Date of Award

1-1-2003

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

College

College of Graduate Studies

First Advisor

Thomas Walle

Second Advisor

Karl J. Karnaky, Jr.

Third Advisor

Jürgen Rohr

Fourth Advisor

Patrick Meier

Fifth Advisor

Bao-Ting Zhu

Abstract

Tea flavonoids, including (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) have been shown to have chemopreventive activity in cancer. Studies in animals and humans suggest that these dietary compounds have a very low oral bioavailability. However, the reason behind this limited bioavailability as well as the mechanisms underlying absorption is not clearly understood. The overall hypothesis of this dissertation project is that the human intestinal absorption of (-)-epicatechin (EC) and its gallate, (-)epicatechin- 3-gallate (ECG) is limited and regulated both by multiple transport mechanisms and metabolism. We used the Caco-2 cell monolayers, a well-established model to study human intestinal absorption together with molecularly-specific HPLC to characterize the transport of EC and ECG. We found that EC showed a direction dependency in transport, with no apical to basolateral transport (absorption) but was preferentially effluxed. Using inhibitors of known efflux transporters, this direction-dependency was further investigated and suggested the involvement of MRP2. Further, EC was also metabolized in the Caco-2 cells to form two isomeric sulfate conjugates. These anionic EC-sulfates were also effluxed by MRP2. Extensive metabolism studies done using various human tissues as well as recombinant enzymes, showed that EC was not glucuronidated in humans, but instead was very efficiently sulfated. EC also showed interesting species variability in the metabolism, with sulfation being the important metabolic pathway in humans as compared to glucuronidation in the rats. In contrast to EC, when cell uptake studies were done using its corresponding gallate ECG, it was very efficiently transported across the apical membrane of the Caco-2 cells by a pH-dependent mechanism presumably by the monocarboxylate transporter (MCT). This was indicated by the use of MCT inhibitors like phloretin and benzoic acid, which resulted in a significant inhibition of ECG uptake. This transport by MCT results in a very high accumulation of ECG in the enterocytes. However, when the role of efflux transporters in ECG accumulation was examined, we found that ECG was a substrate of MRP1, MRP2 as well as P-glycoprotein. This was determined by examining uptake in Caco-2 as well as MRP2 or P-glycoprotein transfected MDCK cells in presence or absence of inhibitors of these efflux pumps. This uptake was not dependent on cellular glutathione levels. Thus, the work described in this dissertation helps to understand the mechanisms governing the intestinal absorption of dietary catechins. This work has also helped identify the major site of action of these flavonoids. The observations made demonstrate that the absorption of these dietary flavonoids is a complex process involving multiple membrane transporters, both absorptive and efflux as well as metabolism.

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