PubMed Health⌕ Search

Biomedical subjects

V M Monnier

Publications and source records attributed to V M Monnier.

At least 19 recordsLinked to original sources
PubMed2003

Methylglyoxal-bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase, nuclear factor kappaB and intracellular reactive oxygen species formation.

Accumulating evidence suggests that the pathophysiology of diabetes is analogous to chronic inflammatory states. Circulating levels of inflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNFalpha) are increased in both type 1 and type 2 diabetes. TNFalpha plays an important role in the pathogenesis of insulin resistance in type 2 diabetes. However, the reason for this increase remains unclear. Levels of the dicarbonyl methylglyoxal (MGO) are elevated in diabetic plasma and MGO-modified bovine serum albumin (MGO-BSA) can trigger cellular uptake of TNF. Therefore we tested the hypothesis that MGO-modified proteins may cause TNFalpha secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFalpha release in a dose-dependent manner. MGO-modified ribonuclease A and chicken egg ovalbumin had similar effects. Cotreatment of cells with antioxidant reagent N-acetylcysteine (NAC) inhibited MGO-BSA-induced TNFalpha secretion. MGO-BSA stimulated the simultaneous activation of p44/42 and p38 mitogen-activated protein kinase. PD98059, a selective MEK inhibitor, inhibited MGO-BSA-induced TNFalpha release as well as ERK phosphorylation. Pretreatment of cells with NAC also resulted in inhibition of MGO-BSA-induced ERK phosphorylation. MGO-BSA induced dose-dependent NFkappaB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFkappaB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFkappaB. Furthermore, the NFkappaB inhibitor parthenolide suppressed MGO-BSA-induced TNFalpha secretion. Confocal microscopy using dichlorofluorescein to demonstrate intracellular reactive oxygen species (ROS) showed that MGO-BSA produced more ROS compared with native BSA. MGO-BSA could also stimulate protein kinase C (PKC) translocation to the cell membrane, considered a key signaling pathway in diabetes. However, there was no evidence that PKC was involved in TNFalpha release based on inhibition by calphostin C and staurosporine. Our findings suggest that the presence of chronically elevated levels of MGO-modified bovine serum albumin may contribute to elevated levels of TNFalpha in diabetes.

Animals↗
PubMed2003

Enzymatic deglycation with amadoriase enzymes from Aspergillus sp. as a potential strategy against the complications of diabetes and aging.

The Amadori compound is the major single modification of the extracellular matrix by the Maillard reaction in vivo. It is also a source of biologically active glycoxidation products and the formation of glucosepane, the major protein cross-link found in biological tissues so far. For this reason, introduction of deglycating enzymes as anti-aging strategy would be desirable. This article provides an update on amadoriase enzymes from fungi which, one day, will hopefully help prevent the in vivo consequences of glycation.

Aging↗
PubMed2001

Kinetic studies, mechanism, and substrate specificity of amadoriase I from Aspergillus sp.

Amadoriase is a flavoenzyme that catalyzes the oxidative deglycation of Amadori products (fructosyl amino acids or aliphatic amines) to yield free amine, glucosone, and hydrogen peroxide. The mechanism of action of amadoriase I from Aspergillus sp. has been investigated by stopped-flow kinetic studies using fructosyl propylamine and O(2) as substrates in 10 mM Tris HCl, pH 7.9, 4 degrees C. Using both substrate analogues and fast kinetic techniques, the active configuration of the substrate was found to be the beta-pyranose form. Stopped-flow studies showed that the reductive half-reaction is triphasic and generates intermediates that absorb at long wavelengths and is consistent either with (i) the reaction of the substrate with the flavin followed by iminium deprotonation or hydrolysis and then product release or with (ii) the formation of flavin reduction intermediates (carbanion equivalents or adducts), followed by product release. The rate of product release after flavin reduction is lower than the aerobic turnover rate, 14.4 s(-1), suggesting that it is not involved in the catalytic cycle and that reoxidation of the reduced enzyme occurs in the E(red)-product complex. In the oxidative half-reaction, the reduced flavin is oxidized by O(2) in a single phase. The observed rate constant has a linear dependence on oxygen concentration, giving a bimolecular rate constant of 4.9 x 10(4) M(-1) s(-1) in the absence of product, and 3.6 x 10(4) M(-1) s(-1) when the product is bound. The redox potentials of amadoriase have been measured at pH 7.0, 25 degrees, giving values of +48 and -52 mV for the oxidized enzyme/anionic semiquinone and anionic semiquinone/reduced enzyme couples, respectively.

Amino Acid Oxidoreductases↗
PubMed2001

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.

Aging↗
PubMed2000

6-Deoxy-6-fluoro-L-ascorbic acid: crystal structure and oxidative degradation.

Ascorbic acid and its oxidation products have been implicated in non-enzymatic modification of proteins in aging and diseases of oxidative stress. We have studied the feasibility of using 6-deoxy-6-fluoroascorbic acid (6) for identification of ascorbic acid degradation products by 19F NMR spectroscopy. Crystals of compound 6 from nitromethane belonged to the space group P2(1) with a = 5.547(2), b = 6.769(3), c = 9.302(2) A, beta = 91.80(3) degrees and Z = 2. Atomic coordinates, bond lengths and angles, hydrogen coordinates, anisotropic and isotropic displacement parameters were similar if not identical with those of native ascorbic acid. Similarly, UV properties and oxidation kinetics by CuCl2 at different pH values were essentially identical with ascorbic acid. Using 750 MHz 19F NMR spectroscopy, five to six new fluorinated products were detected after overnight oxidation of 6 with Cu2+, suggesting that 6 may be a powerful and sensitive tool for assessment of its catabolism in vivo.

Ascorbic Acid↗
PubMed2000

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.

Animals↗
PubMed2000

Cloning of amadoriase I isoenzyme from Aspergillus sp.: evidence of FAD covalently linked to Cys342.

Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H(2)O(2). We previously reported the purification and characterization of two amadoriase isoenzymes from Aspergillus fumigatus and the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization-collisional-activated dissociation tandem mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.

Amino Acid Oxidoreductases↗
PubMed2000

Mechanisms for the formation of glycoxidation products in end-stage renal disease.

BACKGROUND: Advanced glycation end products (AGEs) accumulate on tissue and plasma proteins in patients with renal failure far in excess of normal aging or diabetes. The aim of these studies was to elucidate the nature of the precursors and the pathways that lead to an accelerated formation of two structurally identified AGEs [pentosidine and Nepsilon(carboxymethyl)lysine (CML)] in the uremic milieu. METHODS: Serum levels of the glycoxidation products, pentosidine and CML, were quantitated by high-performance liquid chromatography in uremic patients treated by dialysis. The formation of early glycation products (as furosine) and late glycoxidation products was modeled in uremic serum and in spent peritoneal dialysate. RESULTS: Clinical factors that affect circulating levels of AGEs included dialysis clearance and dialyzer membrane pore size, but not the presence or absence of diabetes. Both pentosidine and CML form at an accelerated rate in serum from uremic patients. Chelating agents most effectively slow the formation in vitro. In uremic fluids, the primary mechanism of formation of pentosidine is through the Amadori pathway. The primary mechanism of formation of CML is through metal-chelated autoxidation of reducing sugars generating reactive carbonyl precursors. In uremic serum, the presence of an unidentified reactive low molecular weight precursor accelerates the formation of pentosidine. CONCLUSIONS: The formation of the two glycoxidation products, pentosidine and CML, proceeds by different pathways and is enhanced by different precursors in the uremic milieu. The formation of both AGEs is markedly enhanced by metal-catalyzed reactions, evidence for the presence of increased metal-ion mediated oxidant stress in uremia.

Adult↗
PubMed2000

Studies of renal injury III: lipid-induced nephropathy in type II diabetes.

UNLABELLED: Studies of renal injury III: Lipid-induced nephropathy in type II diabetes. BACKGROUND: Nephrotoxicity from elevated circulating lipids occurs in experimental and clinical situations. We tested the hypothesis that lipid-induced nephropathy causes advanced renal failure in rats with type II diabetes and dyslipidemia. METHODS: First generation (F1) hybrid rats derived from the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) and the LA/NIH-corpulent rat (LA/N-fa) were studied for 41 weeks while being on specific diets. Group 1 (14 rats) ingested 11.5% protein, 47.9% fat, and 40.6% carbohydrate. Group 2 (8 rats) ingested 26.9% protein, 16.7% animal fat, and 56.4% carbohydrate, and group 3 (20 rats) ingested 20.2% protein, 40.4% soy and coconut oil, and 39.4% carbohydrate. RESULTS: Hyperglycemia was more severe in rat groups 1 and 2 than in group 3. In contrast, circulating cholesterol and hydroperoxide levels were highest in group 3, intermediate in group 2, and lowest in group 1. Group 3 had severe renal failure secondary to glomerulosclerosis and tubulointerstitial disease, with striking deposition of the lipid peroxidation stress biomarker 4-hydroxynonenal in glomeruli and renal microvessels. Moreover, in group 3, increased arterial wall thickness also connoted vascular injury. In contrast, the glycoxidation stress biomarkers pentosidine and carboxymethyl-lysine were preferentially localized to renal tubules of hyperglycemic rats in groups 1 and 2 and did not segregate with the most severe renal injury. Glomerular and interstitial fibrosis was accompanied by proportional increases in renal transforming growth factor-beta1 levels, which were threefold higher in the hypercholesterolemic rats of group 3 than in the hyperglycemic rats of group 1. CONCLUSIONS: Acquisition of non-nodular glomerular sclerosis and tubulointerstitial disease is dependent on lipoxidation stress in rats with type II diabetes. On the other hand, in the absence of hypercholesterolemia, prolonged glycoxidation stress does not appear to be uniquely nephrotoxic.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Transition metal-catalyzed oxidation of ascorbate in human cataract extracts: possible role of advanced glycation end products.

PURPOSE: With age, human lens crystallins become more pigmented, oxidized, modified by ascorbate oxidation and advanced glycation end products (AGEs), and bind copper. The hypothesis was tested that the major AGE and ascorbylation product in the human lens, N(epsilon)-carboxymethyl-L-lysine (CML), has an EDTA-like structure, which may predispose it to bind redox active copper. METHODS: Young, old, and cataractous human lens protein fractions were glycated with ascorbic acid and tested for their ability to bind Cu(II) by atomic absorption spectroscopy and oxidize (14C1)-ascorbate by radiometric thin-layer chromatography method. AGEs were assayed by high-performance liquid chromatography (HPLC). CML-rich proteins were immunoprecipitated from young, old, and cataractous crystallins using affinity-purified CML antibody and tested for their ability to oxidize ascorbate and generate hydroxyl radicals in the presence of H2O2 using 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trap and EPR spectroscopy. RESULTS: Ascorbate oxidizing activity at 24 hours of native crystallins was significantly increased in both the water soluble (WS; P < 0.001) and insoluble (WIS; P < 0.05) fractions from cataractous and normal lenses. The chelator DTPA completely prevented oxidation up to 24 hours of incubation but less effectively thereafter. Mean endogenous Cu content in pooled young, old, and cataract fractions increased from 0.016 to 0.026 nmol/mg protein, respectively, in WS (P < 0.05) and WIS (P < 0.001) fractions, and Cu(II) binding was 20% to 30% increased in cataractous versus old and young lenses in WS (P < 0.01) and WIS (P < 0.001) fractions. Mean levels of the AGEs, CML, and pentosidine were markedly elevated in WS and WIS fractions from cataractous versus old or young crystallins (20% to severalfold, P < 0.05 to P < 0.001). In a separate experiment, protein-bound Fe was not elevated. Crystallins ascorbylated in vitro showed an increase in CML as well as Cu(II) binding. CML-rich proteins (immunoprecipitated from cataractous lenses) oxidized ascorbate approximately 4 times faster than similar proteins from young and old normal lenses (P < 0.01) and generated hydroxyl radicals in the presence of H2O2 and DMPO. CONCLUSIONS: The association between CML formation, copper binding, and generation of free radicals by cataractous lens crystallins can be duplicated by ascorbylation in vitro. These effects are only in part attributable to CML itself, and other modifications (AGEs, conformational changes) may participate in the process. A vicious cycle between AGE formation, lipoxidation, and metal binding may exist in the aging lens, suggesting that chelation therapy could be beneficial in delaying cataractogenesis.

Ascorbic Acid↗
PubMed1999

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, (13)C 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 to N(epsilon)-(carboxymethyl)lysine and pentosidine. Ascorbate, D-pentoses, and D-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.

Adolescent↗
PubMed1999

Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox active copper binding: relevance to diseases of glycoxidative stress.

Aging and age-related diseases are associated with the production of reactive oxygen species which modify lipids, proteins and DNA. Here we hypothesized the glyco- and lipoxidation product N(epsilon)-(carboxymethyl)lysine (CML) in proteins should bind divalent and redox active transition metal binding. CML-rich poly-L-lysine and bovine serum albumin (BSA) were chemically prepared and found to bind non-dialyzable Cu(2+), Zn(2+) and Ca(2+). CML-BSA-copper complexes oxidized ascorbate and depolymerized protein in the presence of H(2)O(2). CML-rich tail tendons implanted for 25 days into the peritoneal cavity of diabetic rats had a 150% increase in copper content and oxidized ascorbate three times faster than controls. CML-rich proteins immunoprecipitated from serum of uremic patients oxidized four times more ascorbate than control and generated spin adducts of DMPO in the presence of H(2)O(2). The chelator DTPA suppressed ascorbate oxidation thereby implicating transition metals in the process. In aging and disease, CML accumulation may result in a deleterious vicious cycle since CML formation itself is catalyzed by lipoxidation and glycoxidation.

Animals↗
PubMed1999

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).

Adolescent↗
PubMed1998

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.

Arginine↗
PubMed1997

Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy.

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.

Adolescent↗