Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.

Increased levels of advanced glycation endproducts in the lenses and blood vessels of cigarette smokers.

BACKGROUND: Advanced glycation endproducts (AGEs) arise from the spontaneous reaction of reducing sugars with the amino groups of macromolecules. AGEs accumulate in tissue as a consequence of diabetes and aging and have been causally implicated in the pathogenesis of several of the end-organ complications of diabetes and aging, including cataract, atherosclerosis, and renal insufficiency. It has been recently proposed that components in mainstream cigarette smoke can react with plasma and extracellular matrix proteins to form covalent adducts with many of the properties of AGEs. We wished to ascertain whether AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers.

MATERIALS AND METHODS: Lens and coronary artery specimens from nondiabetic smokers and nondiabetic nonsmokers were examined by immunohistochemistry, immunoelectron microscopy, and ELISA employing several distinct anti-AGE antibodies. In addition, lenticular extracts were tested for AGE-associated fluorescence by fluorescence spectroscopy.

RESULTS: Immunoreactive AGEs were present at significantly higher levels in the lenses and lenticular extracts of nondiabetic smokers (p < 0.003). Anti-AGE immunogold staining was diffusely distributed throughout lens fiber cells. AGE-associated fluorescence was significantly increased in the lenticular extracts of nondiabetic smokers (p = 0.005). AGE-immunoreactivity was significantly elevated in coronary arteries from nondiabetic smokers compared with nondiabetic nonsmokers (p = 0.015).

CONCLUSIONS: AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers than in nonsmokers, irrespective of diabetes. In view of previous reports implicating AGEs in a causal association with numerous pathologies, these findings have significant ramifications for understanding the etiopathology of diseases associated with smoking, the single greatest preventable cause of morbidity and mortality in the United States.

Acid-catalyzed hydrolysis of cellulose and cellulosic waste using a microwave reactor system

A microwave reactor system was investigated as a potential technique to maximize sugar yield for the hydrolysis of municipal solid waste for ethanol production. Specifically, dilute acid hydrolysis of a-cellulose and waste cellulosic biomass (grass clippings) with phosphoric acid was undertaken within the microwave reactor system. The experimental data and reaction kinetic analysis indicate that the use of a microwave reactor system can successfully facilitate dilute acid hydrolysis of cellulose and waste cellulosic biomass, producing high yields of total sugars in short reaction times. The maximum yield of reducing sugars was obtained at 7.5% (w/v) phosphoric acid and 160 degrees C, corresponding to 60% of the theoretical total sugars, with a reaction time of 5 min. When using a very low acid concentration (0.4% w/v) for the hydrolysis in the microwave reactor, it was found that 10 g of total sugars/100 g dry mass was produced, which is significant considering the low acid concentration. When hydrolyzing grass clippings using the microwave reactor, the optimum conditions were an acid concentration of 2.5% (w/v), 175 degrees C with a 15 min reaction time, giving 18 g/100 g dry mass of total sugars, with xylose being the sugar with the highest yield. It was observed that pentose sugars were more easily formed but also more easily degraded, these being significantly affected by increases in acid concentration and temperature. Kinetic modeling of the data indicated that the use of microwave heating may account for an increase in reaction rate constant, k(1), found in this study in comparison with conventional systems described in the literature.

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.

The Maillard reaction in vivo

The Maillard or browning reaction between reducing sugars and protein contributes to the chemical deterioration and loss of nutritional value of proteins during food processing and storage. This article presents and discusses evidence that the Maillard reaction is also involved in the chemical aging of long-lived proteins in human tissues. While the concentration of the Amadori adduct of glucose to lens protein and skin collagen is relatively constant with age, products of sequential glycation and oxidation of protein, termed glycoxidation products, accumulate in these long-lived proteins with advancing age and at an accelerated rate in diabetes. Among these products are the chemically modified amino acids, N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxymethyl)hydroxylysine (CMhL), and the fluorescent crosslink, pentosidine. While these glycoxidation products are present at only trace levels in tissue proteins, there is strong evidence for the presence of other browning products which remain to be characterized. Mechanisms for detoxifying reactive intermediates in the Maillard reaction and catabolism of extensively browned proteins are also discussed, along with recent approaches for therapeutic modulation of advanced stages of the Maillard reaction.

Post-slaughter changes in ATP metabolites, Reducing and phosphorylated sugars in chicken meat

The formation of ATP breakdown products in chicken M. pectoralis major post-slaughter is reported. The concentrations of metabolites were followed in chicken breast throughout the carcass processing post-slaughter and during chilled storage. The concentration of glucose remains similar throughout the period whilst that of glucose-6-phosphate decreases linearly. Glucose and glucose-6-phosphate concentrations were inversely related to the pHu of the breast meat throughout chilled storage. Rapid post-mortem glycolysis and high pHu values suggest the occurrence of stress at and pre-slaughter. Whilst ATP, ADP and AMP were rapidly broken down, the concentration of IMP rose rapidly and remained high. Concentrations of inosine, ribose and hypoxanthine increased gradually post-slaughter but an initial increase in ribose phosphate was not sustained. Most of the potential ribose present in chicken meat, believed to be important for flavor formation, remains bound in the form of inosine and IMP. There is evidence that additional breakdown pathways for ribose and ribose-5-phosphate may deplete the concentrations of these precursors.

Effect of pH and temperature on the formation of volatile compounds in cysteine/reducing sugar/starch mixtures during extrusion cooking

Mixtures of cysteine, reducing sugar (xylose or glucose), and starch were extrusion cooked using feed pH values of 5.5, 6.5, and 7.5 and target die temperatures of 120, 150, and 180 degreesC. Volatile compounds were isolated by headspace trapping onto Tenax and analyzed by gas chromatography-mass spectrometry. Eighty and 38 compounds, respectively, were identified from extrudates prepared using glucose and xylose. Amounts of most compounds increased with temperature and pH. Aliphatic sulfur compounds, thiophenes, pyrazines, and thiazoles were the most abundant chemical classes for the glucose samples, whereas for xylose extrudates highest levels were obtained for non-sulfur-containing furans, thiophenes, sulfur-containing furans, and pyrazines. 2-Furanmethanethiol and 2-methyl-3-furanthiol were present in extrudates prepared using both sugars, but levels were higher in xylose samples. The profiles of reaction products were different from those obtained from aqueous or reduced-moisture systems based on cysteine and either glucose or ribose.

Facing up to binge drinking: Reducing binge drinking in adolescent males

Objective: To evaluate the effectiveness of an oro-facial trauma-based brief intervention, designed to raise adolescent males’ awareness about the immediate dangers of binge drinking.
Design: Non-randomised controlled exploratory trial
Setting: Secondary level schools
Materials & Methods: Pre, post and follow up validated questionnaires were used to assess a variety of descriptive data and changes in behaviour. Sixty Year 12 students were recruited in the pilot study and 182 in the definitive study.
Intervention: A brief visual presentation containing salient information and anonymised photographs relating to oro-facial injuries.
Main Outcome Measures: Intention to binge drink.
Results: The majority of participants obtained alcohol from off-license or licensed premises. At the commencement of the study, 68% of the participants were regular drinkers. Whilst there was no change in drinking behaviour, the intervention group reported that it was significantly more likely (compared to the control group) that they would reduce their drinking to less than binge levels.
Conclusion: The intervention resulted in participants reporting a more negative attitude towards binge drinking and increased their intention to disengage from binge drinking.