Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Food Science." Includes bibliographical references (p. 103-117).

Tea and coffee are rich in polyphenols with a variety of biological activities. Polyphenols found in tea are predominantly flavonoids, of which up to 15% are present as free or esterified gallic acid. Coffee polyphenols are almost wholly comprised of chlorogenic acids. Many of the demonstrated activities of polyphenols are consistent with favourable effects on the risk of chronic diseases. In investigating the relationships between intake and exposure to such compounds and chronic disease-related endpoints, it is important to be able to identify biomarkers that are specific to the compounds of interest. 4-O-methyl gallic acid (4OMGA) and isoferulic acid have been identified as potential biomarkers of intake and exposure to polyphenols derived from tea and coffee, respectively. 4OMGA is derived from gallic acid in tea, and isoferulic acid from chlorogenic acid in coffee. The major objectives of the research which is the subject of this thesis were (1) to establish a dose-response relationship of 24h urinary excretions of 4OMGA and isoferulic acid following ingestions of black tea and coffee of different strengths, and (2) to explore relationships of tea and coffee intake with 24h urinary excretion of 4OMGA and isoferulic acid in human populations. It was found that there was rapid excretion of both 4OMGA and isoferulic acid in the first 6h after tea and coffee ingestion, respectively. Approximately 60 80% of the ingested dose was excreted during the first 6h after ingestion. Urinary excretion of 4OMGA and isoferulic acid was directly related to the dose of tea and coffee, respectively. That is, higher intake resulted in increased urinary excretion of the metabolites. The relationships of 24h urinary excretion of 4OMGA and isoferulic acid with long-term usual (111 participants) and contemporary recorded current (344 participants) tea and coffee intake were assessed. 4OMGA was strongly related to usual (r = 0.50, P < 0.001) and current (r = 0.57, P < 0.001) tea intake. Isoferulic acid was less strongly, but significantly associated with usual (r = 0.26, P = 0.008) and current (r = 0.18, P < 0.001) coffee intake. Overall, the results are consistent with the proposal that 4OMGA is a good biomarker for black tea derived polyphenol intake and exposure, but isoferulic acid may have only limited use as a biomarker for coffee-derived polyphenol exposure.

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here. Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency. The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.