of the present study was to investigate the effect of MGO on protein, DNA glycation, and oxidative stress markers using S. cerevisiae as a system.

Methods

Saccharomyces cerevisiae cells were incubated with 8 mM of MGO for 4 h and 24 h. After incubation, protein and DNA samples were isolated from the lysed cells. The samples were analyzed for various glycation (fructosamine, β-amyloid, free amino group, free thiol group, and hyperchromic shift analysis) and oxidative stress markers (total antioxidant potential, catalase, glutathione, and lipid peroxidation).

Results

MGO (8 mM) acted as a potent glycating agent, causing protein and DNA glycation in treated yeast cells. The glycation markers fructosamine and β-amyloid were significantly elevated when incubated for 4 h as compared to 24 h. Oxidative stress in the glycated yeast cells alleviated cellular antioxidant capacity and reduced the cell viability.

Conclusion

MGO caused significant glycation modifications of proteins and DNA in yeast cells. It also triggered increase in intracellular oxidative stress. MGO-induced protein, DNA glycation, and oxidative stress in S. cerevisiae indicate the suitability of the yeast model to study various biochemical pathways involved in diabetic complications and even conformational pathologies.

     

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.     

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical‐mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the γ‐linkage, H‐Glu(Cys‐Gly‐OH)‐OH (GSH). Screening for products formed by GSH with D ‐glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N‐(1‐deoxy‐D ‐fructos‐1‐yl)‐pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N‐terminal glutamic acid and the formation of pyroglutamic acid‐related Amadori product were accompanied by generation of Cys‐Gly‐derived Amadori and thiazolidine compounds. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Carnosine disaggregates glycated alpha-crystallin: an in vitro study

Protein glycation, which promotes aggregation, involves the unwanted reaction of carbohydrate oxidation products with proteins. Glycation of lens alpha-crystallin occurs in vivo and may contribute to cataractogenesis. Anti-glycation compounds such as carnosine may be preventive, but interestingly carnosine reverses lens opacity in human trials. The mechanism for this observation may involve carnosine's ability to disaggregate glycated protein. We investigated this hypothesis using glycated alpha-crystallin as our in vitro model. Methylglyoxal-induced glycation of alpha-crystallin caused aggregation as evidenced by increased 90 degrees light scattering. After addition of carnosine, light scattering returned to baseline levels suggesting that the size of the glycation-induced aggregates decreased. Additionally, carnosine decreased tryptophan fluorescence polarization of glycated alpha-crystallin, suggesting that carnosine increased peptide chain mobility, which may contribute to the controlled unfolding of glycated protein. Comparatively, guanidine-HCl and urea had no effect. Our data support the hypothesis that carnosine disaggregates glycated alpha-crystallin.     

Activation of protein-bound copper ions during early glycation: study on two proteins

This study proposes several possible pathways by which hyperglycemia could make protein-bound metal ions more redox active. These mechanisms were tested on bovine serum albumin and calf lens protein. Proteins rich in early glycation products were less capable of competing for copper ions in the presence of other ligands (e.g., glycine and calcein), suggesting that glycated proteins might have diminished stability constants of their copper chelates compared to control counterparts. When protein-copper complexes were tested for their ability to cause the oxidation of ascorbic acid, as well as the reduction of molecular oxygen to hydrogen peroxide, glycated and control proteins differed considerably in their redox abilities. Oxidative damage on proteins documented by protein carbonyl content and amino acid analysis indicates the involvement of Fenton chemistry upon metal chelation. The possible biological consequences of the observed activation of metal ions bound to early glycated proteins are discussed.     

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.