Progress 04/12/99 to 02/12/04
Outputs
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? 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. Objectives 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 ss-carotene into chylomicrons. We will test the hypothesis that SRB1, CD-36, FAT, and other putative lipid transporters mediate the uptake of ss- carotene into the cell. We will test the hypothesis that
increasing the expression of ss-carotene 15,15' dioxygenase increases the total flux of ss- 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 low density lipoprotein (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. 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 United States 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 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 United States 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. The expected technical outcomes of the experiments are detailed above under each specific objective. The overall outcomes are high quality, peer-reviewed publications reporting the results of the research. Major milestones for Objectives 1 and 4 are the construction of the transfected cell lines and RNAi cells, and we will reach this milestone within 2-3 years. The testing of the cell lines will then be accomplished. Objective 3 is fairly straightforward and will be carried out in the first 2 years (with the development of the antibodies as an important preliminary milestone). Objective 2 will be worked on throughout as time permits, with the major focus on this objective coming in the last two years of the 5-year cycle. Year 1 Year 2 Year 3 Year 4 Year 5 Objective 1
--------------------------------------> ------------ ------> Construction of transfected cells Characterization of cells Objective 2 ----------------------------------- Characterization of carotenoid- enriched cells Objective 3 -------------> --------------------------> Ab development Localization studies Objective 4 ----------------------------> -------------------------- > Construction of Characterization of cells transfected & RNAi cells 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substnatially met, briefly explain why not and your plans to do so. For CRIS 1235-51000-049-00D this is a new CRIS and hence there were no milestones achieved this year. For the previous CRIS 1235-51000-040-00D milestones were not specified. B. List the milestones (from the list in
Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? In the next year, our efforts will be focused on constructing of transfected cell lines for use in studies on vitamin A and carotenoid metabolism. In year 2, our efforts in the vitamin A field will be focused on understanding the localization, expression and function carboxylesterases ES-2, ES-3, ES-4 and ES-10 and, in particular, the role these enzymes play in vitamin A metabolism. We will study the cell and tissue-specific expression of these enzymes using real-time PCR and laser capture microdissection. In year 3, we will begin the characterization of the cells so as to define the mechanisms involved in the intestinal absorption and hepatic metabolism of vitamin A and carotenoids. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004
year: We sought to apply our recently developed cell culture model to study the mechanisms of vitamin A absorption in the intestine. Dr. Alexandrine During, a postdoctoral fellow in the Phytonutrients Laboratory, used CACO2 cells in culture to study the kinetics of retinol (vitamin A) absorption. The results showed that there was substantial "absorption" of free (unesterified) retinol and that the transport of retinol across the basolateral membrane was likely to be a facilitated transport. This work should lead to a rethinking of the mechanism by which the essential nutrient, vitamin A, is absorbed in the human intestine. B. Other significant accomplishments: None to report. C. Significant accomplishments/activities that support special targeted populations: 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 Dr. Harrison joined the Diet and Human Performance Laboratory
(DHPL) on August 31, 1998, and has been actively pursued by Dr. Harrison for a number of years. Since joining 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 vitamin is also being recognized as indicated by invitations to prepare invited reviews. The project on carotenoids, also moved to ARS when Dr. Harrison joined DHPL on August 31, 1998, and has been actively pursued by Dr. Harrison 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 the 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 the 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. During, A., Harrison, E.H. Intestinal absorption and metabolism of carotenoids: Insights from cell culture. Presented at the Gordon Research Conference on Carotenoids, Ventura, CA, January 2004 (by invitation). Linke. T., During A., Harrison, E.H. Mechanisms involved in the intestinal and hepatic metabolism of retinoids and carotenoids. Presented at the Federation of American Socities for Experimental Biology (FASEB) Summer Research Conference on Retinoids, Pine Mountain, GA, June 2004 (by invitation).
Impacts
(N/A)
Publications
- Linke, T., Ross, C., Harrison, E.H. 2004. Seldi-tof MS profiling of rat plasma: a novel tool for biomarker discovery in nutrition research. Journal of Chromatography. 1043:65-71.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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. 2. How serious is the problem? Why does it matter? 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 United States 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. 3. How does it relate to the National Program(s) and
National Program Component(s) to which it has been assigned? 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 "a definition of marginal deficiencies... and a 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. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment During FY 2003 Year: We sought to apply our recently developed cell culture model to study the mechanisms of vitamin A absorption in the intestine. Dr. Alexandrine During, a postdoctoral fellow in the Phytonutrients Laboratory, used CaCo2 cells in culture to study the kinetics of retinol (vitamin A) absorption. The results showed that there was substantial "absorption" of free (unesterified) retinol and that the transport of retinol across the basolateral membrane was likely to be a facilitated transport. This work should lead to a rethinking of the mechanism by which the essential nutrient, vitamin A, is absorbed in the human intestine. B. Other Significant Accomplishments: None. C. Significant Accomplishments/Activities that Support Special Targeted Populations: 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 the Agricultural Research Service (ARS) when Dr. Harrison joined the Diet and Human Performance Laboratory (DHPL) on 08/31/98, and has been actively pursued by Dr. Harrison for a number of years. Since joining 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 vitamin is also being recognized as indicated by invitations to prepare invited reviews. The project on carotenoids, also moved to ARS
when Dr. Harrison joined DHPL on 08/31/98, and has been actively pursued by Dr. Harrison 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 do you expect to accomplish, year by year, over the next 3 years? In the next year our efforts in the vitamin A field will be focused on understanding the localization, expression and function carboxylesterases ES-2, ES-3, ES-4 and ES-10, and in particular the role these enzymes play in vitamin A metabolism. We will study the cell and tissue-specific expression of these enzymes using real-time PCR and laser capture microdissection. In the second year our efforts will be focused on the mechanism of cellular uptake of carotenoids using cell culture model systems and stable isotope studies in humans. Finally, in the third year we will develop plasma and tissue proteomics approaches to study the influence of vitamin A and carotenoid nutritional status on global gene expression
in animal models and humans. 7. 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 the 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 the Beltsville Agricultural Research Center (BARC)in this field. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Linke, T., Harrison, E.H. Proteomic and bioinformatic approaches to nutrition research: Plasma profiling of retinol deficient rats by SELDI- TOF mass spectrometry. Presented as a late-breaking abstract at Experimental Biology 2003,San Diego, California, April 2003.
Impacts
(N/A)
Publications
- During, A., Harrison, E.H. Preferential uptake of zeaxanthin and lutein versus beta-carotene by differentiated ARPE-19 cells. The Federation of American Societies of Experimental Biology Journal. 2003. v.17. p.A758.
- Linke, T., Harrison, E.H. Purification, identification, and characterization of an acid retinyl ester hydrolase. The Federation of American Societies of Experimental Biology Journal. 2003. v.17. p.A315.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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. 2. How serious is the problem? Why does it matter? 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 United States 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. 3. How does it relate to the national Program(s) and
National Program Component(s) to which it has been assigned? Our research falls under the National Program on Human Nutrition Requirements, Food Composition, and Intake (NP 107). 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. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment During FY 2002 Year: This year we have isolated an acid retinyl ester hydrolase from rat liver microsomes and we have demonstrated that it is identical to rat liver carboxylesterase ES-10. This new information on the molecular identity of this retinyl ester hydrolase, along with our previous work, sets the stage for future investigations by us and others on the functional role of the carboxylesteraes in vitamin A metabolism. B. Other Significant Accomplishment(s), if any: None. C. Significant Activities that Support Special Targeted Populations: None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? The project on vitamin A, moved to ARS when Dr. Harrison joined the Diet and Human Performance Lab (DHPL) on 08/31/98, and has been actively pursued by Dr. Harrison for a number
of years. Since joining 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 vitamin is also being recognized, as indicated by invitations to prepare invited reviews. The project on carotenoids, also moved to ARS when Dr. Harrison joined DHPL, and has been actively pursued by Dr. Harrison 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 do you expect to accomplish, year by year, over the next 3
years? In the next year our efforts in the vitamin A field will be focused on understanding the localization, expression and function carboxylesterases ES-2, ES-3, ES-4 and ES-10 and in particular the role these enzymes play in vitamin A metabolism. We will study the cell and tissue-specific expression of these enzymes using real-time PCR and laser capture microdissection. In the second year our efforts will be focused on the mechanism of cellular uptake of carotenoids using cell culture model systems. Finally, in year three we will develop tissue proteomics approaches to study the influence of vitamin A and carotenoid nutritional status on global gene expression in animal models and humans. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? 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 the retinyl ester hydrolases (REHs) and for studying intestinal absorption of vitamin A. Our Laboratory is recognized as a leader in this field, as evidenced by an invitation to review our work in the American Society of Nutritional Sciences Journal, The Journal of Nutrition, in May 2001. 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. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace
your review publications which are listed below) During, A., Harrison, E.H. "Cellular uptake, incorporation into chylomicrons, and secretion of all-trans b-carotene by CaCO-2 cells," talk presented at the 13th International Carotenoid Symposium, Honolulu, HI, January 2002. Invited speaker at the Department of Nutrition, University of California at Davis, "Mechanisms involved in the digestion & absorption of vitamin A & carotenoids," March 14, 2002. Linke, T., Harrison, E.H. "Purification and characterization of an acid retinyl ester hydrolase from rat liver microsomes," poster presented at the Federation of American Societies of Experimental Biology Summer Research Conference on Retinoids, Tucson, AZ, June 2002.
Impacts
(N/A)
Publications
- Paik, J., During, A., Harrison, E.H., Mendelsohn, C.L., Lai, K., Blaner, W. S. Expression and characterization of a murine enzyme able to cleave b- carotene: The formation of retinoids. Journal of Biological Chemistry. 2001. v.76. p.32160-32168.
- During, A., Hussain, M.M., Morel, D.W., Harrison, E.H. Carotenoid uptake and secretion by CaCO-2 cells: b-carotene isomer selectivity and carotenoid interactions. Journal of Lipid Research. 2002. v.43. p.1086- 1095.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
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 lipoprotein's content of specific dietary carotenoids and asking whether this inhibits the oxidation of the lipoprotein by human aortic endothelial cells 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.
2. How serious is the problem? Why does it matter?
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 United States 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.
3. How does it relate to the National Program(s) and National Component(s)?
Our research falls under the National Program 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.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment During FY 2001: We sought to develop an in vitro model system to study the mechanisms of intestinal absorption of vitamin A and carotenoids because studies in humans are expensive and certain mechanistic studies are not feasible using human subjects. In collaboration with Dr. Mahmood Hussain of SUNY Downstate Medical Center, we developed a model system using human intestinal cells (CACO2) in culture that accurately mimics the intestinal absorption of fat-soluble nutrients like vitamin A and carotenoids. We used this system to demonstrate that the formation of intestinal lipoproteins called chylomicrons is absolutely required for vitamin A and carotenoid absorption and that the markedly reduced bioavailability of cis-isomers of carotenoids is due to discrimination of these isomers at the level of uptake by the intestinal cell membrane. This model system opens up the possibility of studying the molecular regulation of vitamin A and carotenoid
absorption from the gut. B. Other Significant Accomplishments: None. C. Significant Accomplishments/Activities that Support Special Targeted Populations: 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 Dr. Harrison joined DHPL on 08/31/98, and has been actively pursued by Dr. Harrison for a number of years. Since joining 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 vitamin is also being recognized as indicated by invitations to prepare invited reviews. The project on carotenoids, also moved to ARS when Dr. Harrison joined DHPL on 08/31/98, and has been actively pursued by Dr. Harrison 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. 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 do you expect to accomplish, year by year, over the next 3 years?
In the next 3 years our efforts in the vitamin A field will be focused on isolating and characterizing new membrane-associated REHs with the goal of obtaining pure proteins for molecular analysis in order to determine their relationship with other known lipases and carboxylesterases. We will also study the cell and tissue-specific expression of REHs, using real-time PCR and laser capture microdissection. In the comming years, our efforts in the carotenoid field will be focused on the mechanism of cellular uptake of carotenoids using cell culture model systems. Finally, we will develop tissue proteomics approaches to study the influence of vitamin A and carotenoid nutritional status on global gene expression in animal models and humans.
7. 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 & durability of the technology product?
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 the REHs and for studying intestinal absorption of vitamin A. Our Laboratory is recognized as a leader in this field as evidenced by an invitation to review our work in the American Society of Nutritional Sciences journal, The Journal of Nutrition, in May 2001. 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.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Invited speaker at the Department of Microbiology & Immunology, University of South Carolina School of Medicine. Talk on "Lipases and carboxylesterases: Possible roles in the hepatic utilization of vitamin A", November 6, 2000. During, A. and Harrison, E.H. "Cellular uptake, incorporation into chylomicrons, and secretion of all-trans b-carotene by CaCO2 cells," talk presented at the annual meeting Experimental Biology 2001 in Orlando, FL, April 2001.
Impacts
(N/A)
Publications
- Nayak, N., Harrison, E.H., Hussain, M.M. Retinyl ester secretion by intestinal cells: A specific and regulated process dependent on assembly and secretion of chylomicrons. Journal of Lipid Research. 2001. v. 42. p. 272-280.
- Hargrove, R.L., Etherton, T.D., Pearson, T.A., Harrison, E.H., Kris- Etherton, P.M. Low-fat and high monosaturated fat diets decrease human low density lipoprotein oxidative susceptibility in vitro. Journal of Nutrition. 2001. v. 131 p. 1758-1763.
- Harrison, E.H. Hussain, M.M. Mechanisms involved in the digestion and absorption of dietary vitamin A. Journal of Nutrition. 2001. v. 131 p. 1405 -1408.
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Progress 10/01/99 to 09/30/00
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
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 lipoproteins content of specific dietary carotenoids and asking whether this inhibits the oxidation of the lipoprotein by human aortic endothelial cells in culture. We use this system as a model of what may be occurring in the aortic wall in the intact human.
2. How serious is the problem? Why does it matter?
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, particularly 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 United States 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.
3. How does it relate to the National Program(s) and National Component(s)?
Our research falls under the National Program #107 on Human Nutrition Requirements, Food Composition, and Intake (100%). 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 (ie., 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.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment During FY 2000: Little is known about factor regulating intestional absorption of the essential nutrient, vitamin A. In the area of vitamin A research our major accomplishment in the past year was a definitive study demonstrating a role for the pancreatic enzyme, triglyceride lipase, in the digestion and absorption of dietary vitamin A. This research helped to clarify which of several enzymes is important in vitamin A digestion. B. Other Significant Accomplishments: In the area of carotenoid research our major accomplishment in the past year was the demonstration of the mechanism of endothelial cell-mediated oxidation of LDL by studying in detail the composition of the media required to support oxidation. Our results suggests a crucial importance of either iron or copper in the oxidation and a mechanism involving the Fenton reaction and redox cycling of the metal. This result will be of importance in defining potential targets for
intervention in preventing heart disease (atherosclerosis). C. Significant Accomplishments/Activities that Support Special Targeted Populations: 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 Dr. Harrison joined the Diet and Human Performance Laboratory on 08/31/98, and has been actively pursued by Dr. Harrison for a number of years. Since joining 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. The project on carotenoids, also moved to ARS when Dr. Harrison joined DHPL on 08/31/98, and has been actively pursued by Dr. Harrison 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. The impact of both of the areas of investigation 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 do you expect to accomplish, year by year, over the next 3 years?
In the coming year our efforts in the vitamin A field will be focused on isolating and characterizing new membrane-associated REHs with the goal of obtaining pure proteins for molecular analysis in order to determine their relationship with other known lipases and carboxylesterases. In the coming year our efforts in the carotenoid field will be focused on the mechanism of cellular uptake of carotenoids using cell culture model systems.
7. 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 & durability of the technology product?
None.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
"Lipases and carboxylesterases: Possible roles in the hepatic utilization of vitamin A", invited review for the American Society of Nutritional Sciences Journal, The Journal of Nutrition, in February, 2000. Invited speaker at the Eastern Regional research Laboratory (ARS). Gave talk on "Ipases and carboxylesterases: Possible roles in the hepatic utilization of vitamin A"in May 2000.
Impacts
(N/A)
Publications
- Harrison, E.H. Lipases and Carboxylesterases: Possible Roles in the Hepatic Utilization of Vitamin A. Journal of Nutrition. 2000. v.130. p. 340S-344S.
- Van Bennekum, A.M., Fisher, E.A., Blaner, W.S. and Harrison, E.H. Hydrolysis of retinyl esters by pancreatic triglyceride lipase. Biochemistry. 2000. v.39. p.4900-4906.
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Progress 01/01/99 to 09/30/99
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 lipoprotein's content of specific dietary carotenoids and asking whether this inhibits the oxidation of the lipoprotein by human aortic endothelial cells in culture. We use this system as a model of what may be occurring in the aortic wall in the intact human. 2. How serious is the problem? Why does it matter? 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, particularly 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 United States 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. 3. How does it relate to the National Program(s) and National Component(s) to which it has been assigned? Our research falls under the National Program 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 United States 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. 4. What were the most significant accomplishments this past year? In the area of vitamin A research, our major accomplishment in the past year was a definitive study demonstrating the role for the enzyme, lipoprotein lipase (LPL) in the tissue uptake of dietary vitamin A, using gene
knockout and transgenic systems. This work clarified a problem in assessing the role of that particular REH in vitamin A metabolism. We have also conducted experiments that suggest a role for the pancreatic enzyme, triglyceride lipase, in the digestion and absorption of dietary vitamin A. In the area of carotenoid research, our major accomplishment in the past year was 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 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. Second, we have explored the mechanism of endothelial cell-mediated oxidation of LDL by studying, in detail, the composition of the media required to support oxidation. Our results suggests a crucial importance of either iron or copper in the oxidation and a mechanism involving the Fenton reaction and redox cycling of the metal. 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 Dr. Harrison joined DHPL on August 31, 1998, and has been actively pursued by Dr. Harrison for a number of years. Since joining 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. The project on carotenoids, also moved to ARS when Dr. Harrison joined DHPL on August 31, 1998, and has been actively pursued by Dr. Harrison 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. The impact of both of the areas of investigation 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 do you expect to accomplish, year by year, over the next 3 years? In the coming year, our efforts in the vitamin A field will be focused in two areas. First, we will study the impact of expression of another REH (pancreatic triglyceride lipase or PTL) on the metabolism of dietary vitamin A. Second, we will isolate and characterize new membrane- associated REHs, with the goal of obtaining pure proteins for molecular analysis, in order to determine their relationship with other known lipases and carboxylesterases. In the coming year, our efforts in the
carotenoid field also will be focused in two areas. We will study the impact of carotenoid enrichment of the endothelial cells on their ability to oxidize LDL. Additionally, we will initiate studies on the mechanism of cellular uptake of carotenoids using cell culture model systems. 7. 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 & durability of the technology product? 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 both our methodology for isolating and studying the REHs. Our laboratory is recognized as a leader in this field, as evidenced by an invitation to present our work at the American Society of Nutritional Sciences Symposium on "Mechanistic Aspects of Vitamin
and Coenzyme Utilization and Function," held at the Experimental Biology '99 Meeting in April 1999. 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 both our methodology for enrichment of lipoproteins with individual cartenoids and our new cell culture medium for oxidation experiments. 8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below). Invited participant and speaker at Workshops on Nutrient Metabolism in the Genetic Anemias and Dietary Supplements of Potential Benefit to Patients with Sickle Cell Disease, sponsored by the Division of Blood Diseases & Resources, National Heart, Lung, and Blood Institute, the Office of Rare Diseases, NIH, and the Office of Dietary Supplements, NIH.
Gave talk on "Different antioxidant capacity of specific carotenoids in endothelial cell-mediated LDL oxidation" in May 1999.
Impacts
(N/A)
Publications
- VAN BENNEKUM, A.M., LI, L., HARRISON, E.H. et al. 1999. Carboxyl ester lipase overexpression in rat hepatoma cells and CEL-deficiency in mice ... uptake or metabolism of chylomicron retinyl ester. Biochem. 38:4150-4156.
- DUGAS, T.R., MOREL, D.W. and HARRISON, E.H. 1999. Dietary supplementation with beta-carotene, but not with lycopene, ... cell-mediated oxidation of low-density lipoprotein. Free Radical Biol. Med. 26:1238-1244.
- VAN BENNEKUM, A.M., KAKO, Y., WEINSTOCK, P.H., HARRISON, E.H. et al. 1999. The level of lipoprotein lipase expression influences tissue clearance of chylomicron retinyl ester. J. Lipid Res. 40:565-574.
- WENG, W., LI, L., VAN BENNEKUM, A.M., POTTER, S.H., HARRISON, E.H. et al. 1999. Intestinal absorption of dietary cholesteryl ester is decreased but retinyl ester absorption ... knockout mice. Biochem. 38:4143-4149.
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