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.

Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

A novel single base deletion in the LDLR gene (211delG):Effect on serum lipid profiles and the influence of other genetic polymorphisms in the ACE, APOE and APOB genes

A single base deletion (211delG) in the low density lipoprotein receptor (LDLR) gene was shown to cause familial hypercholesterolaemia (FH) in a large family from Northern Ireland. Twenty-four of 52 family members tested had this mutation, 13 of which were newly diagnosed. Mutation-positive individuals had significantly higher mean total-cholesterol (TC) and LDL-cholesterol (LDL-C) than those without 211delG. LDL-C was a more accurate indicator of disease status than TC, When TC levels alone were considered, in individuals over 16 years, a false negative rate (TC <7.5 mmol/l) of 40% was found; however, this fell to 13% based on inclusion of LDL-C levels. Individuals with coronary artery disease (CAD) had significantly higher TC levels than those without CAD and tended to have tendinous xanthomas (TX) and corneal arcus (CA). Genetic polymorphisms in the angiotensin converting enzyme (ACE) and apolipoprotein (ape) B genes did not appear to be associated with lipid levels or with the clinical severity of the disease; however, the apo E e4 allele did show a lipid-raising effect in individuals with the mutation.

Free radical-mediated lipid peroxidation and systemic nitric oxide bioavailability:implications for postexercise hemodynamics

The metabolic vasodilator mediating postexercise hypotension (PEH) is poorly understood. Recent evidence suggests an exercise-induced reliance on pro-oxidant-stimulated vasodilation in normotensive young human subjects, but the role in the prehypertensive state is not known.

Degradable Polymer Networks and Star Polymers Based on Mixtures of Two Cleavable Dimethacrylate Crosslinkers: Synthesis, Characterization, and Degradation

Mixtures of two cleavable dimethacrylate crosslinkers, the hydrolyzable di(methacryloyloxy-1-ethoxy)methane (DMOEM) and the thermolyzable 1,1-ethylene-diol dimethacrylate (EDDMA), were used for the preparation of neat crosslinker polymer networks, randomly crosslinked polymer networks of methyl methacrylate (MMA), and star polymers of MMA, using group transfer polymerization in tetrahydrofuran (THF). All star polymers and randomly crosslinked polymer networks containing mixtures of the hydrolyzable DMOEM and the thermolyzable EDDMA crosslinkers gave THF-soluble final products when subjected to sequential thermolysis and hydrolysis, in this order. When applying sequential hydrolysis and thermolysis, only the star polymers with an EDDMA crosslinker content equal to or higher than 50% gave THF-soluble final products.

Squirrelpox Virus: Assessing Prevalence, Transmission and Environmental Degradation

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Freeze-Dried Strawberries Lower Serum Cholesterol and Lipid Peroxidation in Adults with Abdominal Adiposity and Elevated Serum Lipids

Dietary flavonoid intake, especially berry flavonoids, has been associated with reduced risks of cardiovascular disease (CVD) in large prospective cohorts. Few clinical studies have examined the effects of dietary berries on CVD risk factors. We examined the hypothesis that freeze-dried strawberries (FDS) improve lipid and lipoprotein profiles and lower biomarkers of inflammation and lipid oxidation in adults with abdominal adiposity and elevated serum lipids. In a randomized dose-response controlled trial, 60 volunteers [5 men and 55 women; aged 49 ± 10 y; BMI: 36 ± 5 kg/m2 (means ± SDs)] were assigned to consume 1 of the following 4 beverages for 12 wk: 1) low-dose FDS (LD-FDS; 25 g/d); 2) low-dose control (LD-C); 3) high-dose FDS (HD-FDS; 50 g/d); and 4) high-dose control (HD-C). Control beverages were matched for calories and total fiber. Blood draws, anthropometrics, blood pressure, and dietary data were collected at screening (0 wk) and after 12-wk intervention. Dose-response analyses revealed significantly greater decreases in serum total and LDL cholesterol and nuclear magnetic resonance (NMR)–derived small LDL particle concentration in HD-FDS [33 ± 6 mg/dL, 28 ± 7 mg/dL, and 301 ± 78 nmol/L, respectively (means ± SEMs)] vs. LD-FDS (−3 ± 11 mg/dL, −3 ± 9 mg/dL, and −28 ± 124 nmol/L, respectively) over 12 wk (0–12 wk; all P < 0.05). Compared with controls, only the decreases in total and LDL cholesterol in HD-FDS remained significant vs. HD-C (0.7 ± 12 and 1.4 ± 9 mg/dL, respectively) over 12 wk (0–12 wk; all P < 0.05). Both doses of strawberries showed a similar decrease in serum malondialdehyde at 12 wk (LD-FDS: 1.3 ± 0.2 μmol/L; HD-FDS: 1.2 ± 0.1 μmol/L) vs. controls (LD-C: 2.1 ± 0.2 μmol/L; HD-C: 2.3 ± 0.2 μmol/L) (P < 0.05). In general, strawberry intervention did not affect any measures of adiposity, blood pressure, glycemia, and serum concentrations of HDL cholesterol and triglycerides, C-reactive protein, and adhesion molecules. Thus, HD-FDS exerted greater effects in lowering serum total and LDL cholesterol and NMR-derived small LDL particles vs. LD-FDS in the 12-wk study. These findings warrant additional investigation in larger trials. This trial was registered at clinicaltrials.gov as NCT01883401.

Comparison of 5% versus 15% sucrose intakes as part of a eucaloric diet in overweight and obese subjects:effects on insulin sensitivity, glucose metabolism, vascular compliance, body composition and lipid profile. A randomised controlled trial

AIMS: The effect of dietary sucrose on insulin resistance and the pathogenesis of diabetes and vascular disease is unclear. We assessed the effect of 5% versus 15% sucrose intakes as part of a weight maintaining, eucaloric diet in overweight/obese subjects.

METHODS: Thirteen subjects took part in a randomised controlled crossover study (M:F 9:4, median age 46 years, range 37-56 years, BMI 31.7±0.9 kg/m(2)). Subjects completed two 6 week dietary periods separated by 4 week washout. Diets were designed to have identical macronutrient profile. Insulin action was assessed using a two-step hyperinsulinaemic euglycaemic clamp; glucose tolerance, vascular compliance, body composition and lipid profiles were also assessed.

RESULTS: There was no change in weight or body composition between diets. There was no difference in peripheral glucose utilization or suppression of endogenous glucose production. Fasting glucose was significantly lower after the 5% diet. There was no demonstrated effect on lipid profiles, blood pressure or vascular compliance.

CONCLUSION: A low-sucrose diet had no beneficial effect on insulin resistance as measured by the euglycaemic glucose clamp. However, reductions in fasting glucose, one hour insulin and insulin area under the curve with the low sucrose diet on glucose tolerance testing may indicate a beneficial effect and further work is required to determine if this is the case. Clinical Trial Registration number ISRCTN50808730.

The Host Defence Peptide LL-37 is Susceptible to Proteolytic Degradation by Wound Fluid Isolated from Foot Ulcers of Diabetic Patients

The pleiotropic effects of host defence peptides (HDPs), including the ability to kill microorganisms, enhance re-epithelialisation and increase angiogenesis, indicates a role for these important peptides as potential therapeutic agents in the treatment of chronic, non-healing wounds. However, the maintenance of peptide integrity, through resistance to degradation by the array of proteinases present at the wound site, is a prerequisite for clinical success. In this study we explored the degradation of exogenous LL-37, one such HDP, by wound fluid from diabetic foot ulcers to determine its susceptibility to proteolytic degradation. Our results suggest that LL-37 is unstable in the diabetic foot ulcer microenvironment. Following overnight treatment with wound fluid, LL-37 was completely degraded. Analysis of cleavage sites suggested potential involvement of both host- and bacterial-derived proteinases. The degradation products were shown to retain some antibacterial activity against Pseudomonas aeruginosa but were inactive against Staphylococcus aureus. In conclusion, our data suggest that stabilising selected peptide bonds within the sequence of LL-37 would represent an avenue for future research prior to clinical studies to address its potential as an exogenously-applied therapeutic in diabetic wounds. 

The Application of Computational Chemistry and Chemometrics to Developing a Method for Online Monitoring of Polymer Degradation in the Manufacture of Bioresorbable Medical Implants

Bioresorbable polymers such as PLA have an important role to play in the development of temporary implantable medical devices with significant benefits over traditional therapies. However, development of new devices is hindered by high manufacturing costs associated with difficulties in processing the material. A major problem is the lack of insight on material degradation during processing. In this work, a method of quantifying degradation of PLA using IR spectroscopy coupled with computational chemistry and chemometric modeling is examined. It is shown that the method can predict the quantity of degradation products in solid-state samples with reasonably good accuracy, indicating the potential to adapt the method to developing an on-line sensor for monitoring PLA degradation in real-time during processing.