Cobalamin (Cb1, B12) deficiency results in severe hematologic and neurologic complications. The enterohepatic circulation of Cb1 is a unique and unexploited model of receptor-regulated processes involving two major organ systems, the liver and gut. Intrinsic factor (IF), transcobalamin II (TCII) and other Cb1-binding proteins play important yet poorly understood roles in absorption, systemic distribution and cellular transport of this essential micronutrient. A number of these Cb1-binding proteins participate in the enterohepatic circulation of the vitamin, a process necessary for conservation of Cb1 body stores. The long-term goal of this project is to gain a mechanistic understanding of how receptors for Cb1-binding proteins regulate Cb1 circulation through the enterohepatic system. Using the rat as an experimental model, the specific aims to be accomplished over the next three years are to: 1) Determine the fate of IF during and after absorption of Cb1 and establish the origin and nature of Cb1-binding proteins in portal circulation that carry newly absorbed Cb1; 2) Localize (e.g. parenchymal vs non-parenchymal) and determine the density, specificity and affinity of hepatic receptors for Cb1-binding proteins; and, 3) Characterize Cb1-binding proteins present in bile and elucidate the effect of bile on absorption of dietary and biliary Cb1. These objectives will be accomplished by: 1) Purifying Cb1-binding proteins from rat tissues using affinity-photodissociative chromatography; 2) Preparing rabbit antibodies against rat IF and TCII for use in recovering radioiodinated Cb1-binding proteins from portal plasma, bile, cells and tissues; and, 3) Utilizing in vitro rat model systems such as the isolated perfused liver to study hepatic uptake and processing of purified radioiodinated Cb1-binding proteins carrying 57Co-labeled Cb1 and in vivo rat model studies to assess the relative physiological importance of pathways and mechanisms derived from in vitro information. These studies should provide a better understanding of the pathogenesis of Cb1-related illness and essential mechanisms of enterohepatic circulation in general.
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