Brush border digestion : development of a physiologically relevant in vitro model : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University, Manawatū, New Zealand

Abstract

Chapter Two has, in part, been published in the Springer publication, Reviews of Physiology, Biochemistry and Pharmacology, but the Author retains the right to publish the material in any collection consisting of the Author's own works: Hooton, D., Lentle, R., Monro, J., Wickham, M., & Simpson, R. (2015). The secretion and action of brush border enzymes in the mammalian small intestine. In: B. Nilius, T. Gudermann, R. Jahn, R. Lill, O. Petersen, & P. de Tombe (Eds.), Reviews of Physiology, Biochemistry and Pharmacology (vol 168, pp. 59-118). Cham: Springer. doi: 10.1007/112_2015_24The majority of current in vitro digestion methods either exclude the small intestinal brush border (BB) phase of digestion or do not incorporate the entire array of BB enzymes that are required to achieve terminal endogenous digestion in vivo. Accordingly, the digestate, and its derivitives, may not be representative of the digestive process in vivo. In order to improve the fidelity of the in vitro digestion process this thesis developed a physiologically relevant small intestinal BB phase using enzymes isolated from rat small intestinal mucosal tissue. The activities of BB enzymes were assessed and compared with known values, and under conditions physiologically representative of the small intestine. Although there were significant differences in BB enzyme activities depending on pH, enzyme solubilisation, and upon prolonged exposure to biliopancreatic secretions the BB preparation was deemed suitable for use in an in vitro digestion method. A rationale for the composition of the BB digestive phase was developed based on published physiological data, and was validated using glycosylated polyphenolic compounds as substrates. Liquid chromatography mass spectrometry (LC-MS) was used to assess the derivatisation products of BB digestion. In the absence of biliopancreatic secretions the onion flesh polyphenolic compounds quercetin-4ʹ-glucoside and isorhamnetin-4ʹ-glucoside, but not quercetin-3-glucoside or quercetin-34ʹ-diglucoside were hydrolysed. The positive control quercetin-3-glucoside was hydrolysed, and the negative control quercetin-3-rutinoside was not hydrolysed. The deglycosylation of quercetin-3-glucoside was monitored under conditions representative of the small intestine, i.e. incorporating bile and pancreatin, while at the appropriate pH. Quercetin-3-glucoside was significantly deglycosylated in BB treatments (no treatment or pancreatin alone) compared to BB treatments with bile (bile alone or pancreatin and bile). The mammalian digestive system is equipped to hydrolyse macronutrients from their polymeric form through to monomers and oligomers suitable for absorption across the epithelial layer. As such the inactivation or degradation of some BB enzymes during the BB digestive phase by bile or pancreatin was not unexpected, and does not preclude its use as an in vitro tool in the future