Considerations for the future

Although the importance of glycation as a marker of disease pathogenesis outside of diabetes is becoming clearer, it is not yet fully understood. More epidemiological studies are required to describe the interactions between oxidative stress and glycation, especially in normoglycaemia. The relatively small effect sizes obtained from the analyses as part of this PhD indicate that future investigations will require large sample sizes to have adequate power and also to allow for successful adjustment for confounders. Studies focusing on glycation have so far only focused on traditional glycation markers and rather neglected the importance of sRAGE and RAGE activation in disease pathogenesis, even though they constitute a major pathway of pathogenesis.

As far as polyphenol and antioxidant trials are concerned, there is still much improvement to be done in terms of study design before conclusions can be reached. If the working hypothesis is that polyphenols will exert health benefits via their antioxidant capacity, at least in susceptible individuals, then markers to document such improvements should be included and results on glycation markers, like HbA1c, should be discussed alongside oxidative stress improvements. The measurement of oxidative stress markers, like F2-isoprostanes, 8-hydroxydeoxyguanosine (8-OH- dG), the Comet assay and measurement of protein carbonyls would allow for sensitive and reproducible measurement of lipid, DNA and protein oxidation, providing a better understanding of the oxidative stress status compared to the commonly used TBARS assay (Freese et al., 1999, Hodgson et al., 2002, Janero, 1990, Frei and Higdon, 2003). Unfortunately, the validity of oxidative stress markers has been a source of disagreement among scientists (Halliwell, 2007, Halliwell, 1994) and no gold-standard method has been identified so far (Dalle-Donne et al., 2006). In that context, it is important to highlight that according to the results of this thesis, HbA1c levels themselves might be a novel marker of oxidative stress. The measurement of HbA1c or fructosamine combined with measurements of blood glucose, ideally both fasting and 2-hour

blood glucose, could be used to identify increased oxidative stress. Future studies should be designed to address this question, as it would valuably inform both research and clinical practice. Sample size and targeting the correct population are two more aspect of study design to be considered. The regulation of redox status is vital for health, with many endogenous regulatory mechanisms, and chronic diseases are likely to reflect only very small perturbations. Dietary factors are likely to play minor parts in regulation of such an important element of the milieu interieur, although subgroups may be more dependent on external factors. Polyphenol supplementation in a relatively healthy population is likely to have at best subtle effect on health markers and hence studies with large sample sizes greater than a hundred are likely to be needed (Cicero et al., 2008). The majority of the studies to-date fall short of that sample size and are hence likely to be underpowered. As a result, beta-errors are likely, and one should be very careful when concluding that polyphenol supplementation has no effect on glycation. The current literature may just describe a lack of power to detect such an effect (if any). Another consideration could be to target the trials towards populations with increased oxidative stress (i.e. obese, smokers), or to identify population subgroups which might have greater susceptibility to oxidative stress with greater dependence on dietary antioxidants. . Such a population may experience a greater benefit in their redox status from polyphenol (or other) supplementation, with the possibility of a knock-on protective effect on glycation markers. This is only a hypothesis and evidence to support this suggestion is not available.

A very important improvement in future study quality will relate to the information available about the supplement used. So far, reports from supplementation studies rarely report in detail the exact composition of the supplements used. Information on the polyphenol composition, the exact dose of each compound, the matrix used to deliver them and even some prior bioavailability data are needed if the results from different trials are to be compared and robust conclusions drawn.

Glycation markers should be picked carefully and the study duration should be sufficient to detect changes. Studies with duration shorter than the biomarker’s half-life are not suitable to represent any changes. Fructosamine measurements for a study with duration less than 14-28 days and HbA1c measurements before a 90-day window are most likely to be unable to detect any changes. The field of proteomics and metabolomics are also of great importance in glycation research, since every protein is ultimately a glycation target analysing changes in patterns of protein damage maybe more effective in drawing conclusion compared to single protein approaches.

Finally, when searching for benefits from a polyphenol supplementation trial, one should keep in mind that physical protein damage is only one pathway of glycation-related pathogenesis. RAGE mediated events are also key in the pathogenesis of diseases and are so far under-studied. Studies on epithelial cells and adipocytes suggest that plant derived polyphenol like silymarin, apigenin, diosmin, epigallocatechin gallate, tyrosol and quercetin have the capacity to reduce glycation related inflammation, especially TNF-alpha levels (Chandler et al., 2010, Kawaguchi et al., 2011, Biesalski, 2007) as well as nitric oxide production as a response to RAGE stimulation (Chandler et al., 2010). Although cell culture experiments can be set-up to study the interaction of polyphenols, glycation and inflammation, this is a more complicated task in-vivo. Measurement of sRAGE alongside the measurement of AGEs is not currently a common practice. Studies in pregnant women and patients with juvenile idiopathic arthritis (JIA) have showed that sRAGE levels may be more important that AGE levels to predict disease activity and complications (Yu et al., 2012, Myles et al., 2011). Also the calculation of a sRAGE/AGE ratio is a new concept that attempts to serve as a proxy for AGEs available to bind with RAGE (McNair et al., 2010, Ng et al., 2013). With little being known about sRAGE modulation, it is becoming evident that new glycation related markers need to be used to effectively describe the impact of glycation on health.