Lipoxidation adducts with peptides and proteins:deleterious modifications or signalling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. © 2013 Elsevier B.V.

Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group. © 2013 The Authors.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles

The association between glycated haemoglobin levels and P100 visual evoked potentials in diabetes mellitus

Diabetes mellitus (DM) is a metabolic disorder which is characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action or both. The long-term specific effects of DM include the development of retinopathy, nephropathy and neuropathy. Cardiac disease, peripheral arterial and cerebrovascular disease are also known to be linked with DM. Type 1 diabetes mellitus (T1DM) accounts for approximately 10% of all individuals with DM, and insulin therapy is the only available treatment. Type 2 diabetes mellitus (T2DM) accounts for 90% of all individuals with DM. Diet, exercise, oral hypoglycaemic agents and occasionally exogenous insulin are used to manage T2DM. The diagnosis of DM is made where the glycated haemoglobin (HbA1c) percentage is greater than 6.5%. Pattern-reversal visual evoked potential (PVEP) testing is an objective means of evaluating impulse conduction along the central nervous pathways. Increased peak time of the visual P100 waveform is an expression of structural damage at the level of myelinated optic nerve fibres. This was an observational cross sectional study. The participants were grouped into two phases. Phase 1, the control group, consisted of 30 healthy non-diabetic participants. Phase 2 comprised of 104 diabetic participants of whom 52 had an HbA1c greater than 10% (poorly controlled DM) and 52 whose HbA1c was 10% and less (moderately controlled DM). The aim of this study was to firstly observe the possible association between glycated haemoglobin levels and P100 peak time of pattern-reversal visual evoked potentials (PVEPs) in DM. Secondly, to assess whether the central nervous system (CNS) and in particular visual function is affected by type and/or duration of DM. The cut-off values to define P100 peak time delay was calculated as the mean P100 peak time plus 2.5 X standard deviations as measured for the non-diabetic control group, and were 110.64 ms for the right eye. The proportion of delayed P100 peak time amounted to 38.5% for both diabetic groups, thus the poorly controlled group (HbA1c > 10%) did not pose an increased risk for delayed P100 peak time, relative to the moderately controlled group (HbA1c ≤ 10%). The P100 PVEP results for this study, do however, reflect significant delay (p < 0.001) of the DM group as compared to the non-diabetic group; thus, subclincal neuropathy of the CNS occurs in 38.5% of cases. The duration of DM and type of DM had no influence on the P100 peak time measurements.

Biomarkers

Introduction – Why do we need ‘biomarkers? Biomarkers of protein oxidation Introduction Major issues/questions Protein carbonyl biomarkers Biochemistry Methods of measurement Storage, stability and limitations in use Protein thiol biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Aliphatic amino acid biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Oxidised Tryptophan Biomarkers Biochemistry Method of measurement Storage, stability and limitations on use Oxidised tyrosine biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Formation of neoepitopes on oxidised proteins Validation of assays for protein oxidation biomarkers Relationship of protein oxidation to disease Modulation of protein oxidation biomarkers by antioxidants Future perspectives Introduction to lipid peroxidation biomarkers Introduction: biochemistry of lipid peroxidation Malondialdehyde Methods of measurement Storage, stability and limitations on use Conjugated dienes Method of measurement Storage, stability and limitations of use LDL lag phase Method of measurement Storage, stability and limitations of use Hydrocarbon gases Biochemistry Method of measurement Storage, stability and limitations on use Lipofuscin Biochemistry Method of measurement Storage, stability and limitation on use Lipid peroxides Biochemistry Method of measurement Storage, stability and limitations on use Isoprostanes Biochemistry Method of measurement Storage, stability and limitations on use Possible new biomarkers of lipid oxidation Relationship of lipid peroxidation to disease Modulation of lipid peroxidation biomarkers by antioxidants Functional consequences of lipid peroxidation Contribution of dietary intake to lipid peroxidation products Biomarkers of DNA oxidation Introduction Confounding factors Units and terminology Nuclear and mitochondrial DNA damage Lymphocytes as surrogate tissues Measurement of DNA damage with the comet assay Practical details Storage, stability, and limitations of the assay Measurement of DNA base oxidation by HPLC Practical details Storage, stability and limitations of the method Measurement of DNA base oxidation by GC–MS Biochemistry of 8-oxoguanine, adenine and fapy derivatives Methods of measurement Storage, stability and limitations of the method Analysis of guanine oxidation products in urine Method of measurement Limitations and criticisms Immunochemical methods Methods of measurement Storage, stability, and limitations of the assay 32P post-labelling Method of measurement Limitations and criticisms Validation of assays for DNA oxidation Oxo-dGuo in lymphocyte DNA Urinary measurements DNA–aldehyde adducts Biochemistry Method of measurement Products of reactive nitrogen species Endpoints arising from oxidative DNA damage Mutations Chromosome aberrations Micronuclei Site-specific DNA damage Relationship of DNA oxidation to disease Modulation of DNA oxidation biomarkers by antioxidants Direct and indirect effects of oxidative stress: measures of total oxidant/antioxidant levels Visualisation of cellular oxidants Biochemistry: histochemical detection of ROS Method of measurement Limitations, storage and stability Measurement of hydrogen peroxide Biochemistry Methods of measurement Storage, stability and limitations on use Measurement of the ratio of antioxidant/oxidised antioxidant Biochemistry Method of measurement Storage, stability and limitations on use Total antioxidant capacity Biochemistry Terminology Methods of measurement Storage, stability and limitations on use Validation of assays for direct oxidant and antioxidant biomarkers Relationship of oxidant/antioxidant measurement to disease Modulation of oxidant/antioxidant biomarkers by dietary antioxidants Induction of genes in response to oxidative stress Background Measurement of antioxidant responsive genes and proteins Effects of antioxidant intake on the activity of antioxidant enzymes

Immunochemical detection of glyoxal DNA damage

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Self monitoring of blood glucose in type 2 diabetes:Longitudinal qualitative study of patients' perspectives

Objective: To explore views of patients with type 2 diabetes about self monitoring of blood glucose over time. Design: Longitudinal, qualitative study. Setting: Primary and secondary care settings across Lothian, Scotland. Participants: 18 patients with type 2 diabetes. Main outcome measures: Results from repeat in-depth interviews with patients over four years after clinical diagnosis. Results: Analysis revealed three main themes - the role of health professionals, interpreting readings and managing high values, and the ongoing role of blood glucose self monitoring. Self monitoring decreased over time, and health professionals' behaviour seemed crucial in this: participants interpreted doctors' focus on levels of haemoglobin A1c, and lack of perceived interest in meter readings, as indicating that self monitoring was not worth continuing. Some participants saw readings as a proxy measure of good and bad behaviour - with women especially, chastising themselves when readings were high. Some participants continued to find readings difficult to interpret, with uncertainty about how to respond to high readings. Reassurance and habit were key reasons for continuing. There was little indication that participants were using self monitoring to effect and maintain behaviour change. Conclusions: Clinical uncertainty about the efficacy and role of blood glucose self monitoring in patients with type 2 diabetes is mirrored in patients' own accounts. Patients tended not to act on their self monitoring results, in part because of a lack of education about the appropriate response to readings. Health professionals should be explicit about whether and when such patients should self monitor and how they should interpret and act upon the results, especially high readings.

Incorporation of N-heterocyclic cations into proteins with a highly directed chemical modification

N-Heterocyclic cations are incorporated into proteins using 5-(2-bromoethyl)phenanthridinium bromide, which selectively reacts with either cysteine or lysine residues, resulting in ethylphenanthridinium (Phen) or highly stable cyclised dihydro-imidazo-phenanthridinium (DIP) adducts respectively; these modifications have been found to manipulate the observed structure of lysozyme and bovine serum albumin by AFM.

A study of mechanochemical reactions of spin traps in hydrocarbon polymers

The identification and quantification of spin adducts and their reduction products (>NOH, >NOR) formed from nitroso compounds and nitrones in EPR and PP during spin trapping techniques have been examined. The nitroxyl yield and polymer bound nitroxyl percentage formed from these spin traps were found to be strongly dependent on the nature of spin trap and radical generator, processing temperature, and irradiation time. The nitroxyl yield and % bound nitroxyl of the spin traps improved significantly in the presence of Trigonox 101 and 2-0H benzophenone. The effect of these spin traps used as normal additive and their spin adducts in the form of EPR-masterbatch on the photo and thermal-oxidation of PP have been studied. Aliphatic nitroso compounds were found to have much better photo-antioxidant activity than nitrones and aromatic nitroso compounds, and their antioxidant activity improved appreciably in the presence of, a free radical generator, Trigonox 101, before and after extraction. The effect of heat, light and oxidising agent (meta-dichloro per benzoic acid) on the nitroxyl yield of nitroso tertiary butane in solution as a model study has been investigated and a cyclic regenerative process involving both chain breaking acceptor and chain breaking donor process has been proposed.

Parasitic infections and storage iron deficiency in children in improverished regions of Mauritius

Using a combination of techniques including relative deprivation index developed by the Government, school performance, household survey and drawing of lots, a sample frame constituting of poverty areas of Mauritius was constructed and four test areas identified. Relevant haematological, parasitological and biochemical parameters of all school-going children living in the four test areas were determined so as to study the possibility of correlation between parasitic infections, plasma ferritin, haemoglobin concentration, white blood cells count, packed cells volume and blood group. It was found that there is a negative correlation between the number of parasites and haemoglobin concentration, packed cells volume of blood and degree of infestation, number of parasites and ferritin and number of parasites and age of subject. It has also been found that, children with blood group 'A' and blood group '0' tend to harbour the most parasites. As regards to storage iron depletion, this is significant only with hookworm infestation. Additionally it has been noted that hookworm infestation is directly related to age contrary to other parasitic infestations.

Enhanced diabetes care to patients of south Asian ethnic origin (the United Kingdom Asian Diabetes Study):a cluster randomised controlled trial

Background - Delivery of high-quality, evidence-based health care to deprived sectors of the community is a major goal for society. We investigated the effectiveness of a culturally sensitive, enhanced care package in UK general practices for improvement of cardiovascular risk factors in patients of south Asian origin with type 2 diabetes. Methods - In this cluster randomised controlled trial, 21 inner-city practices in the UK were assigned by simple randomisation to intervention (enhanced care including additional time with practice nurse and support from a link worker and diabetes-specialist nurse [nine practices; n=868]) or control (standard care [12 practices; n=618]) groups. All adult patients of south Asian origin with type 2 diabetes were eligible. Prescribing algorithms with clearly defined targets were provided for all practices. Primary outcomes were changes in blood pressure, total cholesterol, and glycaemic control (haemoglobin A1c) after 2 years. Analysis was by intention to treat. This trial is registered, number ISRCTN 38297969. Findings - We recorded significant differences between treatment groups in diastolic blood pressure (1·91 [95% CI -2·88 to -0·94] mm?Hg, p=0·0001) and mean arterial pressure (1·36 [-2·49 to -0·23] mm?Hg, p=0·0180), after adjustment for confounders and clustering. We noted no significant differences between groups for total cholesterol (0·03 [-0·04 to 0·11] mmol/L), systolic blood pressure (-0·33 [-2·41 to 1·75] mm?Hg), or HbA1c (-0·15% [-0·33 to 0·03]). Economic analysis suggests that the nurse-led intervention was not cost effective (incremental cost-effectiveness ratio £28?933 per QALY gained). Across the whole study population over the 2 years of the trial, systolic blood pressure, diastolic blood pressure, and cholesterol decreased significantly by 4·9 (95% CI 4·0–5·9) mm?Hg, 3·8 (3·2–4·4) mm?Hg, and 0·45 (0·40–0·51) mmol/L, respectively, and we recorded a small and non-significant increase for haemoglobin A1c (0·04% [-0·04 to 0·13]), p=0·290). Interpretation - We recorded additional, although small, benefits from our culturally tailored care package that were greater than the secular changes achieved in the UK in recent years. Stricter targets in general practice and further measures to motivate patients are needed to achieve best possible health-care outcomes in south Asian patients with diabetes. Funding - Pfizer, Sanofi-Aventis, Servier Laboratories UK, Merck Sharp & Dohme/Schering-Plough, Takeda UK, Roche, Merck Pharma, Daiichi-Sankyo UK, Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Bristol-Myers Squibb, Solvay Health Care, and Assurance Medical Society UK.

Chemistry of phospholipid oxidation

The oxidation of lipids has long been a topic of interest in biological and food sciences, and the fundamental principles of non-enzymatic free radical attack on phospholipids are well established, although questions about detail of the mechanisms remain. The number of end products that are formed following the initiation of phospholipid peroxidation is large, and is continually growing as new structures of oxidized phospholipids are elucidated. Common products are phospholipids with esterified isoprostane-like structures and chain-shortened products containing hydroxy, carbonyl or carboxylic acid groups; the carbonyl-containing compounds are reactive and readily form adducts with proteins and other biomolecules. Phospholipids can also be attacked by reactive nitrogen and chlorine species, further expanding the range of products to nitrated and chlorinated phospholipids. Key to understanding the mechanisms of oxidation is the development of advanced and sensitive technologies that enable structural elucidation. Tandem mass spectrometry has proved invaluable in this respect and is generally the method of choice for structural work. A number of studies have investigated whether individual oxidized phospholipid products occur in vivo, and mass spectrometry techniques have been instrumental in detecting a variety of oxidation products in biological samples such as atherosclerotic plaque material, brain tissue, intestinal tissue and plasma, although relatively few have achieved an absolute quantitative analysis. The levels of oxidized phospholipids in vivo is a critical question, as there is now substantial evidence that many of these compounds are bioactive and could contribute to pathology. The challenges for the future will be to adopt lipidomic approaches to map the profile of oxidized phospholipid formation in different biological conditions, and relate this to their effects in vivo. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

Patients' perceptions and experiences of taking oral glucose-lowering agents: a longitudinal qualitative study

Aims The aims of this study were to examine Type 2 diabetic patients' expectations, perceptions and experiences of oral glucose-lowering agents (OGLAs), including their reasons for taking/not taking these drugs as prescribed and to provide recommendations for developing interventions to improve OGLA adherence. Methods Longitudinal, qualitative study using repeat in-depth interviews with patients (n = 20) over 4 years following clinical diagnosis. Respondents were recruited from primary and secondary care settings across Lothian, Scotland, UK. Results Despite experiences of side-effects, dislikes and concerns about taking multiple drugs and a belief that OGLAs could themselves cause one's diabetes to progress, most respondents appeared motivated to take these drugs as prescribed. This motivation seemed to arise from respondents' experiences of taking OGLAs and observing them to 'work'. Some respondents described feeling better after taking OGLAs, others, typically those who were asymptomatic, used blood glucose self-monitoring and/or glycated haemoglobin results to observe and evidence the effects of their OGLAs. Most respondents demonstrated a 'passive' expectation that health professionals should be responsible for decisions about medications. Hence, non-adherence typically resulted from forgetfulness rather than ambivalence about either medication or consultation style. Respondent concern about OGLA's largely centred upon lack of knowledge about the medication and what to do when doses were missed. Conclusion The findings call for multifaceted strategies to promote adherence. These could include education to address misconceptions and advise patients how to respond to missed doses; reminders to help patients remember to take their drugs; and structured feedback on the impact of OGLAs on glycaemic control.

Use of narrow mass-window, high-resolution extracted product ion chromatograms for the sensitive and selective identification of protein modifications

Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid-protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL. © 2013 American Chemical Society.