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United States Department of Agriculture

Agricultural Research Service

Title: Characterization of Beta Carotene 15,15-Dioxygenase Activity in Tc7 Clone of Human Intestinal Cell Line Caco-2

Authors
item During, Alexandrine
item ALBAUGH, GEORGE
item Smith Jr, James

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 17, 1998
Publication Date: N/A

Interpretive Summary: Dietary recommendations for Americans stress increased consumption of fruits and vegetables. Because these dietary guidelines are being heeded there is not a greater need for the body to manufacture its own vitamin A since it is not found, preformed, in fruits or vegetables. Luckily, cells in the human digestive tract can make vitamin A from the beta-carotene which is widely distributed in fruits and vegetables. However, study of how beta-carotene is changed to vitamin A using human cell lines has not been successful. Therefore, we screened several cell types in an effort to find one which possesses the ability to consistently change beta-caroten vitamin A; in addition to learning more about how this happens, this would allow identification of substances in food which can affect this conversion (i.e., fat, fiber, micronutrients) and hopefully how to maximize it. Five different cell lines available in the United States (four from humans and one from pig) did not form vitamin A when "fed" beta-carotene. However, an offspring (clone) of one of the human intestinal cell lines available to us from a colleague scientist in France, showed marked and consistent activity. To our knowledge, this is the first time that human cells from the intestine, grown outside the body, have been shown to change beta-carotene to vitamin A. This finding will allow us and other scientists to more easily and quickly find out what to recommend to those who eat diets of mainly plant-derived foods so that they will get adequate vitamin A. In addition, these results will benefit nutritionists, dieticians, and health care professionals. Hopefully, the end result will be better vitamin A nutriture for Americans and others.

Technical Abstract: The purpose of this study was to identify mammalian cell line(s) which possess intrinsic enzymatic activity of beta-carotene 15, 15'-dioxygenase. This enzyme (EC1.13.11.21) converts beta-carotene to retinal (precursor of vitamin A and retinoic acid). To assess activity, cellular enzyme preparations were incubated with beta-carotene for 60 min; the retinal formed was quantitated by HPLC. Activity was not detected in IPEC-1, HepG2, HL60, Wurzburg, or parent Caco-2 cell lines. However, two subclones of Caco-2, PF11 and TC7, possessed activity (2.5 and 14.7 pmol/hr.mg, respectively). Using the enzyme preparation of TC7 cells, retinal formation was linear with incubation time and protein concentration; Km and Vm values were 1.6 uM and 23.8 pmol/hr.mg, respectively. In addition, when TC7 cells were maintained in serum-free medium, activity was increased 8.2-fold after 19d of confluency. Finally, 48 hr incubation with beta-carotene (delivered to the TC7 cells in Tween 40) resulted in a 1.7-fold increase of the dioxygenase activity and the appearance of vitamin A (9.3 pmol/mg protein). However, retinoic acid was not detected under our experimental conditions. In sum, the TC7 subclone of the Caco-2 cell line possesses intrinsic beta-carotene 15, 15'-dioxygenase activity and thus can be useful in future investigations of human carotenoid metabolism.

Last Modified: 9/10/2014
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