Implications of anthocyanin instability and metabolism: impact on discovery of intake biomarkers and in vitro mechanisms of action
Smith M J.
University of East Anglia, 2016.
Anthocyanins are polyphenol pigments responsible for the blue/red colouring of many fruits and vegetables whose consumption is associated with reduced risk of cardiovascular disease. Due to instability at neutral pH and reported low bioavailability of anthocyanins, the focus has shifted to their phenolic metabolites to explain the observed bioactivity. The hypothesis of the present study was that greater sensitivity and higher throughput could be achieved by developing existing analytical methodology, metabolite biomarkers of anthocyanin intake could be identified, and physiologically relevant anthocyanin metabolite profiles could reduce expression of proinflammatory cytokines in vitro. Stability of cyanidin-3-glucoside and 17 metabolites in serum was assessed. Unlike the parent anthocyanin, metabolite recovery was unaffected by either pH or freeze/thaw cycling suggesting that acidification and aliquoting of samples may be unnecessary for quantifying anthocyanin metabolites only. HPLC-ESI-MS/MS methodology was developed to increase sensitivity and sample throughput. Initial and developed methods were validated against FDA guidelines and found to have acceptable linearity, specificity and sensitivity with limits of detection of 9.5 ± 13.0 nM and 96.7 ± 541.7 nM respectively. Accuracy and precision were acceptable at 50 µM but outside guidelines at 1.56 µM and 0.098 µM for both methods. Biomarkers of anthocyanin intake were explored using samples from a 13C-labelled cyanidin-3-glucoside trial to compare 13C-labelled metabolites against 12C metabolites from the background diet. Gallic acid was consistently found above background levels in urine and represented the best biomarker candidate. Finally, effect of anthocyanin metabolite profiles on proinflammatory cytokines in CD40L and TNF-α-stimulated HUVEC was investigated. Incubation with metabolite profiles reduced expression of sVCAM-1 by 47-58% and IL-6 by 16-37% following TNF-α stimulation. In conclusion, the present thesis develops methodology for the quantification of anthocyanin metabolites in biological matrices and provides insight into their use as biomarkers of anthocyanin intake and role in maintaining cardiovascular health.
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