Effect of chronic hypoxia on RAGE and its soluble forms in lungs and plasma of mice.

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor. Alternative splicing and enzymatic shedding produce soluble forms that protect against damage by ligands including Advanced Glycation End products (AGEs). A link between RAGE and oxygen levels is evident from studies showing RAGE-mediated injury following hyperoxia. The effect of hypoxia on pulmonary RAGE expression and circulating sRAGE levels is however unknown. Therefore mice were exposed to chronic hypoxia for 21 d and expression of RAGE, sheddases in lungs and circulating sRAGE were determined. In addition, accumulation of AGEs in lungs and expression of the AGE detoxifying enzyme GLO1 and receptors were evaluated. In lung tissue gene expression of total RAGE, variants 1 and 3 were elevated in mice exposed to hypoxia, whereas mRAGE and sRAGE protein levels were decreased. In the hypoxic group plasma sRAGE levels were enhanced. Although the levels of pro-ADAM10 were elevated in lungs of hypoxia exposed mice, the relative amount of the active form was decreased and gelatinase activity unaffected. In the lungs, the RAGE ligand HMGB1 was decreased and of the AGEs, only LW-1 was increased by chronic hypoxia. Gene expression of AGE receptors 2 and 3 was significantly upregulated. Chronic hypoxia is associated with downregulation of pulmonary RAGE protein levels, but a relative increase in sRAGE. These alterations might be part of the adaptive and protective response mechanism to chronic hypoxia and are not associated with AGE formation except for the fluorophore LW-1 which emerges as a novel marker of tissue hypoxia.

Effect of chronic aminoguanidine treatment on age-related glycation, glycoxidation, and collagen cross-linking in the Fischer 344 rat.

Aminoguanidine (AG) is an inhibitor of protein modification by the advanced Maillard reaction. We evaluated its effects in preventing age-related collagen cross-linking, glycation, and glycoxidation in Fischer 344 rats by administering the drug in their drinking water at 1 g/l from the time they were 6 months until they were 24 months of age. Body weight and food and water consumption were consistently recorded throughout the study. Plasma glucose was measured by the glucose oxidase method, and collagen cross-linking was assessed by tail tendon break time (TBT) in urea. Glycation (furosine) and glycoxidation (pentosidine and carboxymethyllysine) were assessed by high-performance liquid chromatography in acid hydrolysates of skin and tendon collagen. Water consumption dramatically increased (p <.0001) after 20 months of age and was accelerated in the control versus AG-treated rats (p <.0001). Plasma glucose increased approximately 20% at age 19 months in both groups (p <.0001). TBT, glycation, and glycoxidation all increased significantly (p <.0001) with age. However, except for a modest decrease of TBT at all ages that approached significance (p =.077), AG had no effect on collagen glycation or glycoxidation. These results are important because they suggest that alpha,beta-dicarbonyl compounds that can be trapped by aminoguanidine do not play a major role in collagen aging in the rat. Instead, post-Amadori pathways involving oxidative or nonoxidative fragmentation of the Amadori product emerge as the more likely mechanism of collagen cross-linking in aging.

A syndrome of molybdenosis, copper deficiency, and type 2 diabetes in the moose population of south-west Sweden.

Since the mid-1980s, a 'mysterious' disease has been afflicting the moose (Alces alces L.) population of south-western Sweden. Molybdenosis combined with secondary copper deficiency syndrome has been suggested as the cause of the clinical signs and of necropsy findings, supported by trace element analysis. Copper deficiency has long been associated with disturbed carbohydrate metabolism and also with oxidative stress. When testing the oxidative stress hypothesis, we found increased concentrations of the glycoxidation products pentosidine and carboxymethyl-lysine (CML), both in plasma proteins and in renal tissue, when compared with control values. The concentration of glycated lysine (furosine), a marker of hyperglycaemia, was also increased. These data, together with elevated insulin levels in affected moose, strongly suggest that they are suffering from an environmentally-induced, non-insulin-dependent type 2 diabetes.

Longitudinal determination of skin collagen glycation and glycoxidation rates predicts early death in C57BL/6NNIA mice.

In 1988, the National Institute on Aging launched a 10-year program aimed at identification of biomarkers of aging. Previous results from our laboratory showed that pentosidine, an advanced glycation product, formed in skin collagen at a rate inversely related to maximum life span across several mammalian species. As part of the Biomarkers Program, we investigated the hypothesis that longitudinal determination of glycation and glycoxidation rates in skin collagen could predict longevities in ad libitum-fed (AL) and caloric restricted (CR) mice. C57BL/6NNia male mice were biopsied at age 20 months and at natural death. Glycation (furosine method) was assessed by gas chromatography/mass spectrometry (GC/MS) and the glycoxidation products carboxymethyllysine (CML) and pentosidine were determined by GC/MS and HPLC, respectively. CR vs. AL significantly (P<0.0001) increased both mean (34 vs. 27 months) and maximum (47 vs. 31 months) life spans. Skin collagen levels of furosine (pmol/micromol lysine) were approximately 2.5-fold greater than CML levels and 100-fold greater than pentosidine. Individual accumulation rates modeled as linear equations were significantly (P<0.001) inhibited by CR vs. AL for all parameters and in all cases varied inversely with longevity (P<0.1 to <0.0001). The incidence of three tissue pathologies (lymphoma, dermatitis, and seminal vesiculitis) was found to be attenuated by CR and the latter pathology correlated significantly with longevities (r=0.54, P=0. 002). The finding that markers of skin collagen glycation and glycoxidation rates can predict early deaths in AL and CR C57BL/6NNia mice strongly suggests that an age-related deterioration in glucose tolerance is a life span-determining process.

Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes: relevance of glycated collagen products versus HbA1c as markers of diabetic complications. DCCT Skin Collagen Ancillary Study Group. Diabetes Control and Complications Trial.

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).

Cell-associated pentosidine as a marker of aging in human diploid cells in vitro and in vivo.

Cellular aging is characterized by alterations at both the morphological and molecular levels, some of which are decreased mitotic rate, increased cytoplasmic vacuolization, and changes in intrinsic cellular constituents (Stanulis-Praeger, 1987. Mech. Ageing Dev. 38, 1-48). In the present investigation, glycoxidation is studied as a marker for cellular aging by measuring cell-associated pentosidine levels in human skin fibroblasts as a function of replicative life span and in human peripheral blood T lymphocytes as a function of chronological age. Fibroblasts were isolated from culture by detachment/centrifugation while lymphocytes were isolated from blood by a Ficoll-Paque/Lympho-Kwik T-Cell Prep technique. Pentosidine levels were measured in acid-hydrolyzed cell pellet suspensions by high-pressure liquid chromatography. Results show that pentosidine was detected in early and late cultured reticular and papillary fibroblasts. Pentosidine, expressed as either protein, DNA, or cell number, significantly (P < 0.0006) increased with in vitro passage and was significantly (P < 0.01) related to cell proliferation as measured by cell density and cell doublings per day during culture. Cell-associated pentosidine was measured in T lymphocytes isolated from healthy, diabetic, and uremic individuals. In healthy controls, levels significantly (P < 0.0003) increased with age. In uremic individuals, a large variation was observed with many values above the 95% confidence intervals determined for controls. Since a previous study showed that plasma pentosidine in healthy subjects does not increase with age, these results suggest that cellular turnover perhaps coupled to a deterioration in cellular anti-glycoxidation defensive mechanisms play a substantial role in explaining increased pentosidine concentrations during cellular aging.

Age-related association of tail tendon break time with tissue pentosidine in DBA/2 vs C57BL/6 mice: the effect of dietary restriction.

In recent years, there has been a growing interest in the development of a panel of biomarkers useful in the evaluation of interventions on aging processes. An ideal marker should change with age, be related to species longevity, and respond to the effects of dietary restriction, which is the only intervention currently known to increase species longevity. In the present study; we compared parameters of collagen aging (i.e., tail tendon break time [TBT] and the glycoxidation product pentosidine) in tendon, ear, and skin of two species of rodents with different life spans: the shorter-lived DBA/2 versus the longer-lived C57BL/6 mouse strain. Both TBT and tissue pentosidine significantly increased with age in both strains of mice. The rate of increase for TBT And pentosidine occurred faster for the DBA/2 compared with the C57BL/6 strain. Dietary restriction significantly inhibited the age-related increase of TBT and pentosidine formation rte in DBA/2 mice. In C57BL/6 mice, the age-related increase of TBT was significantly inhibited by dietary restriction. However, except for tendon at 24 months, pentosidine level was not affected by dietary restriction. These studies show that the rate of collagen aging, as reflected by TBT and glycoxidation, increases proportionally with age, and that these rate increases are related to longevity in two strains of mice. Pentosidine can be monitored with age just as well in a piece of easily accessible ear tissue as in skin or tendon. Thus, pentosidine is expected to be a useful and easily measurable noninvasive marker in future intervention studies on aging.

Ageing promotes the increase of early glycation Amadori product as assessed by epsilon-N-(2-furoylmethyl)-L-lysine (furosine) levels in rodent skin collagen. The relationship to dietary restriction and glycoxidation.

Glucose has been implicated in the aging process by its ability to react nonenzymatically with long-lived proteins like collagen to produce advanced glycosylated end-products (AGEs). In the initial phase of this reaction, referred to as glycation, glucose reacts with the free amino group of proteins resulting in Schiff base formation followed by rearrangement to an Amadori product. Since the Amadori product is transient due to its conversion to other products as well as its reversibility to the initial products, glycation as an age-related marker in collagen has questionable significance. In human studies, glycation of collagen has been found to increase modestly with age. In rodent studies, results are conflicting due to differences in methodology. Thus, it has been concluded that collagen glycation either does not vary or increases modestly with age. In the present study, a C8 HPLC column was used to measure Amadori product formation as the acid-hydrolyzed breakdown product furosine in the skin of rats and mice. Surprisingly, levels were found to increase at a rapid rate during aging of rodents. Impurity of the furosine peak from the use of crude acid-hydrolyzed skin samples was ruled-out because reductive properties and spectroscopic profiles matched those previously described for furosine. In the present study, glycemia was found important in furosine formation as shown by the glycation lowering effects of dietary restriction on collagen. Decreased collagen turnover probably plays a substantial role in explaining the age-related increase in furosine levels in rodent skin collagen.

The mechanism of collagen cross-linking in diabetes: a puzzle nearing resolution.

Considerable interest has been focused in recent years on the mechanism of collagen cross-linking by high glucose in vitro and in vivo. Experiments in both diabetic humans and in animals have shown that over time collagen becomes less soluble, less digestible by collagenase, more stable to heat-induced denaturation, and more glycated. In addition, collagen becomes more modified by advanced products of the Maillard reaction, i.e., immunoreactive advanced glycation end products and the glycoxidation markers carboxymethyllysine and pentosidine. Mechanistic studies have shown that collagen cross-linking in vitro can be uncoupled from glycation by the use of antioxidants and chelating agents. Experiments in the authors' laboratory revealed that approximately 50% of carboxymethyllysine formed in vitro originates from pathways other than oxidation of Amadori products, i.e., most likely the oxidation of Schiff base-linked glucose. In addition, the increase in thermal stability of rat tail tendons exposed to high glucose in vitro or in vivo was found to strongly depend on H2O2 formation. The final missing piece of the puzzle is that of the structure of the major cross-link. We speculate that it is a nonfluorescent nonultraviolet active cross-link between two lysine residues, which includes a fragmentation product of glucose linked in a nonreducible bond labile to both strong acids and bases.

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Structure of advanced Maillard reaction products and their pathological role.

In this article we review recent progress and controversies relating to three areas of the field of advanced glycosylation end-products (AGE). A controversy exists as to whether pyrraline, an AGE detectable by immunohistochemistry in kidneys from patients with renal failure, exists in vivo. Recent data from the authors' laboratory revealed that pyrraline is present in alkaline or protease digests from human skin and plasma. However, the amounts are very low and pyrraline was found to undergo further reactions to form an ether with itself (dipyrraline) as well as a thioether with cysteine. This high reactivity of pyrraline may explain the difficulty of quantitating it accurately in biological material. In contrast, the glycoxidation products carboxymethyllysine (CML) and pentosidine are stable, very resistant to acid hydrolysis and easy to quantitate. They are present in elevated concentrations in the extracellular matrix in diabetes mellitus and ageing. In the diabetic human lens, CML is not elevated, in contrast to pentosidine, suggesting a different mechanism of formation. Recent data in diabetic dogs have shown that pentosidine is elevated only in lenses from poorly controlled dogs, in contrast to LM-1, a fluorophore thought to arise from ascorbate. Further studies are needed to clarify the intracellular mechanism of glycoxidation. The greatest concentrations of AGEs and glycoxidation products are found in patients with end-stage renal disease, and they are almost completely normalized by renal transplantation. Comparison of peritoneal dialysis (PD) with haemodialysis (HD) showed that PD is associated with lower plasma protein pentosidine, possibly due to selective transport of pentosidine-rich protein across the peritoneal wall. Fractionation of plasma proteins from ESRD patients by size showed that 90% of pentosidine is linked to HMW protein and 1-2% is in free form. The mechanism of accelerated glycoxidation in ESRD is still not understood.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.

Differential effects of type 2 (non-insulin-dependent) diabetes mellitus on pentosidine formation in skin and glomerular basement membrane.

Pentosidine is an advanced Maillard/glycation reaction product the formation of which in human skin is significantly increased in Type 1 (insulin-dependent) diabetes mellitus and correlates with the severity of diabetic complications. Preliminary data in a limited number of Type 2 (non-insulin-dependent) diabetic individuals showed that skin pentosidine was not significantly elevated, raising the question of whether statistical power was insufficient for differences to be revealed, or whether pentosidine did not form because biological factors intrinsic to Type 2 diabetes affected the advanced Maillard reaction altogether. To resolve this question, pentosidine levels were measured in 209 human skin samples obtained at autopsy and in purified glomerular basement membranes from 45 subjects of various ages, with and without Type 1 and Type 2 diabetes and uraemia. Pentosidine increased exponentially in skin but curvilinearly in glomerular basement membranes, and reached 75 and 50 pmol/mg collagen at projected 100 years, respectively. Skin levels were not significantly elevated in individuals with Type 2 diabetes (p > 0.05). In contrast, pentosidine levels in glomerular basement membranes were elevated above the 95% confidence interval in the majority of diabetic patients regardless of the type of diabetes and in all individuals on haemodialysis. These data clearly demonstrate that the advanced Maillard reaction is indeed accelerated in Type 2 diabetes and strongly suggest that differences in pentosidine accumulation rates may be due to differences in collagen turnover. In diabetes and uraemia, accelerated Maillard reaction mediated protein crosslinking, as reflected by pentosidine, may contribute to decreased turnover of the extracellular matrix, sclerosis and thickening of basement membranes.

Effects of kidney or kidney-pancreas transplantation on plasma pentosidine.

Tissue and plasma concentrations of pentose-derived glycation end-products ("pentosidine") are elevated in diabetic patients with normal renal function and in both diabetic and nondiabetic patients with end-stage renal disease. To determine the effects of correcting hyperglycemia and/or renal failure on the accumulation of pentosidine, we used reverse phase and ion exchange high performance liquid chromatography to measure this advanced glycation end-product in plasma proteins of diabetic and nondiabetic transplant recipients at various time intervals after kidney-pancreas or kidney transplantation. Changes in plasma pentosidine levels after transplantation were compared to changes in simultaneously obtained glycohemoglobin levels. Both kidney and kidney-pancreas transplantation were accompanied by a dramatic, but incomplete, reduction of plasma pentosidine concentrations within three months of transplantation. Kidney-pancreas transplantation resulted in normal glycohemoglobin levels within three months but offered no advantage over kidney transplantation alone in the partial correction of plasma pentosidine levels. There was no correlation between posttransplant plasma pentosidine and glycohemoglobin levels in either diabetic or nondiabetic transplant recipients. We conclude that renal failure is the major factor accounting for the accumulation of pentosidine in both diabetic and nondiabetic patients with end-stage renal disease. Restoration of euglycemia after kidney-pancreas transplantation provides no additional benefit in reducing plasma pentosidine levels to that achieved by correction of renal failure after kidney transplantation alone.

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 greater than 17 yr. Pentosidine was significantly elevated in all IDDM patients versus control subjects (P less than 0.0001). It correlated strongly with age (P less than 0.0001) and weakly with duration (P less than 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 less than 0.014) and joint stiffness (P less than 0.041). The highest degree of association was with the cumulative grade of individual complication (P less than 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 less than 0.0001), but levels were not significantly correlated with age, diabetes duration, complication, or skin collagen pentosidine (P greater than 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 less than 0.001) and a 23-fold (P less than 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 less than 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.

Pentosidine: a molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia.

Collagen undergoes progressive browning with age and diabetes characterized by yellowing, fluorescence, and cross-linking. The present research was undertaken in order to investigate the nature of the collagen-linked fluorescence. Human collagen was exhaustively cleaved into peptides by enzymatic digestion. Upon purification, a highly fluorescent chromophore was identified and purified from old human collagen. Structure elucidation revealed the presence of an imidazo [4,5-b] pyridinium-type structure acting as a cross-link between arginine, lysine, and a pentose. This advanced glycosylation end-product and protein cross-link results from the reaction of pentoses with proteins and was named pentosidine. Further work indicated that long-term glycosylation of proteins with hexoses also leads to pentosidine formation through sugar fragmentation. The proposed mechanism of pentosidine formation involves the dehydration of the pentose-derived Amadori compound to form an intermediate which is attacked under base catalysis by the guanido group of arginine. The strict requirement for the Amadori rearrangement is uncertain. However, oxidation is definitely involved since pentosidine is not formed in the absence of oxygen. Five-carbon sugars contributing to pentosidine formation could be formed from larger sugars by oxidative fragmentation or from trioses, tetroses, and ketoses by condensation and/or reverse aldol reactions. Pentosidine increases exponentially in human skin at autopsy. Mean age-adjusted skin levels were significantly increased in subjects with uremia and especially in type 1 diabetics with uremia vs. controls. In skin biopsy, levels were significantly elevated in all diabetic (type 1) vs. control subjects. The highest degree of association was with the cumulative grade of diabetic complication (retinopathy, nephropathy, arterial stiffness, and joint stiffness). Pentosidine also forms in various proteins other than collagen, although to a much lesser extent. In blood, pentosidine is mainly associated with plasma proteins and is highly elevated during uremia. In the lens, it is associated with both water-soluble and -insoluble protein fractions and is especially elevated during brunescent cataract formation. The origin of pentosidine in vivo is uncertain. Evidence suggests that the pentoses are the most reactive sugars in pentosidine formation in vitro; however, the origin and importance of free pentoses in vivo, especially during the diabetic state, are not certain. Possible origins include hemolysis and/or a defect in the primary pentose metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)