Vlassopoulos, Antonios (2014) Drivers of redox status & protein glycation. PhD thesis. http://theses.gla.ac.uk/5607/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] DRIVERS OF REDOX STATUS & PROTEIN GLYCATION Antonios Vlassopoulos BSc, MSc A thesis submitted for the degree of Doctor of Philosophy to The University of Glasgow April, 2014 From research conducted at the School of Medicine, Human Nutrition University of Glasgow Glasgow, Scotland © A. Vlassopoulos 2014 ii To my aunt who showed me the way iii Abstract Background: In the past 60 years, the median age of the entire world population has increased and ageing and chronic diseases are now the main medical concerns in the developed world. The identification of early signs of disease pathogenesis is vital for prevention and targeting populations at risk in order to reduce morbidity and mortality. Glycation is well-established as an index of control, or otherwise, and a predictor of end-organ damage, for people with type 2 diabetes. At the beginning of the work for this thesis (2010), evidence was beginning to be raised to suggest that since glycation levels vary considerably, in normoglycaemic, non-diabetic individuals, glycation cannot be solely related to glucose levels and early reports allowed for speculations about a relationship between early glycation and oxidative stress. The aim of this thesis was to establish the relationship between early glycation and oxidative stress in normoglycaemia using techniques from the full breadth of Human Nutrition Research. Methods: In study 1, existing epidemiological data were used to identify relationships between proxies for redox status and early glycation in non-diabetic individuals. One-way ANOVA, Chi- squared and multivariate linear regressions, adjusted for all known confounders were used to explore associations of HbA1c with self-reported smoking status and fruit & vegetables consumptions in the Scottish Health Surveys 2003-2010, among individuals without known diabetes and HbA1c<6.5%. In studies 2 and 3, the associations from epidemiology were explored in mechanistic laboratory studies with high physiological relevance (using physiological concentrations and conditions) to better characterise the effect of oxidative stress and antioxidants on early glycation. In study 2, bovine serum albumin (BSA), reduced BSA (mercaptalbumin) (both 40g/L), and human plasma were incubated with glucose concentrations 0-30 mM for 4 weeks at 37oC. All were tested pre-oxidized for 8 hours prior to glycation with 10nM H 0 or continuously exposed to 10nM H O throughout the 2 2, 2 2 incubation period. Fructosamine was measured (nitroblue tetrazolium method) at two and four weeks. In study 3, Bovine Serum Albumin (BSA) was pre-treated prior to in vitro glycation: either no treatment (native), pre-oxidised (incubated with 10nM H O for 8 hours) or incubated with a 2 2, mixture of phenolic acids at physiologically relevant concentrations, for 8 hours). In-vitro glycation was carried out in presence of i) glucose only (0, 5 or 10mM), ii) glucose (0, 5 or 10mM) plus H O 2 2 (10nM), or iii) glucose (0, 5 or 10mM) plus phenolic acids (10-160nM). Fructosamine was again measured using the nitroblue tetrazolium method. Prior to the experimental study we carried out a iv systematic literature review of dietary interventions reporting plasma concentrations polyphenol metabolites, to inform the design of a physiologically relevant in-vitro study. In study 4, clinical trial data and biological samples were analysed from a randomised controlled dietary advice trial in obese pregnant women, a group at risk from higher glycation and oxidative stress. Samples and data from the UPBEAT study trial (n=117) were analysed. Plasma fructosamine, plasma sRAGE, urinary Ferric Reducing Ability of Plasma (FRAP), urinary Total Phenol (TP) and urinary Advanced Oxidised Protein Products (AOPPs) were measured at 16-18+6 and 27-28+6 weeks gestation. Dietary recalls were used to calculate fruit and vegetable and polyphenol intake at the same timepoints. Data were analysed to identify associations between dietary variables and biochemical markers, as well as their relationships with diagnosis of complications. Associations between maternal variables and neonatal anthropometry were also investigated. Results: In study 1, HbA1c was higher in smokers by 0.25 SDs (0.08%), and 0.38 SDs higher (0.14%) in heavy smokers (>20cigarettes/day) than non-smokers (p<0.001 both). Smokers were twice as likely to have HbA1c in the ‘pre-diabetic’ range (5.7-6.4%) (p<0.001, adj.model). Pre-diabetes and low grade inflammation did not affect the associations. For every extra 80g vegetable portion consumed, HbA1c was 0.03 SDs (0.01%) lower (p=0.02), but fruit consumption did not impact on HbA1c, within the low range of consumptions in this population. In study 2, oxidized BSA (both pre-oxidised and continuously exposed to H O ) was more readily 2 2 glycated than native BSA at all glucose concentrations (p=0.03). Moreover, only oxidized BSA was glycated at physiological glucose concentration (5mM) compared to glucose-free control (glycation increased by 35% compared to native albumin p<0.05). Both 5mM and 10mM glucose led to higher glycation when mercaptalbumin was oxidised than un-oxidised (p<0.05). Fructosamine concentration in human plasma was also significantly higher when oxidized and exposed to 5mM glucose, compared to non-oxidised plasma (p=0.03). The interaction between glucose concentration and oxidation was found to be significant in all protein models (p<0.05). In study 3, the presence of six phenolic acids with BSA during in-vitro glycation did not lower fructosamine formation. However, when BSA was pre-incubated with phenolic acids, significantly lower concentration of fructosamine was detected under glycoxidative conditions (glucose 5 or 10mM plus H O 10nM) (p<0.001 vs. native BSA). 2 2 In study 4, women in the lowest quartile of total polyphenol intake had 8% greater fructosamine levels compared to those in the top quartile. Total polyphenol intake was negatively correlated with sRAGE levels. Diagnosis of severe preeclampsia was associated with elevated AOPPs. Maternal v polyphenol intake was positively correlated with birth weight, while maternal glycoxidation showed the opposite relationship. Conclusions: Study 1 added evidence to relate smoking (an oxidative stress proxy) to protein glycation in normoglycaemic subjects. This association has implications for individuals exposed to ROS and for epidemiological interpretation of HbA1c and its clinical usefulness. Study 2 offered a mechanistic background to the previously shown epidemiological association. This study demonstrated for the first time albumin glycation in-vitro, using physiological concentrations of albumin, glucose and hydrogen peroxide. These results identified low-grade oxidative stress as a key element early in the glycation process, especially in glucose concentrations relative to normoglycaemia. Furthering those findings, study 3 showed that protein-phenolic acid interactions are important regulators of protein glycation. Together those studies highlighted that protein pre- treatment, either with oxidants or phenolic acids, is an important regulator of subsequent glycation in a physiologically relevant system. An important outcome of those studies is that high quality in- vitro studies under conditions closer to physiology are feasible and should be employed more frequently. Finally, study 4 demonstrated an association between polyphenol intake and glycation during pregnancy, with an impact on neonatal outcome measures. Maternal glycoxidation is a promising marker of pre-eclampsia and neonatal anthropometry and could be modulated by maternal lifestyle and dietary habits. Overall, the results of this thesis implicate that drivers of redox status have the capacity to modulate protein glycation in normoglycaemia. These results challenge the assumption that glycation levels are solely dependent on circulating glucose levels and suggest a useful application of glycation outside the field of diabetes. vi Acknowledgements I want to thank my supervisors Dr Emilie Combet and Prof Mike Lean for all the help and outstanding support all along the way. They believed in me from the beginning and pushed me to become better. It is their efforts that helped become who I am today and that I will remember and value throughout my life. It was my pleasure to work and learn from you and I hope that this is not the end for our collaboration. I like to think that along the way we’ve built a relationship that goes beyond the strictly professional limits and that I would be very sorry to lose. I would like to thank the Yorkhill Children Charity, for providing the funding for this PhD, and also Prof Scott Nelson and Prof Lucilla Poston for kindly offering us access to the UPBEAT samples and data. A special thank you goes to all the laboratory and administrative staff in the Department of Human Nutrition, for dealing with all my bizarre and last minute requests. Also to Mr Philip Mcloone for his guidance in analysis data and dealing with large datasets. I should also thank the academic staff of the Department for they entrusted me with teaching lab practicals, journal clubs and all sorts of things to our MSc students from year to year. I hope I did a good job and you are not regretting your choice. The help of our interns and students involved in any part of my projects is highly appreciated and I should thank you for that. A big thank you goes to all the people that were next to me day after day. They were there when I laughed, complained and felt like giving up. Jeremy Jones, Areej Alkhaldy, Martin Bertz and Friederike Vollmer-Bertz thank you for everything. My very close friend Bahareh Mansoorian, who is submitting today with me, we’ve gone a long way with ups and downs but we made it at the end. I’ve left for the end those that worked like my safety net/my guardians, my family, my aunts and Ilektra. You were always my last resort, the place I knew I would find safety and peace, there not words big enough to thank you. vii Author’s Declaration I declare that the work contained in this thesis is original, and is the work of the author Antonios Vlassopoulos. I have been solely responsible for the organisation and day to day running of this study as well as the laboratory analysis and data processing, unless otherwise referenced. Antonios Vlassopoulos Supervisors’ declaration I certify that the work reported in this thesis has been performed by Antonios Vlassopoulos and that during the period of study he has fulfilled the conditions of the ordinances and regulations governing the Degree of Doctor of Philosophy, University of Glasgow Dr. Emilie Combet Aspray Prof. Michael EJ Lean viii Table of Contents Abstract .............................................................................................................................................. iii Acknowledgements ............................................................................................................................ vi Author’s Declaration ......................................................................................................................... vii List of Tables ....................................................................................................................................... x List of Figures ..................................................................................................................................... xi Publications achieved during the present thesis .............................................................................. xii List of Abbreviations ........................................................................................................................ xiii Chapter 1: Introduction ...................................................................................................................... 1 1.1 General Introduction ........................................................................................................... 2 1.2 The glycation reaction - historical background ................................................................... 4 1.3 The importance of glycation for health .............................................................................. 7 1.3.1 Glycation and the AGE-RAGE axis ...................................................................................... 7 1.3.2 Glycation and health throughout the lifecycle .................................................................. 8 1.3.3 HbA1c and risk of chronic diseases .................................................................................. 10 1.4 Oxidative stress and glycation .......................................................................................... 12 1.5 Antiglycative capacity of antioxidants and polyphenols ................................................... 13 1.6 Hypothesis and research questions to be tested in this thesis ........................................ 18 Chapter 2: General Methods ............................................................................................................ 20 2.1 Mixed methods research design ....................................................................................... 21 2.2 Epidemiological data ......................................................................................................... 22 2.3 In-vitro design ................................................................................................................... 23 2.4 Experimental data from randomised controlled trials ..................................................... 24 2.5 Statistical analysis ............................................................................................................. 25 Chapter 3: Influence of smoking and diet on glycated haemoglobin and ‘pre-diabetes’ categorisation: a cross-sectional analysis .................................................................................................................. 27 Abstract ......................................................................................................................................... 28 3.1 Background ............................................................................................................................. 29 3.2 Methods .................................................................................................................................. 30 3.3 Results ..................................................................................................................................... 33 Chapter 4: Role of oxidative stress in physiological albumin glycation: a neglected interaction ..... 42 Abstract ......................................................................................................................................... 43 4.1 Introduction ............................................................................................................................ 44 4.2 Material and methods ............................................................................................................ 45 ix 4.3 Results ..................................................................................................................................... 47 4.4 Discussion ................................................................................................................................ 55 4.5 Conclusion ............................................................................................................................... 58 Chapter 5: Inhibition of protein glycation by phenolic acids: physiological relevance and implication of protein-phenolic interactions ....................................................................................................... 59 Abstract ......................................................................................................................................... 60 5.1 Introduction ............................................................................................................................ 61 5.2 Material and methods ............................................................................................................ 63 5.3 Results ..................................................................................................................................... 65 5.4 Discussion ................................................................................................................................ 77 5.5 Conclusion ............................................................................................................................... 79 Chapter 6: Polyphenol intake and maternal glycation during pregnancy. Maternal diet and glycoxidation as risk factors for complications and neonatal anthropometry in obese pregnancies80 Abstract ......................................................................................................................................... 81 6.1 Introduction ............................................................................................................................ 82 6.2 Methods .................................................................................................................................. 84 6.3 Results ..................................................................................................................................... 87 6.4 Discussion ................................................................................................................................ 93 6.5 Conclusion ............................................................................................................................... 96 Chapter 7: General discussion and implications for future research ............................................... 98 7.1 Research questions and answers .......................................................................................... 100 7.2 Considerations for the future ............................................................................................... 105 7.3 Research questions arising from this thesis .......................................................................... 107 References ...................................................................................................................................... 108 Appendix ......................................................................................................................................... 136