David R. Sell

Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia.

Recent progress in structure elucidation of products of the advanced Maillard reaction now allows probing specifically for the role of this reaction in the pathogenesis of age- and diabetes-related complications. Pyrraline is a glucose-derived advanced glycation end product against which polyclonal and monoclonal antibodies have been raised. Immunohistochemical localization studies revealed that pyrraline is found predominantly in the sclerosed extracellular matrix of glomerular and arteriolar renal tissues from both diabetic and aged nondiabetic individuals. Pentosidine and carboxymethyllysine are Maillard end products derived from both glucose and ascorbate. In addition, pentosidine can be formed from several other sugars under oxidative conditions, and in vitro studies suggest that a common intermediate involving a pentose is a necessary precursor molecule. The highest levels of these advanced Maillard products are generally found in the extracellular matrix, but these products are also present in lens proteins and in proteins with a fast turnover such as plasma proteins. Diabetes, and especially uremia, greatly catalyzes pentosidine formation. Both conditions are characterized by accelerated cataractogenesis, atherosclerosis, and neuropathy, suggesting that molecular damage by advanced Maillard reaction products may be a common mechanism in their development.

Pentosidine formation in skin correlates with severity of complications in individuals with long-standing IDDM

Pentosidine is an advanced glycosylation end product and protein cross-link that results from the reaction of pentoses with proteins. Recent data indicate that long-term glycation of proteins with glucose also leads to pentosidine formation through sugar fragmentation. In this study, the relationship between the severity of diabetic complications and pentosidine formation was investigated in collagen from skin-punch biopsies from 25 nondiabetic control subjects and 41 IDDM patients with diabetes duration >17 yr. Pentosidine was significantly elevated in all IDDM patients versus control subjects (P < 0.0001). It correlated strongly with age (P < 0.0001) and weakly with duration (P < 0.082). Age-adjusted pentosidine levels were highest in grade 2 (severe) versus grade 1 and 0 complication in all four parameters tested (retinopathy, proteinuria, arterial stiffness, and joint stiffness). Significant differences were found for retinopathy (P < 0.014) and joint stiffness (P < 0.041). The highest degree of association was with the cumulative grade of individual complication (P < 0.005), determined by summing indexes of all four parameters. Pentosidine also was significantly elevated in the serum of IDDM patients compared with control subjects (P < 0.0001), but levels were not significantly correlated with age, diabetes duration, complication, or skin collagen pentosidine (P > 0.05). A high correlation between pentosidine levels and long-wave collagen-linked fluorescence also was observed, suggesting that pentosidine is a generalized marker of accelerated tissue modification by the advanced glycosylation/Maillard reaction, which is enhanced in IDDM patients with severe complications.

Chromatographic Quantitation of Plasma and Erythrocyte Pentosidine in Diabetic and Uremic Subjects

Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P < 0.001) and a 23-fold (P < 0.001) elevation of mean ± SD plasma protein pentosidine in diabetic (2.4 ± 1.2 pmol/mg) and uremic (21.5 ± 10.8 pmol/mg) subjects compared with healthy (0.95 ± 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 ± 0.4 pmol/mg hemoglobin, P < 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.

End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen.

Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.

Cross‐Linking of the Extracellular Matrix by the Maillard Reaction in Aging and Diabetes: An Update on “a Puzzle Nearing Resolution”

Abstract: 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.

Glucosepane Is a Major Protein Cross-link of the Senescent Human Extracellular Matrix RELATIONSHIP WITH DIABETES

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 lysine-arginine 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.

Glycation Products as Markers and Predictors of the Progression of Diabetic Complications

Abstract: The structure of a growing number of glycation and advanced glycation end products has been elucidated. Measuring these products can be used to assess cumulative glycemic and glycoxidative damage in diabetes and other chronic conditions. The predictive power of a given glycation product can be tested in large prospective studies that evaluate the risk of developing diabetic micro‐ and macrovascular disease over years following the quantitative determination of that marker. This article provides a comprehensive review of the field, comparing the merits of each marker, whether in skin, serum, or other tissue. Several conclusions are drawn, one of which identifies skin glycation products as powerful predictors of the risk of developing diabetic complications.

Molecular basis of arterial stiffening: role of glycation - a mini-review.

Arterial stiffening is a progressive, ubiquitous and irreversible aging process that is interwoven with and accelerated by various diseases such as diabetes, atherosclerosis and hypertension. In large arteries, aging is characterized by decreased turnover of collagen and elastin and increased advanced glycation end-products (AGEs) and cross-links. Elastic fibers undergo lysis and disorganization subsequent to their replacement by collagen and other matrix components. These events cause the loss of elasticity and induce stiffening. Conceptual approaches to minimize AGE accumulation in arteries include caloric restriction, exercise, low dietary intake of AGEs, deglycation enzymes, increased clearance of AGEs, antagonists of AGE receptors and pharmaceutical interventions. Much optimism exists in the ability of ‘AGE breakers’ such as alagebrium (ALT-711) to cleave AGE cross-links and reverse the age-related stiffening of arteries. However, there is little evidence that these agents actually break pre-existing AGE cross-links in vivo. In contrast, many of these anti-AGE agents share in common the ability to chelate metals, thus acting as inhibitors of metal-catalyzed AGE and protein carbonyl formation. Future work on interventions into the causes of arterial stiffness in aging needs to address more rigorously the relationship between stochastic forms of damage, such a glycation and oxidation, and the changes in elastic fiber structure thought to contribute to loss of arterial elasticity.

Protection From Retinopathy and Other Complications in Patients With Type 1 Diabetes of Extreme Duration: The Joslin 50-Year Medalist Study

OBJECTIVE To assess complication prevalence and identify protective factors in patients with diabetes duration of ≥50 years. Characterization of a complication-free subgroup in this cohort would suggest that some individuals are protected from diabetes complications and allow identification of endogenous protective factors. RESEARCH DESIGN AND METHODS Cross-sectional, observational study of 351 U.S. residents who have survived with type 1 diabetes for ≥50 years (Medalists). Retinopathy, nephropathy, neuropathy, and cardiovascular disease were assessed in relation to HbA1c, lipids, and advanced glycation end products (AGEs). Retrospective chart review provided longitudinal ophthalmic data for a subgroup. RESULTS A high proportion of Medalists remain free from proliferative diabetic retinopathy (PDR) (42.6%), nephropathy (86.9%), neuropathy (39.4%), or cardiovascular disease (51.5%). Current and longitudinal (the past 15 years) glycemic control were unrelated to complications. Subjects with high plasma carboxyethyl-lysine and pentosidine were 7.2-fold more likely to have any complication. Of Medalists without PDR, 96% with no retinopathy progression over the first 17 years of follow-up did not experience retinopathy worsening thereafter. CONCLUSIONS The Medalist population is likely enriched for protective factors against complications. These factors might prove useful to the general population with diabetes if they can be used to induce protection against long-term complications. Specific AGE combinations were strongly associated with complications, indicating a link between AGE formation or processing with development of diabetic vasculopathy.

Isolation, Purification and Partial Characterization of Novel Fluorophores from aging Human Insoluble Collagen-Rich Tissue

Collagen undergoes progressive browning with aging and diabetes characterized by yellowing, fluorescence and crosslinking, the cause of which remains unelucidated. As an initial step towards understanding the mechanism(s) of insolubilization of collagen in aging, the major fluorophores/chromophores from the insoluble fraction of human dura mater were isolated and their spectroscopic properties were characterized. High molecular weight tryptic peptides of insoluble collagen were cleaved by sequential enzymatic digestion followed by separation into high (HMW) and low molecular weight (LMW) fractions by gel filtration chromatography. LMW was further separated by paper and reverse phase chromatography (HPLC). Two fluorescent peaks, nicknamed P and M, were obtained from LMW which had UV maxima at 325 and 350 nm and excitation/fluorescence maxima at 335/385 and 360/460 nm, respectively. Fluorophore M was borohydride reducible and unstable to acid-hydrolysis, while P remained unaffected. Large quantities of fluorophore M and pyridinoline were found in the highly crosslinked HMW fraction remaining following exhaustive proteolytic digestion. Fluorophore P and M were the major fluorophores recovered from the tryptic digest of insoluble dura mater. Fluorescence spectra of M suggest an iminopropene type of configuration which could result from nonenzymatic browning of collagen with, e.g., glucose or malonyldialdehyde, as a result of lipid peroxidation. Spectroscopic and chemical properties of fluorophore P were reminiscent but not identical with those of pyridinium crosslinks. Structure elucidation of these fluorophores is expected to provide important insight into the aging processes of the extracellular matrix.