Development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease

This paper presented at the European Meeting of the Society-for-Free-Radical-Research-Europe 2007, discusses the development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease.

Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

Usual medical treatments or levonorgestrel-IUS for women with heavy menstrual bleeding:long-term randomised pragmatic trial in primary care

Background: Heavy menstrual bleeding (HMB) is a common, chronic problem affecting women and health services. However, long-term evidence on treatment in primary care is lacking. Aim: To assess the effectiveness of commencing the levonorgestrel-releasing intrauterine system (LNG-IUS) or usual medical treatments for women presenting with HMB in general practice. Design and setting: A pragmatic, multicentre, parallel, open-label, long term, randomised controlled trial in 63 primary care practices across the English Midlands. Method: In total, 571 women aged 25–50 years, with HMB were randomised to LNG-IUS or usual medical treatment (tranexamic/mefenamic acid, combined oestrogen–progestogen, or progesterone alone). The primary outcome was the patient reported Menorrhagia Multi-Attribute Scale (MMAS, measuring effect of HMB on practical difficulties, social life, psychological and physical health, and work and family life; scores from 0 to 100). Secondary outcomes included surgical intervention (endometrial ablation/hysterectomy), general quality of life, sexual activity, and safety. Results: At 5 years post-randomisation, 424 (74%) women provided data. While the difference between LNG-IUS and usual treatment groups was not significant (3.9 points; 95% confidence interval = −0.6 to 8.3; P = 0.09), MMAS scores improved significantly in both groups from baseline (mean increase, 44.9 and 43.4 points, respectively; P<0.001 for both comparisons). Rates of surgical intervention were low in both groups (surgery-free survival was 80% and 77%; hazard ratio 0.90; 95% CI = 0.62 to 1.31; P = 0.6). There was no difference in generic quality of life, sexual activity scores, or serious adverse events. Conclusion: Large improvements in symptom relief across both groups show treatment for HMB can be successfully initiated with long-term benefit and with only modest need for surgery.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the me0chanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

The relationship between high-sensitivity CRP and polyclonal Free Light Chains as markers of inflammation in chronic disease

Introduction: Serum concentrations of polyclonal free light chains (FLC) represent the activity of the adaptive immune system. This study assessed the relationship between polyclonal FLC and the established marker of innate immunity, C-reactive protein (CRP), in chronic and acute disease. Methods: We utilized four cross-sectional chronic disease patient cohorts: chronic kidney disease (CKD), diabetes, vasculitis and kidney transplantation; and a longitudinal intensive care case series to assess the kinetics of production in acute disease. Results: There was a weak association between polyclonal FLC and high-sensitivity CRP (hs-CRP) in the study cohorts. A longitudinal assessment in acute disease showed a gradual increase in FLC concentrations over time, often when CRP levels were falling, demonstrating clear differences in the response kinetics of CRP and FLC in this setting. Conclusion: Polyclonal FLC and hs-CRP provide independent information as to inflammatory status. Prospective studies are now required to assess the utility of hs-CRP and polyclonal FLC in combination for risk stratification in disease populations. © 2013 John Wiley & Sons Ltd.

Luminescent techniques for studying free radical production and cell viability in relation to cardiovascular disease and HRT

Epidemiological studies have suggested that hormone replacement therapy (HRT) offers protection from atherosclerosis, a precursor of cardiovascular disease (CVD), in postmenopausal women. There is good evidence that oxidation of low-density lipoprotein (LDL) by leucocyte-derived reactive oxygen species plays a key role in development of an atherosclerotic plaque. Therefore we have investigated whether the possible protection against CVD by HRT could be due to immunomodulation, specifically of free radical production. The study involves 2 approaches: I) analysing the production of free radicals by leucocytes from women on HRT, 2) investigating the effect of I7p-oestradiol and progesterone on cultured myeloid cells (HL60 and U937). Free radical production by leucocytes was determined using a recently developed bioluminescent assay. In the assay, Pholasin® emits light in the presence of free radicals produced by the NADPH oxidase system of leucocytes stimulated with PMA or fMLP. Cell viability was also investigated using a bioluminescent assay (Cell Titer-Glo®) in which cytosolic ATP levels were measured by the production of luminescence in the presence of Luciferin/Luciferase reagent. Studies of leucocytes from HRT patients showed considerable variation in free radical production, which appeared to be dependent on HRT regime. Studies on the cultured cells showed that there was no cell proliferation at low hormone concentrations, while high concentrations caused cytotoxicity. The effect of hormones on free radical production in this in vitro model system is currently being investigated. The results show that the effects of the hormones on cells of the immune system are very dose dependent, and that both beneficial and adverse effects may occur. In conclusion, luminescent techniques offer a valuable and sensitive approach to studying inflammatory and oxidative processes both in vivo and in vitro.