2007 Annual Report 1a.Objectives (from AD-416) The overall objective is to further knowledge of carotenoids and vitamin A. We seek to define the mechanism(s) of intestinal absorption of dietary carotenoids and their distribution into blood cells. Objectives are to study:. 1) Mechanisms of intestinal absorption of carotenoids & their incorporation into chylomicrons in CACO-2 cells in culture and. 2)Modulation of macrophage function by dietary carotenoids. Dietary vitamin A enters the liver as retinyl esters (RE) in chylomicron remnants. Retinyl ester hydrolases (REHs) have been purified from rat liver and shown to be carboxylesterases, ES-2 and ES-10. We seek to define the role of ES-2, ES 10, and the related ES-3 and ES-4, in the metabolism of vitamin A and to study: . 3) Cell type distribution in liver and . 4) Physiologic roles of the carboxylesterases in the metabolism of RE in cultured cells. We also need to apply proteomics to define new biomarkers of vitamin A status in experimental animals and then extend these studies to humans. 1b.Approach (from AD-416) 1. Mechanisms of intestinal absorption of carotenoids & their incorporation into chylomicrons in CACO2 cells in culture. We will test the hypothesis that other fat-soluble nutrients inhibit the incorporation of -carotene into chylomicrons. We will test the hypothesis that SRB1, CD-36, FAT, and other putative lipid transporters mediate the uptake of -carotene into the cell. We will test the hypothesis that increasing the expression of -carotene 15,15' dioxygenase increases the total flux of -carotene through the cell. 2. Modulation of macrophage function by dietary carotenoids. We will use the U937 cell line as well as human peripheral blood monocyte-macrophages to examine the partitioning of carotenoids into blood-borne cells and the effects of carotenoid enrichment on monocyte macrophage function. We will test the hypothesis that plasma carotenoids partition into various blood cells as well as into plasma lipoproteins. We will test the hypothesis that carotenoid enrichment alters monocyte-macrophage phenotype, either enhancing or inhibiting potentially pro-atherosclerotic aspects of cell function including response to chemotactic stimuli, adhesion to endothelial cells, differentiation into macrophages, ability to oxidize LDL, and ability to form foam cells. 3. Cell type distribution of retinyl ester hydrolases (carboxylesterases) in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells. 4. Physiologic roles of the carboxylesterases in the metabolism of chylomicron retinyl esters in cultured cells. cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE. 5. Application of proteomics to nutrition research. We will use SELDI-TOF mass spectrometry of plasma to define new biomarkers of nutrient exposure in animal models and humans. 3.Progress Report This research falls under National Program 107, Human Nutrition, Component 1: Nutrition Requirements, and Component 7: Bioavailability of Nutrients and Food Components. Its aim is to better understand the basic biochemistry involved in the storage, release, and bioavailability of Vitamin A in the body. With the resignation of the two key scientists working on this project, many of the proposed activities have been delayed. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer