2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This research falls under National Program 107 - Human Nutrition and, specifically, component #1 - "nutrition requirements," and #7 - "bioavailability of nutrients and food components." Specific program components are focused also on "definition of marginal deficiencies..." and "bioavailability of nutrients in foods". Our research on vitamin A exemplifies these foci by examining the basic biochemistry of enzymes involved in the storage, release, and bioavailability of vitamin A in the body. We are studying the liver enzymes that hydrolyze retinyl esters, the form in which newly absorbed dietary vitamin A is delivered to the liver. These enzymes (retinyl ester hydrolases or REHs) are important in both the uptake of dietary vitamin A by the liver and in the mobilization of stored vitamin A from the liver when dietary input is inadequate to meet the body's need for this essential nutrient. Although a number of liver enzymes can catalyze the hydrolysis of vitamin A esters, the molecular identity and physiological role of these enzymes is unclear. Our research attempts to fill these gaps in knowledge by isolating these enzymes in pure form using classical techniques of enzymology, and by using gene "knockout" techniques to study the physiological role of these enzymes in animal models. We are also studying the impact of dietary carotenoids on the oxidation of human plasma lipoproteins, a process thought to be involved in the etiology of atherosclerosis. We approach this by altering the cellular content of specific dietary carotenoids, and asking whether this inhibits the oxidation of the lipoprotein by human aortic endothelial cells and macrophages in culture. We use this system as a model of what may be occurring in the aortic wall in the intact human. We are also studying the basic mechanisms involved in the intestinal absorption of vitamin A and carotenoids. Vitamin A deficiency is the leading cause of blindness in the world. More marginal deficiency is associated with increased risk of infections such as measles. Our work is on the basic biochemical mechanisms involved in the metabolism of vitamin A in the intestine and in the liver, the major site of vitamin A storage in the body. Cardiovascular disease, resulting from the underlying process called atherosclerosis, is a leading cause of death and morbidity in the U. S. population. There is a good deal of evidence that this disease process is related in part to the oxidation of plasma lipoproteins by cells of the blood vessel wall. There is also strong epidemiological evidence relating the consumption of fruits and vegetables (which contain high amount of antioxidant phytonutrients, such as carotenoids) to a decreased incidence of cardiovascular disease. It is important to define the effects of specific antioxidant nutrients on lipoprotein oxidation to be able to make reasonable recommendations for the consumption of these nutrients. It is also important to understand the basic mechanisms of the disease process in order to define potential targets for therapeutic intervention. Finally, it is important to understand the mechanisms of intestinal absorption of carotenoids, because the extent of bioavailability needs to be considered when formulating recommended intakes. Our research falls under the National Program 107 on Human Nutrition Requirements, Food Composition, and Intake. As indicated in the Program Statement, "human nutrition science has moved from a focus on the prevention of nutrient deficiencies to an emphasis on health-maintenance and reduced risks of chronic diseases." Our research on carotenoids exemplifies that focus by examining the effects of components of the diet (i.e., carotenoids) that are not nutritionally essential, but that may have impact on a major chronic disease. As indicated in the Program Statement, the annual economic impact of cardiovascular disease in the U.S. exceeds $80 billion. Specific program components are focused also on "definition of marginal deficiencies..." and "bioavailability of nutrients in foods." Our research on vitamin A exemplifies those foci by examining the basic biochemistry of enzymes involved in the storage, release, and bioavailability of vitamin A in the body. 2.List the milestones (indicators of progress) from your Project Plan. FY 2005 Objective 1 - Begin construction of transfected cells Objective 3 - Begin antibody development Objective 4 - Begin construction of transfected and RNAi cells FY 2006 Objective 1 - Continue construction of transfected cells Objective 2 - Begin characterization of carotenoid-enriched cells Objective 3 - Complete antibody development - Begin localization studies Objective 4 - Continue construction of transfected RNAi cells FY 2007 Objective 1 - Complete construction of transfected cells - Begin characterization of transfected cells Objective 2 - Continue characterization of carotenoid-enriched cells Objective 3 - Complete localization studies Objective 4 - Complete construction of transfected an RNAi cells - Begin characterization of transfected and RNAi cells FY 2008 Objective 1 - Continue characterization of transfected cells Objective 2 - Continue characterization of carotenoid-enriched cells Objective 4 - Continue characterization of transfected and RNAi cells FY 2009 Objective 1 - Complete characterization of transfected cells Objective 2 - Complete characterization of carotenoid-enriched cells Objective 4 - Complete characterization of transfected and RNAi cells 4a.What was the single most significant accomplishment this past year? The role that different carboxylesterases play in vitamin A metabolism has been a matter of debate for many years. We identified carboxylesterase ES-10 as the most important contributor to retinyl ester hydrolase activity in liver. The protein was purified from liver and characterized in terms of its substrate specificity and enzyme kinetics. The tissue distribution of this enzyme was analyzed on both the gene and protein level, and demonstrated that ES-10 plays a major role in hepatic vitamin A metabolism. This finding will allow the vitamin A research community to investigate which influence drugs and xenobiotics have on the expression of ES-10 and thus on vitamin A metabolism. 4b.List other significant accomplishments, if any. We have completed a proof-of-principle study that proteomic methods can be used for biomarker discovery in nutrition research. Using plasma profiling of retinol deficient rats we used to Surface Enhanced Laser Desorption and Ionization Time of Flight Mass Spectrometer (SELDI-TOFMS) and two-dimensional liquid chromatograph (2D LC)/offline Matrix Enhanced Laser Desorption and Ionization Time of Flight Mass Spectrometer (MALDI-TOFMS) analysis to detect novel biomarkers of vitamin A-deficiency. It is expected that this approach will be applied to other nutrition deficiencies and metabolic imbalance disorders as well. Especially the SELDI-TOF MS approach is of great interest to the nutrition research community, due to its ability to screen larger number of samples in a high throughput fashion. This approach, in combination with bioinformatics methods, would allow nutrition researchers to create a database of biomarker patterns that can be correlated to nutritional status and would allow detection of nutritional imbalances by a simple blood test. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project on vitamin A, moved to ARS when the investigator joined the Diet and Human Performance Laboratory (DHPL) on 08/31/98, and has been actively pursued by the investigator for a number of years. Since joining the Agricultural Research Service (ARS), the major accomplishments of this project were defining the role or lack of role of three specific enzymes, namely carboxylester lipase (CEL), lipoprotein lipase (LPL), and pancreatic triglyceride lipase (PTL) on the metabolism of dietary vitamin A. This work has had major impact in the field of vitamin A metabolism, by defining which of the many enzymes (lipases and carboxylesterases) that can hydrolyse vitamin A esters actually do have a physiological role in the metabolism of this essential nutrient. Our recent work on mechanisms of intestinal absorption of vitamins is also being recognized, as indicated by invitations to prepare invited reviews. The project on carotenoids, also moved to ARS when the investigator joined DHPL on 08/31/98, and has been actively pursued by the investigator for a number of years. Since joining ARS, the major accomplishments have been the demonstration of the specificity of the effects of dietary carotenoids on the oxidation of human low-density lipoprotein (LDL) by endothelial cells in culture. We found that enrichment of LDL with beta-carotene protected LDL from oxidation. However, enrichment with the two other major carotenoids in the diet (and in plasma), namely, lutein and lycopene, actually enhanced cell-mediated oxidation of the LDL. Thus, the specific content of carotenoids in LDL clearly modulates its susceptibility to oxidation, but individual carotenoids may either inhibit or promote LDL oxidation. This underscores the importance of studying individual dietary carotenoids, and not just beta-carotene. We also found that enrichment of LDL with beta-carotene by supplementation of the diet protected LDL from oxidation. However, enrichment with another major carotenoid in the diet (and in plasma), namely lycopene (achieved by ingestion of tomato juice), had no effect on cell-mediated oxidation of the LDL. By studying the impact of enriching lipoproteins with carotenoids by dietary supplementation and comparing this method with the in vitro enrichment methods previously developed we clearly showed that dietary supplementation is more effective in inhibiting LDL oxidation than is in vitro enrichment. These results have shown us and other scientists that in vitro supplementation methods are not appropriate for studying the impact of carotenoid content of lipoproteins on their oxidation. Our work on the molecular mechanisms of intestinal absorption of carotenoids has demonstrated an important in vitro model system for studying bioavailability of these important phytonutrients. We have also reported the cloning of the enzyme that converts beta-carotene to vitamin A. The impact of our work both in vitamin A and in carotenoids is indicated by our continuing (1) publication in high impact journals, (2) invitations to present the work at major scientific meetings, and (3) continued grant support from the National Institutes of Health. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Our work in vitamin A has been presented at scientific conferences and published in the peer-reviewed literature. Other scientists are aware of our work and will likely adopt our methodology for isolating and studying retinyl ester hydrolases (REHs) and for studying intestinal absorption of vitamin A. Our work on carotenoids has been presented at scientific conferences and published in the peer-reviewed literature. Other scientists are aware of our work and will likely adopt our methodology for enrichment of lipoproteins with individual carotenoids, our new cell culture medium for oxidation experiments, and our new model system for studying carotenoid intestinal absorption using a cell culture model. Our work on proteomics has been extended to other investigators at the Beltsville Human Nutrition Research Center (BHNRC), and we serve as a resource for Beltsville Agricultural Research Center (BARC) in this field. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). During, A., Harrison, E.H. 2004. An in vitro model to study the intestinal absorption of carotenoids. In: Pigments in Food, More Than Colours (Dufosse, L., editor) Universite de Bretagne Occidentale, Quimper, France. p. 262-264. Linke,T., Ross, A.C., Harrison, E.R. 2005. Protein expression profiling of retinol-deficient rat plasma by 2D-LC and SELDI-TOF MS. Presented at the Baltimore Proteomics Symposium, Baltimore, Maryland, June 2005 (by invitation). Review Publications Linke, T.N., Dawson, H.D., Harrison, E.H. 2005. Microsomal acid retinyl ester hydrolase isolation, characterization, substrate and tissue specificity. Journal of Biological Chemistry. 280:23287-23294. During, A., Harrison, E.H. 2004. Intestinal absorption and metabolism of carotenoids: insights from cell culture. Archives Of Biochemistry and Biophysics. 430:77-88. Mcdevitt, T.M., Tchao, R., Harrison, E.H., Morel, D.W., 2005. Carotenoids normally present in serum inhibit proliferation and induce differentiation of a human monocyte/macrophage cell line (U937). Journal of Nutrition. 135:160-164.