PubMed Health⌕ Search

Biomedical subjects

J W Baynes

Publications and source records attributed to J W Baynes.

At least 55 records · Page 3Linked to original sources
PubMed1997

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes.

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.

Adolescent↗
PubMed1997

Carboxymethylethanolamine, a biomarker of phospholipid modification during the maillard reaction in vivo.

Nepsilon-(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, approximately 0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n = 22, p >> 0.5). Levels of CML in erythrocyte membrane proteins were approximately 0.2 mmol/mol of lysine for both control and diabetic subjects (p >> 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p = 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.

Adult↗
PubMed1997

N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compounds and in the proteins RNase and collagen. CEL was also detected in human lens proteins at a concentration similar to that of CML, and increased with age in parallel with the concentration of CML. Although CEL was formed in highest yields during the reaction of methylglyoxal and triose phosphates with lysine and protein, it was also formed in reactions of pentoses, ascorbate and other sugars with lysine and RNase. We propose that levels of CML and CEL and their ratio to one another in tissue proteins and in urine will provide an index of glyoxal and methylglyoxal concentrations in tissues, alterations in glutathione homoeostasis and dicarbonyl metabolism in disease, and sources of advanced glycation end-products in tissue proteins in aging and disease.

Adolescent↗
PubMed1997

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Aldehydes↗
PubMed1997

Technical note. The serum concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine is increased in uremia.

Advanced glycation end products (AGEs) such as pentosidine and N epsilon-(carboxymethyl)lysine (CML) have been traditionally quantified by HPLC or gas chromatography--mass spectrometry (GC/MS). Enzyme-linked immunosorbent assays (ELISA) have been introduced as a convenient alternative to simplify the detection and measurement of AGEs in proteins and tissues, but some of these studies are limited by the lack of information on the structure of the epitopes recognized by antibodies to AGE-proteins. In this work we demonstrate that an antibody used in a previous study, reporting increased levels of AGEs in patients with diabetes or on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD), recognizes CML as its major epitope. We also show that there is a significant correlation between the concentration of AGEs in serum measured by ELISA and a GC/MS assay for CML in serum proteins. Both analyses yielded comparable results, with patients on CAPD and HD having about threefold higher AGE- or CML-concentrations in their serum. Our data suggest that ELISA assays for CML should be useful for the clinical measurement of AGEs in serum proteins.

Antibodies↗
PubMed1996

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions.

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

Arachidonic Acid↗
PubMed1996

New biomarkers of Maillard reaction damage to proteins.

The amount of advanced glycation end-products (AGE) in tissue proteins increases in diabetes mellitus, and the concentration of a subclass of AGEs, known as glycoxidation products, also increases with chronological age in proteins. The rate of accumulation of glycoxidation products is accelerated in diabetes and age-adjusted concentrations of two glycoxidation products, N epsilon-(carboxymethyl)lysine (CML) and pentosidine, correlate with the severity of complication in diabetic patients. Although AGEs and glycoxidation products are implicated in the development of diabetic complications, these compounds are present at only trace concentrations in tissue proteins and account for only a fraction of the chemical modifications in AGE proteins prepared in vitro. The future of the AGE hypothesis depends on the chemical characterization of a significant fraction of the total AGEs in tissue proteins, a quantitative assessment of their effects on protein structure and function, and an assessment of their role as mediators of biological responses. In this manuscript we describe recent work leading to characterization of new AGEs and glycoxidation products. These compounds include: (1) the imidazolone adduct formed by reaction of 3-deoxyglucosone with arginine residues in protein; (2) N epsilon-(carboxyethyl)lysine, an analogue of CML formed on reaction of methylglyoxal with lysine; (3) glyoxal-lysine dimer; and (4) methyl-glyoxal-lysine dimer, which are imidazolium crosslinks formed by reaction of glyoxal or methylglyoxal with lysine residues in protein. The presence of 3-deoxyglucosone, methylglyoxal and glyoxal in vivo and the formation of the above AGEs in model carbonyl-amine reaction systems suggests that these AGEs are also formed in vivo and contribute to tissue damage resulting from the Maillard reaction.

Aging↗
PubMed1996

Role of the Maillard reaction in diabetes mellitus and diseases of aging.

Advanced glycation end-products (AGEs) are formed by spontaneous chemical reactions between carbohydrates and tissue proteins. The accumulation of AGEs in long-lived proteins contributes to the age-related increase in brown colour, fluorescence and insolubilisation of lens crystallins and to the gradual crosslinking and decrease in elasticity of connective tissue collagens with age. These nonenzymatic reactions, known collectively as Maillard or browning reactions, are also implicated in the development of pathophysiology in age-related diseases such as diabetes mellitus, atherosclerosis, Alzheimer's disease, and in dialysis-related amyloidosis. Oxygen and oxidation reactions accelerates Maillard reactions in vitro, and the structurally characterised AGEs that accumulate in long-lived tissue proteins are in fact glycoxidation products, formed by sequential glycation and oxidation reactions. In addition to their immediate effects on protein structure and function, AGEs also induce oxidative stress, leading to inflammation and propagation of tissue damage. Thus, glycation of protein, formation of AGEs and resultant oxidative stress, which accelerate Maillard reactions, can initiate an autocatalytic cycle of deleterious reactions in tissues. Pharmacological inhibition of the Maillard reaction should improve the prognosis for a broad range of age-related diseases. The role of oxidative stress as a catalyst and the consequences of Maillard reaction damage in tissues suggests that antioxidant therapy may also retard the progression of age-related pathology.

Aging↗
PubMed1995

Pathways of formation of glycoxidation products during glycation of collagen.

Glycoxidation products (GOPs), such as N epsilon-(carboxymethyl)lysine (CML) and pentosidine, are formed during reaction of glucose with protein under oxidative conditions in vitro. It is uncertain whether these GOPs are derived from oxidation of Amadori adducts on protein or from oxidation of glucose or intermediates formed prior to the Amadori rearrangement. To address this question, we reacted collagen with 250 mM glucose in 200 mM phosphate buffer, pH 7.4, under antioxidative conditions, yielding a protein rich in Amadori adducts, but with only traces of GOPs. This "preglycated" collagen was then exposed to [13C6]glucose under oxidative conditions, producing both natural and [13C2]-CML. At 200 mM phosphate buffer, [13C2]-CML was the major product, even at low (5 mM) [13C6]glucose concentration, indicating a limited role for Amadori compounds in formation of CML in high phosphate. The relative yields of natural and [13C2]-CML varied with phosphate concentration, becoming similar at more physiological (10 mM) phosphate. We conclude that during glycation of proteins at high phosphate concentrations in vitro, GOPs are formed primarily by oxidation of free glucose or rapidly-formed intermediates preceding the Amadori rearrangement, such as carbinolamine or Schiff base adducts. In contrast, at lower phosphate and glucose concentrations in vivo, the Amadori adduct may be the more significant precursor of GOPs. The fact that glycoxidation reactions proceed by multiple routes must be considered in the development of therapeutic approaches for inhibiting the Maillard reaction in diabetes.

Animals↗
PubMed1995

N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins.

Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.

Aging↗
PubMed1995

Identification of liver endothelial cells as the primary site of IgM catabolism in the rat.

Rat IgMs, both monoclonal protein from ascites fluid and total serum IgM, were purified by sequential gel filtration and metal chelate affinity chromatography on immobilized zinc-iminodiacetate. Two monoclonal IgMs, IR202 and IR968, chromatographed identically on gel filtration, but required different pHs for elution from the zinc affinity column. IR202 behaved like a euglobulin, being readily precipitated in low-ionic-strength buffers, while IR968 remained soluble under these conditions. IgM was isolated from serum in 30-50% yield by chromatographic procedures similar to those used for the monoclonal proteins, and 20-30% of the isolated serum IgM was precipitable as a euglobulin. The half-life of both monoclonal and serum euglobulin IgMs was 0.8 days, while the polyclonal globulin and IR968 had half-lives of 1.8 and 2.8 days, respectively, in the rat circulation. The tissue and cellular sites of catabolism of the monoclonal IgMs were determined after labeling with the residualizing label, dilactitol-[125I]tyramine. For both proteins the liver was identified as the major tissue site of catabolism, accounting for 60-80% of degraded protein in the body. When liver was fractionated into parenchymal and nonparenchymal cells (NPC), the NPC were found to account for 86 and 69% of protein recovered in liver, for IR202 and IR968, respectively. Separation of NPC into endothelial (EC) and Kupffer cell populations by elutriation centrifugation revealed that EC contained the majority, approximately 70% of total NPC radioactivity from either IgM. Based on the ratios of endocytic indices (microliter of plasma/10(6) cells/day) for each cell type, the EC also had a higher efficiency for uptake of both IgMs, approximately threefold greater, than for the fluid phase marker, polyvinylpyrrolidone, or for rat serum albumin. We conclude that hepatic EC are a major site of IgM catabolism, regardless of the heterogeneity in physical and biological properties of various IgM populations.

Animals↗
PubMed1995

Metabolism of fructose-3-phosphate in the diabetic rat lens.

Fructose-3-phosphate and sorbitol-3-phosphate are produced in diabetic rat lenses by a 3-phosphokinase. While sorbitol-3-phosphate appears to be an inert polyol phosphate, fructose-3-phosphate is a potent cross-linking agent and a potential in vivo source of 3-deoxyglucosone. The objective of this study was to investigate the production and decomposition of fructose-3-phosphate in the diabetic rat lens. The results indicate that this metabolite achieves a steady-state concentration of almost 1 mumol/g wet weight within 2 weeks after the onset of diabetes. These steady-state levels appear to be a consequence of a balance between its production from fructose and its further decomposition to 3-deoxyglucosone. This conclusion is supported by results from disappearance of fructose-3-phosphate in insulin-treated diabetic rats and in vitro incubations of fructose-3-phosphate with amines where production of 3-deoxyglucosone was detected using a number of different methods including mass spectrometry. In agreement with these results, elevated concentrations of 3-deoxyglucosone along with its detoxification product, 3-deoxyfructose, were detected in the diabetic rat lenses. Other sugars and sugar phosphates which were detectable in the diabetic rat lenses were glucose, fructose, glucose-6-phosphate, fructose-6-phosphate, and sedoheptulose-7-phosphate. In conclusion, results from this study suggest that fructose-3-phosphate and 3-deoxyglucosone are likely to be important contributors to the process of nonenzymatic glycation in diabetic rat lenses.

Animals↗
PubMed1995

Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose.

Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.

Arabinose↗
PubMed1995

Formation of reactive intermediates from Amadori compounds under physiological conditions.

The Maillard or browning reaction between reducing sugars and proteins contributes to the chemical aging of tissue proteins in vivo and to the accelerated aging of proteins in diabetes. To identify reactive carbohydrate intermediates formed in the Maillard reaction under physiological conditions, we studied the decomposition of the model Amadori compound, N alpha-formyl-N epsilon-fructoselysine (fFL) and of Amadori compounds on glycated collagen at pH 7.4 and 37 degrees C. Because of effects of buffer and oxidative conditions on the decomposition of Amadori compounds, the kinetics and products of decomposition were studied in varying phosphate concentrations and in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer under both aerobic and anaerobic conditions. The half-life of fFL was significantly shorter in phosphate, compared to Hepes buffer, and under aerobic, compared to anaerobic, conditions. The decomposition of both fFL and Amadori adducts on glycated collagen was accelerated by increasing the phosphate concentration and/or pH. Glucose and mannose were identified as major products formed by reversal of the Amadori rearrangement, along with tetroses, pentoses, and 3-deoxyglucosone, formed by reverse aldol, rearrangement, and hydrolysis reactions. The tetrose and pentose products included both aldose and ketose sugars. These same products were also formed in similar yields on decomposition of Amadori adducts on glycated collagen in vitro. The spontaneous decomposition of Amadori compounds to more reactive sugars in vivo, including tetroses, pentoses, and 3-deoxyglucosone, provides a mechanism for generating reactive intermediates under physiological conditions and for propagating damage to protein as a result of glycation of proteins by glucose in vivo.

Aerobiosis↗
PubMed1995

Caloric restriction decreases age-dependent accumulation of the glycoxidation products, N epsilon-(carboxymethyl)lysine and pentosidine, in rat skin collagen.

Nonenzymatic glycation of body proteins and subsequent advanced glycation reactions have been implicated in the aging process, while caloric restriction (CR) in rodents results in an increase in both mean and maximum life span. We have evaluated the effect of chronic (25 months) CR on glycation of blood proteins and accumulation of advanced glycation and oxidation (glycoxidation) products, N epsilon-(carboxymethyl)lysine (CML), and pentosidine, in skin collagen. Brown-Norway rats, fed ad libitum (AL) from birth, were divided into two equal groups at 4 months of age and placed on AL or CR diets (CR = 60% of AL diet). Cohorts of animals were sacrificed at 7, 13, and 25 months after the initiation of CR. At necropsy glycated hemoglobin was measured by affinity HPLC and glycated plasma protein by the fructosamine assay; extracts of skin collagen were analyzed by gas chromatography-mass spectrometry for CML and by reversed-phase HPLC for pentosidine. Glycation of hemoglobin, plasma proteins, and skin collagen was decreased significantly (18-33%) by CR. Concentrations of CML and pentosidine increased significantly with age in skin collagen in both AL and CR animals; however, CR significantly reduced levels of CML (25%), pentosidine (50%), and fluorescence (15%) in collagen in the oldest rats. We conclude that CR reduces the extent of glycation of blood and tissue proteins and the age-related accumulation of glycoxidation products in skin collagen.

Aging↗
PubMed1994

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction.

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Animals↗