Vincent M. Monnier

Radical AGEing in Alzheimer's disease

The pathological presentation of Alzheimers disease, the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intracellular and extracellular filamentous protein aggregates that form lesions termed neurofibrillary tangles and senile plaques, respectively. Several independent parameters have been suggested as the primary factor that is responsible for this pathogenesis, including apolipoprotein epsilon genotype, hyperphosphorylation of cytoskeletal proteins, or metabolism of amyloid beta. However, at present, no one theory explains adequately the host of complex biochemical and pathological facets of the disease. Recent findings suggest that age-related increases in oxidative stress and protein glycation either individually, or more probably in a synergistic manner, could, exclusive of the other theories or in concert with them, account for all aspects of Alzheimers disease.

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.

Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline.

Pyrraline is one of the major Maillard compounds resulting from the reaction of glucose with amino compounds at slightly acidic pH. For in vivo studies, monoclonal pyrraline antibodies were raised after immunization of Balb/c mice with keyhole limpet hemocyamin-caproyl pyrraline conjugate. Of 660 hybridoma clones from one donor, 260 produced an antibody to the free hapten, two of which named Pyr-A and Pyr-B also cross-reacted with L-lysyl pyrraline. Using Pyr-B antibody and an ELISA, a gradual increase in pyrraline immunoreactivity was observed in serum albumin incubated with glucose or 3-deoxyglucosone. Plasma pyrraline levels increased fourfold (P less than 0.001) in Sprague-Dawley rats upon induction of diabetes with streptozotocin and were twofold increased in randomly selected plasmas from diabetic humans. Highly specific pyrraline immunoreactivity was detected in sclerosed glomeruli from diabetic and old normal kidneys as well as in renal arteries with arteriolosclerosis and in perivascular and peritubular sclerosed extracellular matrix and basement membranes. The preferential localization of pyrraline immunoreactivity in the extracellular matrix strengthens the notion that the advanced glycosylation reaction may contribute to decreased turnover and thickening of the extracellular matrix in diabetes and aging.

Structure and Mechanism of Formation of Human Lens Fluorophore LM-1 RELATIONSHIP TO VESPERLYSINE A AND THE ADVANCED MAILLARD REACTION IN AGING, DIABETES, AND CATARACTOGENESIS

Human lens crystallins become progressively yellow-brown pigmented with age. Both fluorescent and non-fluorescent protein adducts and cross-links are formed, many of which result from the advanced Maillard reaction. One of them, LM-1, is a blue fluorophore that was earlier tentatively identified as a cross-link involving lysine residues (1). A two-step chromatographic system was used to unequivocally identify and quantitatively prepare a synthetic fluorescent cross-link with lysine residues that had identical UV, fluorescent, and chromatographic properties with both acetylated and non-acetylated LM-1. Proton, 13C NMR, and molecular mass of the synthetic compound were identical with vesperlysine A, a fluorescent cross-link discovered by Nakamura et al. (2). The fragmentation patterns of vesperlysine A and LM-1 were identical as determined by NMR/mass spectrometry. Lenticular levels of vesperlysine A increase curvilinearly with age and reach 20 pmol/mg at 90 years. Levels correlate with degree of lens crystallin pigmentation and fluorescence and are increased in diabetes, in contrast toN ε-(carboxymethyl)lysine and pentosidine. Ascorbate, d-pentoses, andd-threose, but neither d-glucose under oxidative conditions, dl-glyceraldehyde, methylglyoxal, glyoxal, nor glycolaldehyde, are precursors. However, addition of C-2 compounds greatly catalyzes vesperlysine A formation from ribose. Thus, vesperlysine A/LM-1 is a novel product of the advanced Maillard reaction in vivo and a specific marker of a diabetic process in the lens that is different from glyco- and lipoxidation.

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.