Abstract
The purpose of this study was to identify mammalian cell line(s) which possess intrinsic enzymatic activity of β-carotene 15,15′-dioxygenase. This enzyme (EC1.13.11.21) converts β-carotene to retinal (precursor of retinol and retinoic acid). To assess activity, cellular enzyme preparations were incubated with β-carotene for 60 min; retinal formed was quantified 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/h.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 μM and 23.8 pmol/h.mg, respectively. In addition, when TC7 cells were maintained in serum-free medium, activity was increased 8.2-fold after 19 days of postconfluency. Finally, 48 h incubation with β-carotene (delivered to TC7 cells in Tween 40) resulted in a 1.7-fold increase of 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 β-carotene 15,15′-dioxygenase activity and thus can be useful in future investigations of human carotenoid metabolism.
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