Beta-Carotene protects against oxidative stress, heart disease and cancer by quenching singlet oxygen and enhancing the immune response; humans world-wide derive most of their vitamin A from it. Despite its key physiologic roles, the quantitative aspects of Beta-carotene absorption and metabolism to retinol and other retinoids (especially retinoic acid) in humans is poorly understood. Further, the effects of Beta-carotene status on these processes is virtually unknown. Progress here has been hindered by the lack of a suitable animal model, the scarcity of stable isotope labeled carotenoids and retinoids, and by the analytical difficulties associated with measuring them in biological tissues. We are now in a position to conduct isotope studies on Beta- carotene in vivo in human because we can make the Beta-carotene-d8 and we are experienced with stable isotopes. Our objective is to characterize the dynamics of Beta-carotene metabolism in women and men under conditions of carotene depletion and repletion. These studies will be performed in the metabolic research unit (MRU) at USDA Western Human Nutrition Research Center (WHNRC) in San Francisco, CA. Specifically, we will accomplish the following objectives: 1) determine the kinetics of appearance and disappearance of Beta-carotene-d8 in plasma and its conversion into the plasma retinol pools as retinol--d4; 2) follow the metabolism of Beta-carotene-d8 into retinoic acid-d4, retinyl and retinoyl-d4- glucuronide and possibly other retinoids in plasma, urine and feces; 3) determine the influence of body storage reserves of Beta- carotene-Beta on its metabolism; 4) document the biological behavior of the deuterated carotene for any isotopic discrimination. Two 125-day carotene depletion/repletion studies are proposed in the MRU using the same crossover design. Study one will be with 12 adult women volunteers, and study 2 will be 12 adult men. The study's length is 125 days, and includes 50 day intervals for the depletion of carotene body reserves and 25 days for repletion with 15 mg Beta-carotene daily supplements. A single 40 mg Beta- carotene-d8 dose will be given at two time points; one time point at carotene depletion and the other at carotene repletion. After the WHNRC studies a dual label experiment designed to assess the biological behavior of the Beta-carotene-d8 for isotopic discrimination is proposed. We will administer a single 40 mg dose of stable isotope labeled Beta-carotene (20 mg Beta-carotene-d8 plus 20 mg 13C-Beta-carotene) to 2 subjects (1 male, 1 female). The identity of the 13C and deuterated Beta-carotenes and their key metabolites (retinol-d4 and 13C-retinol) will be distinguished using mass spectrometry. Plasma kinetic curves for the carotene and retinol isotopomers will be obtained. The degree of correlation between the two data sets will reflect the degree of the isotope discrimination. A quantitative understanding of the dynamics of Beta-carotene metabolism in vivo in humans is crucial to understanding the overall role of Beta-carotene in health and disease.
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