Drivers of redox status & protein glycation

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 H202, or continuously exposed to 10nM H2O2 throughout the 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 H2O2, for 8 hours) or incubated with a 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 H2O2 (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 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 H2O2) was more readily 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 H2O2 10nM) (p<0.001 vs. native BSA). 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 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.