Active glycation in neurofibrillary pathology of Alzheimer disease: N(epsilon)-(carboxymethyl) lysine and hexitol-lysine.

Advanced glycation end products are a diverse class of posttranslational modifications, stemming from reactive aldehyde reactions, that have been implicated in the pathogenesis of a number of degenerative diseases. Because advanced glycation end products are accelerated by, and result in formation of, oxygen-derived free radicals, they represent an important component of the oxidative stress hypothesis of Alzheimer disease (AD). In this study, we used in situ techniques to assess N(epsilon)-(Carboxymethyl)lysine (CML), the predominant advanced glycation end product that accumulates in vivo, along with its glycation-specific precursor hexitol-lysine, in patients with AD as well as in young and aged-matched control cases. Both CML and hexitol-lysine were increased in neurons, especially those containing intracellular neurofibrillary pathology in cases of AD. The increase in hexitol-lysine and CML in AD suggests that glycation is an early event in disease pathogenesis. In addition, because CML can result from either lipid peroxidation or advanced glycation, while hexitol-lysine is solely a product of glycation, this study, together with studies demonstrating the presence of 4-hydroxy-2-nonenal adducts and pentosidine, provides evidence of two distinct oxidative processes acting in concert in AD neuropathology. Our findings support the notion that aldehyde-mediated modifications, together with oxyradical-mediated modifications, are critical pathogenic factors in AD.

Therapeutic potential of AGE inhibitors and breakers of AGE protein cross-links.

Glucose and other reducing sugars react non-enzymatically with proteins leading to the formation of advanced glycosylation end products (AGEs) and AGE-derived protein cross-linking. Formation of AGEs is a normal physiological process, which is accelerated under the hyperglycaemic condition in diabetes. Under normal conditions, AGEs build up slowly and accumulate as one ages. Numerous studies have indicated that AGEs contribute to the pathological events leading to diabetic complications, such as age-related diseases, including nephropathy, retinopathy, vasculopathy and neuropathy. Potential therapeutic approaches to prevent these complications include pharmacological inhibition of AGE formation and disruption of pre-formed AGE-protein cross-links. Studies using animal models and preliminary clinical trials have shown the ability of the AGE-inhibitor, pimagedine and the cross-link breaker, ALT-711, to reduce the severity of pathologies of advanced glycosylation. These agents offer potential treatments for glucose-derived complications of diabetes and ageing.

Inhibition of advanced glycation endproduct formation by acetaldehyde: role in the cardioprotective effect of ethanol.

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called "French paradox," (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

Elevated AGE-modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs.

BACKGROUND: Advanced glycation endproducts (AGEs) are implicated in the pathogenesis of atherosclerotic vascular disease of diabetic and nondiabetic etiology. Recent research suggests that advanced glycation of ApoB contributes to the development of hyperlipidemia. AGE-specific receptors, expressed on vascular endothelium and mononuclear cells, may be involved in both the clearance of, and the inflammatory responses to AGEs. The aim of this study was to examine whether there is a relationship between serum AGE-ApoB and AGEs in arterial tissue of older normolipidemic nondiabetic patients with occlusive atherosclerotic disease, compared with age-matched and younger asymptomatic persons. MATERIALS AND METHODS: Serum AGE-ApoB was measured by ELISA in 21 cardiac bypass patients. Furthermore, an AGE-specific monoclonal antibody, and polyclonal antibodies against anti-AGE-receptor (anti-AGE-R) 1 and 2 were used to explore the localization and distribution of AGEs and AGE-R immunoreactivity (IR) in arterial segments excised from these patients. RESULTS: Serum AGE-ApoB levels were significantly elevated in the asymptomatic, older population, compared with those in young healthy persons (259 +/- 24 versus 180 +/- 21 AGE U/mg of ApoB, p < 0.01). Higher AGE-ApoB levels were observed in those patients with atherosclerosis (329 +/- 23 versus 259 +/- 24 AGE U/mg ApoB, p < 0.05). Comparisons of tissue AGE-collagen with serum AGE-ApoB levels showed a significant correlation (r = 0.707, p < 0.01). In early lesions, AGE-IR occurred mostly extracellularly. In fatty streaks and dense, cellular atheromatous lesions, AGE-IR was visible within lipid-containing smooth muscle cells and macrophages, while in late-stage, acellular plaques, AGE-IR occurred mostly extracellularly. AGE-R1 and -R2 were observed on vascular endothelial and smooth-muscle cells and on infiltrating mononuclear cells in the early-stage lesions, whereas in dense, late-stage plaques, they colocalized mostly with lipid-laden macrophages. On tissue sections, scoring of AGE-immunofluorescence correlated with tissue AGE and plasma AGE-ApoB. CONCLUSIONS: (1) The correlation between arterial tissue AGEs and circulating AGE-ApoB suggests a causal link between AGE modification of lipoproteins and atherosclerosis. AGE-specific receptors may contribute to this process. (2) Serum AGE-ApoB may serve to predict atherosclerosis in asymptomatic patients.

Formation of plasma advanced glycosylation end products (AGEs) has no influence on plasma viscosity.

Plasma viscosity is mainly determined by large non-spherical proteins. In Type 1 diabetes mellitus, plasma viscosity increases with deterioration of diabetic control. Since protein glycation and formation of advanced glycosylation end products (AGEs) alter the structural and functional properties of proteins, AGEs might influence the rheological properties of plasma proteins. Therefore, we investigated the influence of plasma-AGEs on plasma viscosity in 34 normoalbuminuric diabetic patients (17 Type 1, 17 Type 2) with normal renal and liver function. In an additional experiment, 6 ml plasma of 9 healthy volunteers were incubated under sterile conditions for 14 days at 37.5 degrees C in the presence of 5.2 and 32.9 mmol l(-1) glucose. In diabetic patients, plasma-AGE levels were not correlated with plasma viscosity. Plasma-AGE levels in healthy controls (246 +/- 37 U ml[-1], mean +/- SD) were raised significantly (p<0.001) after the incubation at 37.5 degrees C (392 +/- 57 U ml[-1] and 552 +/- 58 U ml[-1], respectively). However, no difference was found in plasma viscosity pre- and post-incubation (pre-incubation: 1.25 +/- 0.04 mPas, post-incubation: 1.23 +/- 0.03 and 1.24 +/- 0.03, respectively). We conclude that there is no influence of plasma-AGEs on plasma viscosity.

Standardizing the immunological measurement of advanced glycation endproducts using normal human serum.

Advanced glycation endproducts (AGEs) have been linked to many sequelae of diabetes, renal disease and aging. To detect AGE levels in human tissues and blood samples, a competitive enzyme-linked immunosorbent assay (ELISA) has been widely used. As no consensus or standard research method for the quantitation of AGEs currently exists, nor a universally defined AGE unit available, the comparative quantitation of AGEs between research laboratories is problematic and restricts the usefulness of interlaboratory clinical data. By comparing the cross-reactivities of five different anti-AGE antisera with five different in vitro AGE-modified proteins, we found that the immunological recognition of AGEs by competitive ELISA is both AGE-carrier protein- and anti-AGE antibody-dependent. This suggests that in vitro AGE-modified proteins might not be appropriate standards for AGEs that occur naturally in vivo. Based on our observation that serum AGE levels in the normal human population are consistently within a narrow range and several folds lower than in diabetics, we propose a method to standardize AGE units against normal human serum (NHS). In this new method, one AGE unit is defined as the inhibition that results from 1:5 diluted NHS in the competitive AGE-ELISA; thus the AGE value in NHS is 5 units/ml. This NHS method requires a competitive AGE-ELISA with reasonable sensitivity such that 1:5 NHS produces a 25 to 40% inhibition of anti-AGE antibody binding to immobilized AGE-proteins. By using this standardized method we found that the AGE levels in normal human serum (5.0 +/- 2.2 units/ml; mean +/- SD, n = 34) fit a normal distribution (chi 2-test, p < 0.01), and the serum AGE levels in diabetic patients (20.3 +/- 3.8 units/ml, n = 7) are significantly higher than that of the normal population (p < 0.0001). Since AGE units can now be defined against a universally available standard, NHS, the results of quantitative AGE measurements using this method should be comparable between assays and between different laboratories. Taken together, standardizing the AGE-ELISA protocol as described here provides a simple and quantitative method that should facilitate the expanded application of clinical AGE data.

Tobacco smoke is a source of toxic reactive glycation products.

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products "glycotoxins." Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

Reduction of plasma apolipoprotein-B by effective removal of circulating glycation derivatives in uremia.

Patients with diabetes and renal insufficiency (Db/ESRD), a group subject to accelerated atherosclerosis exhibit marked increases in the levels of circulating, glycation-derived reactive substances, termed advanced glycation endoproducts (AGEs). These products have been previously shown to react covalently with apoliprotein B (ApoB) to form AGE-ApoB, a modification that results in delayed low density lipoprotein (LDL) clearance and possibly to dyslipidemia. Because the effect of hemodialysis on AGE removal was shown to be unsatisfactory, based on single intradialytic studies, we examined the effect of long-term hemodialysis therapy on serum AGE-ApoB levels, as well as on total serum ApoB of 25 Db/ESRD patients treated by two types of hemodialysis filters, the Fresenius Inc. F8, as the low flux (LF), or high-flux polysulfone AN69 (HF) for two months using an AGE-specific ELISA. At the end of eight weeks, circulating AGE-ApoB levels were reduced significantly (by 35%) from baseline (P = 0.039) in patients treated by HF compared to a modest 16% reduction noted in patients treated by LF (P = 0.05) N = 12, P = 0.047). Of note, total plasma ApoB was reduced by 27% from baseline (P = 0.02) in patients treated by HF compared to a 6% reduction noted in those treated with LF (P = 0.8). In vitro comparison of AGE mass balance, and mass adsorption by the different filters revealed that the higher efficiency of HF filter was due to greater adsorption. The association of reduced AGE-ApoB levels with a decrease in total circulating ApoB by HF and not by LF dialysis suggests: (1) a causal link between AGE clearance and dyslipidemia in diabetic ESRD, and, (2) that more efficient modes of renal replacement treatment and AGE removal could significantly benefit clinical outcome.

Long-term assessment of glucose control by haemoglobin-AGE measurement.

BACKGROUND: Control of blood glucose is important in reducing both the incidence and the severity of complications in diabetes mellitus. One consequence of long- term hyperglycaemia is the formation and accumulation of advanced glycation end-products (AGEs) on tissue macromolecules. An AGE-modified form of human haemoglobin (Hb-AGE) present at high levels in the red cells of diabetic patients, differs from glucose-derived Amadori product HbA1c in being chemically irreversible and thus persisting for the circulating life of the red cell. We therefore compared Hb- AGE with HbA1c as indicators of long-term blood glucose control. METHODS: In an open study we measured circulating HbA1c and Hb-AGE concentrations in eight patients with poorly controlled non-insulin-dependent diabetes after a switch to subcutaneous insulin therapy and careful blood glucose monitoring. RESULTS: After 16 weeks of insulin therapy, the mean HbA1c had decreased from 13.3 (SD 1.2) to 7.3 (0.9)% and the mean Hb-AGE from 12.1 (1.5) to 7.3 (1.3) U/mg Hb. The rate of Hb-AGE decline was 23% slower than that of HbA1c (p=0.044). INTERPRETATION: The observation that Hb-AGE declines more slowly than HbA1c is consistent with the irreversible nature of the AGE product. Because of this property, Hb-AGE may prove superior to HbA1c as a long-term index of circulating glucose concentrations.

Serum advanced glycosylation end-products in patients on hemodialysis and CAPD.

OBJECTIVES: Part I: To evaluate the long-term effects of daily glucose absorption from the peritoneal dialysis fluid on the formation of low-molecular-weight advanced glycosylation end-products (AGE-peptides) in nondiabetic continuous ambulatory peritoneal dialysis (CAPD) patients. Part II: To determine the acute effect of CAPD on serum AGE-peptide concentrations. DESIGN: Part I: Noninterventional, parallel, cross-sectional clinical trial. Part II: Crossover clinical trial. SETTING: A university-based hospital, and clinics. PATIENTS: Part I: Sixty nondiabetic subjects recruited into three age-matched (+/- 5 years) groups, as follows: 20 healthy volunteers (controls); 20 hemodialysis patients; and 20 CAPD patients. Part II: Eight patients with diabetes mellitus (type I or II) and chronic renal failure who were about to undergo CAPD. INTERVENTION: Part I: None. Part II: Uninterrupted CAPD, as medically required. MEASUREMENTS: Part I: To determine serum AGE-peptide concentrations blood samples were obtained randomly from controls and CAPD patients, and predialysis from hemodialysis patients. Hemoglobin A1c was also measured in all subjects. Part II: To determine serum AGE-peptide concentrations, blood samples were collected within one month prior to initiation of CAPD (predialysis) and, again, one week after initiation of uninterrupted CAPD (postdialysis). Hemoglobin A1c was measured predialysis. RESULTS: Part I: Mean hemoglobin A1c values for all groups were within the normal range; however, the mean value for CAPD patients was significantly higher than for both hemodialysis patients and healthy controls (controls, 5.21% +/- 0.6%; hemodialysis, 5.12% +/- 0.5%; CAPD, 5.78% +/- 0.6%; p < 0.01). The dialysis patients had a significantly higher mean serum AGE-peptide concentration than the control subjects (controls, 7.02 +/- 3.4 units/mL; hemodialysis, 11.9 +/- 3.6 units/mL; CAPD, 11.1 +/- 4.5 units/mL; p < 0.01). There was no difference in the mean serum AGE-peptide concentration of patients in the hemodialysis and CAPD groups. Part II: The mean hemoglobin A1c value in the diabetic predialysis patients was 9.2% +/- 1.9%. There was no difference between the predialysis and postdialysis serum AGE-peptide concentrations (predialysis, 16.9 +/- 9.6 units/mL; postdialysis, 16.0 +/- 2.9 units/mL; p = 0.78). CONCLUSIONS: Despite the increased glucose load and the higher hemoglobin A1c values, indicating poor glycemic control, nondiabetic CAPD patients did not have higher serum AGE-peptide concentrations than the nondiabetic hemodialysis patients. In diabetic patients, CAPD did not further increase the serum concentrations of AGE-peptides.