The role of glyoxal and glycolaldehyde in protein cross-linking and N epsilon-(carboxymethyl)lysine (CML) formation during Maillard reaction under physiological conditions was investigated. Incubation of bovine serum albumin with these reagents lead to rapid formation of C-2-imine cross-links and CML. Initial CML formation rate from glyoxal was not dependent on oxidation, suggesting an intramolecular Cannizzaro reaction. CML formation from glucose/lysine or Amadori product of both was strongly dependent on oxidation. Blocking of Amadori product by boric acid totally suppressed CML formation from Amadori product, but only by 37% in the glucose/lysine system. Trapping of glyoxal with aminoguanidine hardly suppressed CML formation from Amadori product, whereas it blocked 50% of CML production in the glucose/lysine system. While these results would support a significant role for glucose autoxidation in CML formation, the addition of lysine to a glucose/aminoguanidine incubation system catalyzed glyoxal-triazine formation 7-fold, thereby strongly suggesting that glucose autoxidation is not a factor for glyoxal-mediated CML formation. Based on these results, it can be estimated that approximately 50% of the CML forming in a glucose/lysine system originates from oxidation of Amadori product, and 40-50% originates from a pre-Amadori stage largely independent from glucose autoxidation. This step may be related to the so-called Namiki pathway of the Maillard reaction.

During aging long-lived proteins accumulate specific post-translational modifications. One family of modifications, termed Maillard reaction products, are initiated by the condensation between amino groups of proteins and reducing sugars. Protein modification by the Maillard reaction is associated with crosslink formation, decreased protein solubil-

The nonenzymatic glycosylation reaction that is accelerated in diabetes is the first step of the Maillard or nonenzymatic browning reaction that occurs in stored food. The glucose-protein adduct rearranges and dehydrates to form brown and fluorescent pigments, which can act as crosslinks, resulting in decreased protein solubility and altered mechanical properties. Evidence suggesting that this process occurs in vivo has been found in lens crystallins. The observation that nonenzymatic glycosylation and insolubility increases in collagen with age and diabetes led us to investigate the possible browning of human collagen. Several complications of diabetes mellitus occur in collagenrich tissues and resemble processes and diseases characteristic of aging. These include earlier onset and greater severity of atherosclerosis (1), stiffening of lungs (2) and large arteries (3, 4), thickening of capillary and glomerular basement membranes (5, 6), periarticular rigidity (7), and osteoarthritis (8). With age, collagen becomes less soluble (9, 10), more crosslinked (11), and more glycosylated (10,12), and it accumulates yellow and fluorescent pigments (13,14). In diabetes, several of these changes occur at an earlier age, suggesting an apparent acceleration of the aging process. Compared with age-matched nondiabetics, collagen from diabetics is less soluble (10), is more resistant to digestion by collagenase (15) and cyanogen bromide (16), has more pepsin-releasable high molecular weight peptides (10), and is more nonenzymatically glycosylated (10,15,17). Glycosylation and subsequent crosslinking occur in food proteins that are stored or heated in the presence of reducing sugars and are due to the Maillard or nonenzymatic browning reaction. Glucose, for example, first reacts nonenzymatically with free amino groups on proteins to form a stable amino 1-deoxyketosyl adduct, also called the Amadori product. Nonenzymatic glycosylation has been shown to occur with hemoglobin to form hemoglobin Al, and with a variety of other body proteins-e.g., lens crystallins, collagen, and nerve proteins (18 proteins (20). The recent demonstration that insolubility and resistance to enzymatic digestion of human collagen increase with age and diabetes in parallel with nonenzymatic glycosylation (10) led to the present investigation of the browning process in the same subject population. Insoluble dura mater collagen from nondiabetics and subjects with type I and type II diabetes was examined for the presence of yellow and fluorescent pigments similar to those that form in the nonenzymatic browning reaction with glucose. MATERIAL AND METHODSThe samples of dura mater used in this study were obtained at autopsy from the same subjects in whom age-and diabetes-related changes in skin collagen solubility and glycosylation were reported by Schnider and Kohn (10). These include 17 samples of dura mater from subjects without clinical or pathological evidence of connective tissue disease or diabetes mellitus and similar samples from 3 subjects w...

The incubation of lens proteins with reducing sugars leads to the formation of fluorescent yellow pigments and cross-like similar to those reported in aging and cataractous human lenses. Called nonenzymatic browning or the Maillard reaction, this aging process also occurs in stored foods. Reducing sugars condense with the free amino group of proteins, then rearrange and dehydrate to form unsaturated pigments and cross-linked products. Although most proteins in living systems turn over with sufficient rapidity to avoid nonenzymatic browning, some, such as lens crystallins and skin collagen, are exceptionally long-lived and may be vulnerable.

The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N(epsilon)-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the primary prevention and secondary intervention cohorts of the Diabetes Control and Complications Trial. By comparison with conventional treatment, 5 years of intensive treatment was associated with 30-32% lower furosine, 9% lower pentosidine, 9-13% lower CML, 24% higher acid-soluble collagen, and 50% higher pepsin-soluble collagen. All of these differences were statistically significant in the subjects of the primary prevention cohort (P < 0.006-0.001) and also of the secondary intervention cohort (P < 0.015-0.001) with the exception of CML and acid-soluble collagen. Age- and duration-adjusted collagen variables were significantly associated with the HbA1c value nearest the biopsy and with cumulative prior HbA1c values. Multiple logistic regression analyses with six nonredundant collagen parameters as independent variables and various expressions of retinopathy, nephropathy, and neuropathy outcomes as dependent variables showed that the complications were significantly associated with the full set of collagen variables. Surprisingly, the percentage of total variance (R2) in complications explained by the collagen variables ranged from 19 to 36% with the intensive treatment and from 14 to 51% with conventional treatment. These associations generally remained significant even after adjustment for HbA1c, and, most unexpectedly, in conventionally treated subjects, glycated collagen was the parameter most consistently associated with diabetic complications. Continued monitoring of these subjects may determine whether glycation products in the skin, and especially the early Amadori product (furosine), have the potential to be predictors of the future risk of developing complications, and perhaps be even better predictors than glycated hemoglobin (HbA1c).

Nonenzymatically glycosylated proteins gradually form fluorescent cross-linked protein adducts--a process termed "browning." The rate of this reaction increases with the glucose concentration. Assaying for the presence of browning products in long-lived proteins should therefore provide information on long-term metabolic control. We measured collagen-linked fluorescence typical for nonenzymatic browning in skin-biopsy specimens from 41 subjects with longstanding Type I diabetes and from 25 controls. Fluorescence correlated with age and (weakly) with the duration of diabetes. Mean age-adjusted fluorescence values were twice as high in diabetic subjects as in control subjects (P less than 0.0001) and increased with the severity of retinopathy, nephropathy, and arterial and joint stiffness. The correlation was significant for retinopathy (r = 0.42; P less than 0.01), arterial stiffness (r = 0.41; P less than 0.01), joint stiffness (r = 0.34; P less than 0.05), and the sum of all complications (r = 0.47; P less than 0.01). Fluorescence also correlated with systolic (r = 0.42; P less than 0.01) and diastolic (r = 0.36; P less than 0.05) blood pressures. If one can assume that the fluorescence results from a browning product of glucose, our data suggest that there is an overall correlation between the severity of diabetic complications and cumulative glycemia over many years.

Several mechanistic pathways linking hyperglycemia to diabetes complications, including glycation of proteins and formation of advanced glycation end products (AGEs), have been proposed. We investigated the hypothesis that skin collagen glycation and AGEs predict the risk of progression of microvascular disease. We measured glycation products in the skin collagen of 211 Diabetes Control and Complications Trial (DCCT) volunteers in 1992 who continued to be followed in the Epidemiology of Diabetes Interventions and Complications study for 10 years. We determined whether the earlier measurements of glycated collagen and AGE levels correlated with the risk of progression of retinopathy and nephropathy from the end of the DCCT to 10 years later. In multivariate analyses, the combination of furosine (glycated collagen) and carboxymethyllysine (CML) predicted the progression of retinopathy ( 2 ‫؍‬ 59.4, P < 0.0001) and nephropathy ( 2 ‫؍‬ 18.2, P ‫؍‬ 0.0001), even after adjustment for mean HbA 1c (A1C) ( 2 ‫؍‬ 32.7, P < 0.0001 for retinopathy) and ( 2 ‫؍‬ 12.8, P ‫؍‬ 0.0016 for nephropathy). The predictive effect of A1C vanished after adjustment for furosine and CML ( 2 ‫؍‬ 0.0002, P ‫؍‬ 0.987 for retinopathy and 2 ‫؍‬ 0.0002, P ‫؍‬ 0.964 for nephropathy). Furosine explained more of the variation in the 10-year progression of retinopathy and nephropathy than did CML. These results strengthen the role of glycation of proteins and AGE formation in the pathogenesis of retinopathy and nephropathy. Glycation and subsequent AGE formation may explain the risk of these complications associated with prior A1C and provide a rational basis for the phenomenon of "metabolic memory" in the pathogenesis of these diabetes complications. Diabetes 54:3103-3111, 2005 N onenzymatic glycation of proteins and subsequent formation of advanced glycation end products (AGEs) is one of the pathogenetic mechanisms thought to link hyperglycemia to diabetic retinopathy and nephropathy (1,2). Inhibitors of AGE formation (3-8) and breakers of AGE-protein crosslinks (9,10) reduce both microvascular complications in experimental diabetic models. The relationship of longterm intensive control of glycemia and its effect on these complications with indicators of skin collagen glycation (furosine), glycoxidation and AGE formation (pentosidine and carboxymethyllysine [CML]), and cross-linking (acid and pepsin solubility) were previously examined in 215 patients with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT) (11) who underwent a skin biopsy ϳ1 year before the close of the trial. Compared with conventional treatment, intensive treatment was associated with significantly lower levels of furosine, pentosidine, CML, and relative fluorescence and with higher levels of acid-and pepsin-soluble collagen (11). Age-and duration-adjusted collagen variables were significantly associated with the A1C value closest in time to the biopsy and with mean DCCT A1C. Retinopathy, nephropathy, and neuropathy outcomes as dependent variables were sign...

Advanced glycation end products (AGEs) include a variety of protein adducts whose accumulation alters the structure and function of tissue proteins and stimulates cellular responses. They have been implicated in tissue damage associated with diabetic complications. To assess the possible link between AGE accumulation and the development of diabetic nephropathy (DN), we have examined the immunohistochemical localization of various AGE structures postulated to date, i.e., pentosidine, Nepsilon-(carboxymethyl)lysine (CML), and pyrraline, in diabetic and control kidneys. CML and pentosidine accumulate in the expanded mesangial matrix and thickened glomerular capillary walls of early DN and in nodular lesions and arterial walls of advanced DN, but were absent in control kidneys. By contrast, pyrraline was not found within diabetic glomeruli but was detected in the interstitial connective tissue of both normal and diabetic kidneys. Although the distribution of pyrraline was topographically identical to type III collagen, distribution of pentosidine and CML was not specific for collagen type, suggesting that difference in matrix protein composition per se could not explain heterogeneous AGE localization. Since oxidation is linked closely to the formation of pentosidine and CML, we also immunostained malondialdehyde (MDA), a lipid peroxidation product whose formation is accelerated by oxidative stress, assuming that local oxidative stress may serve as a mechanism of pentosidine and CML accumulation. Consistent with our assumption, diabetic nodular lesions were stained positive for MDA. These findings show that AGE localization in DN varies according to AGE structure, and suggest that the colocalization of markers of glycoxidation (pentosidine and CML) with a marker of lipid peroxidation reflects a local oxidative stress in association with the pathogenesis of diabetic glomerular lesions. Thus, glycoxidation markers may serve as useful biomarkers of oxidative damage in DN.