Effect of collagen turnover on the accumulation of advanced glycation end products

Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p <0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p <0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p <0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p <0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells

We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes.

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine:two products of nonenzymatic modification of aminophospholipids formed in vivo

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

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.

Lipoprotein glycation and its metabolic consequences

Glycation of lipoproteins is implicated in the development of the macro- and microvascular complications of diabetes, atherosclerosis in general, and other disease processes including aging. Enhanced glycation may have direct effects, and may also amplify the effects of oxidative stress on lipoproteins. Most studies have examined the effects of glycation of LDL, particularly with respect to its atherogenicity. Other lipoproteins are more difficult to study because their several apolipoproteins, being of varying age, are not uniformly exposed to glucose. Inhibition of the combined stresses of glycation and oxidation towards lipoproteins may have beneficial effects on health.

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.

Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia reperfusion injury and rheumatoid arthritis. Chemical modification of amino acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative stress in vivo. The focus of the studies described here was initially to identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (HNE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays for the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diimine cross-link and the Michael addition product of HNE to lysine. Using these assays, we showed that the concentrations of all three compounds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine (CML) also increased during LDL oxidation, while that of its putative carbohydrate precursor the Amadori compound N epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating that CML is a marker of both glycoxidation and lipoxidation reactions. These results suggest that MDA and HNE adducts to lysine residues should serve as biomarkers of lipid modification resulting from lipid peroxidation reactions, while CML may serve as a biomarker of general oxidative stress resulting from both carbohydrate and lipid oxidation reactions.

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.

Accumulation of Maillard reaction products in skin collagen in diabetes and aging

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins.

Glycation and oxidation:a role in the pathogenesis of atherosclerosis

Reactions involving glycation and oxidation of proteins and lipids are believed to contribute to atherogenesis. Glycation, the nonenzymatic binding of glucose to protein molecules, can increase the atherogenic potential of certain plasma constituents, including low-density lipoprotein (LDL). Glycation of LDL is significantly increased in diabetic patients compared with normal subjects, even in the presence of good glycemic control. Metabolic abnormalities associated with glycation of LDL include diminished recognition of LDL by the classic LDL receptor; increased covalent binding of LDL in vessel walls; enhanced uptake of LDL by macrophages, thus stimulating foam cell formation; increased platelet aggregation; formation of LDL-immune complexes; and generation of oxygen free radicals, resulting in oxidative damage to both the lipid and protein components of LDL and to any nearby macromolecules. Oxidized lipoproteins are characterized by cytotoxicity, potent stimulation of foam cell formation by macrophages, and procoagulant effects. Combined glycation and oxidation, "glycoxidation," occurs when oxidative reactions affect the initial products of glycation, and results in irreversible structural alterations of proteins. Glycoxidation is of greatest significance in long-lived proteins such as collagen. In these proteins, glycoxidation products, believed to be atherogenic, accumulate with advancing age: in diabetes, their rate of accumulation is accelerated. Inhibition of glycation, oxidation, and glycoxidation may form the basis of future antiatherogenic strategies in both diabetic and nondiabetic individuals.

Lipoprotein glycation and its metabolic consequences

In people with diabetes, glycation of apolipoproteins correlates with other indices of recent glycemic control, including HbA1. For several reasons, increased glycation of apolipoproteins may play a role in the accelerated development of atherosclerosis in diabetic patients. Recognition of glycated LDL by the classical LDL receptor is impaired, whereas its uptake by human monocyte-macrophages is enhanced. These alterations may contribute to hyperlipidemia and accelerated foam-cell formation, respectively. Glycation of LDL also enhances its capacity to stimulate platelet aggregation. The uptake of VLDL from diabetic patients by human monocyte-macrophages is enhanced. This enhancement may be due, at least in part, to increased glycation of its lipoproteins. Glycation of HDL impairs its recognition by cells and reduces its effectiveness in reverse cholesterol transport. Glycation of apolipoproteins may also generate free radicals, increasing oxidative damage to the apolipoproteins themselves, the lipids in the particle core, and any neighboring macromolecules. This effect may be most significant in extravasated lipoproteins. In these, increased glycation promotes covalent binding to vascular structural proteins, and oxidative reactions may cause direct damage to the vessel wall. Glycoxidation, or browning, of sequestered lipoproteins may further enhance their atherogenicity. Finally, glycated or glycoxidized lipoproteins may be immunogenic, and lipoprotein-immune complexes are potent stimulators of foam-cell formation.

Role of glycation in modification of lens crystallins in diabetic and nondiabetic senile cataracts

To assess the significance of glycation, nonenzymatic browning, and oxidation of lens crystallins in cataract formation in elderly diabetic patients, we measured three distinct products of glycation, browning, and oxidation reactions in cataractous lens crystallins from 29 diabetic patients (mean +/- SD age 72.8 +/- 8.8 yr) and 24 nondiabetic patients (age 73.5 +/- 8.3 yr). Compounds measured included 1) fructoselysine (FL), the first stable product of glycation; 2) pentosidine, a fluorescent, carbohydrate-derived protein cross-link between lysine and arginine residues formed during nonenzymatic browning; and 3) N epsilon-(carboxymethyl)lysine (CML), a product of autoxidation of sugar adducts to protein. In diabetic compared with nondiabetic patients, there were significant increases (P less than 0.001) in HbA1 (10.2 +/- 3.1 vs. 7.1 +/- 0.7%), FL (7.6 +/- 5.4 vs. 1.7 +/- 1.2 mmol/mol lysine), and pentosidine (6.3 +/- 2.8 vs. 3.8 +/- 1.9 mumol/mol lysine). The disproportionate elevation of FL compared with HbA1 suggests a breakdown in the lens barrier to glucose in diabetes, whereas the increase in pentosidine is indicative of accelerated nonenzymatic browning of diabetic lens crystallins. CML levels were similar in the two groups (7.1 +/- 2.4 vs. 6.8 +/- 3.0 mmol/mol lysine), providing no evidence for increased oxidative stress in the diabetic cataract. Thus, although the modification of lens crystallins by autoxidation reactions was not increased in diabetes, the increase in glycation and nonenzymatic browning suggests that these processes may acclerate the development of cataracts in diabetic patients.

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)