In view of the growing evidences for the anticarcinogenic actions of carotenoids per se,a clear understanding of the mechanism of absorption, metabolism and distribution of carotenoids is essential. Recent studies including ours strongly support that ferret may be a suitable model for carotenoid research since it can absorb substantial amounts of intact beta-carotene just like humans. Therefore, it is proposed to carry out the proposed studies using ferret as the experimental animal model. Preferential localization of carotenoids in specific susceptible organs may protect against malignancy. The possibility of the formation of products other than retinal from the cleavage of beta-carotene in vivo and in vitro should be investigated because some of these carotenoid products may possess greater anticarcinogenic properties. It is important to prove whether or not retinal is the obligatory intermediate in the formation of retinoids from beta-carotene and to pinpoint the role of CRBP II in these processes. It is equally important to characterize a specific protein complex for beta-carotene inside the cell and for its transport. The experimental approaches described should help to accomplish the following specific aims using ferret as the experimental animal model: 1. To increase the absorption and distribution of beta-carotene in various organs. 2. To test the dietary inducibility of beta-carotene cleavage (BCC) enzyme. 3. To correlate the in vivo products of beta-carotene metabolism with those in vitro. 4. To prove unequivocally whether or not retinal is a normal intermediate during the conversion of beta-carotene into retinol and retinoic acid using the BCC enzyme. 5. To define the roles of CRBP II in the conversion of beta-carotene to retinoids using the BCC enzyme. 6. To identify a specific protein which forms a complex with beta-carotene for its transport and storage. The PI has expertise in the following systems and techniques to accomplish these specific aims: 1. Thoracic duct cannulation technique to quantitatively recover the products of beta-carotene cleavage. 2. HPLC procedures for separation and identification of various metabolites of beta-carotene. 3. Scanning spectrophotometry for characterization of carotenoids and retinoids. 5. Centrifugal elutriation and discontinuous gradient methods for purification of liver cells. 6. Ultracentrifugation, ion-exchange, gel- filtration and affinity column chromatography and gel electrophoretic techniques for the characterization of beta-carotene- protein complex.
