The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysinearginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n ‫؍‬ 110) and glomerular basement membrane (GBM, n ‫؍‬ 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p ‫؍‬ 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes.Reducing sugars react nonenzymatically with proteins to form adducts and cross-links referred to as advanced glycation end products (1). Their accumulation is particularly high in long-lived proteins, such as lens crystallins and collagen, and results in intra-and intermolecular cross-linking. The latter has been hypothesized to result in an age-and diabetes-related stiffening of collagenous tissues and is believed to play an important role in the etiology of atherosclerosis and cardiovascular disease (3) as well as the loss of elasticity in lungs, joints, and skin (4 -6). These age-related processes are markedly accelerated by diabetes and are associated with morbidity and mortality in diabetic individuals (7,8). In skin, protein crosslinking is associated with an age-related loss of elasticity, increased stiffening, and wrinkling (9). However, this process occurs ubiquitously with age, suggesting a fundamental underlying mechanism.Although most of the age-related changes in the extracellular matrix could be duplicated by incubating reducing sugars with proteins, Eble et al. (10) suggested that the major cross-links are not stable to conventional conditions of acid hydrolysis. Indeed, Biemel et al. (11) recently showed that glucose and sugar-derived dicarbonyl intermediates react with human serum albumin to form various acid...

The aging extracellular matrix is characterized by an age-related increase in insolubilization, yellowing, and stiffening, all of which can be mimicked by the Maillard reaction in vitro. These phenomena are accelerated in metabolic diseases such as diabetes and end-stage renal disease, which have in common with physiological aging the accumulation of various glycation products and cross-links. Eight years ago we concluded that the evidence favored oxidative cross-linking in experimental diabetes [Monnier, V.M. et al. 1996. The mechanism of collagen cross-linking in diabetes: a puzzle nearing completion. Diabetes 45(Suppl. 3): 67-72] and proposed a major role for a putative non-UV active cross-link derived from glucose. Below, we provide an update of the field that leads to the conclusion that, while oxidation might be important for Maillard reaction-mediated cross-linking via Strecker degradation and allysine formation, the single most important collagen cross-link known to date in diabetes and aging is glucosepane, a lysyl-arginine cross-link that forms under nonoxidative conditions.

. For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9 -13. The bicyclic structures 9 -11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9 -13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.

We tested the hypothesis that green tea prevents diabetes-related tissue dysfunctions attributable to oxidation. Diabetic rats were treated daily with tap water, vitamins C and E, or fresh Japanese green tea extract. After 12 months, body weights were decreased, whereas glycated lysine in aorta, tendon, and plasma were increased by diabetes (P < 0.001) but unaffected by treatment. Erythrocyte glutathione and plasma hydroperoxides were improved by the vitamins (P < 0.05) and green tea (P < 0.001). Retinal superoxide production, acellular capillaries, and pericyte ghosts were increased by diabetes (P < 0.001) and improved by green tea and the vitamins (P variable). Lens crystallin fluorescence at 370/440 nm was ameliorated by green tea (P < 0.05) but not the vitamins. Marginal effects on nephropathy parameters were noted. However, suppressed renal mitochondrial NADH-linked ADP-dependent and dinitrophenol-dependent respiration and complex III activity were improved by green tea (P variable). Green tea also suppressed the methylglyoxal hydroimidazolone immunostaining of a 28-kDa mitochondrial protein. Surprising, glycoxidation in tendon, aorta, and plasma was either worsened or not significantly improved by the vitamins and green tea. Glucosepane cross-links were increased by diabetes (P < 0.001), and green tea worsened total cross-linking. In conclusion, green tea and antioxidant vitamins improved several diabetes-related cellular dysfunctions but worsened matrix glycoxidation in selected tissues, suggesting that antioxidant treatment tilts the balance from oxidative to carbonyl stress in the extracellular compartment. Diabetes 54:517-526, 2005

The presence of the various protein crosslinks GOLD 2, MOLD3, GODIC4, MODIC5, DODIC6, and glucosepan 7in foods has been established for the first time by liquid chromatography – mass spectrometry (LC‐MS) with electrospray ionization (ESI). In compounds2 and3two lysine moieties, in4–7a lysine and an arginine side chain are joined by the crosslink. Unequivocal identification of2–7 was achieved with independently synthesized reference material. The quantitative results for the investigated foodstuffs show MODIC 5to be the most important Maillard crosslink. The concentrations of 5 and GODIC 4are 5–10 fold higher than those of the corresponding imidazolium compounds3 and 2, establishing5and 4as the major food protein crosslinks derived from methylglyoxal and glyoxal, respectively. The maximum value of 151 mg MODIC 5/kg protein (equivalent to 0.42 mmol/kg protein) was found in a butter biscuit sample which also shows the highest overall Maillard crosslink content with 0.71 mmol47/kg protein. These first quantitative results suggest that compounds4–7 can be jointly responsible for protein polymerization in the course of food processing.