ABSTRACT
Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.
Subject(s)
Caco-2 Cells , Micelles , Vitamin E/chemistry , alpha-Tocopherol/analogs & derivatives , alpha-Tocopherol/chemistry , Absorption , Caco-2 Cells/chemistry , Caco-2 Cells/metabolism , Enterocytes/chemistry , Enterocytes/metabolism , Humans , Hydrolysis , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Microscopy, Confocal , Models, Biological , Solubility , Sterol Esterase/chemistry , Sterol Esterase/metabolism , Taurocholic Acid/chemistry , Taurocholic Acid/metabolism , Time Factors , Tocopherols , Vitamin E/metabolism , alpha-Tocopherol/metabolismABSTRACT
Although data on the behavior of tocopheryl acetate (Tac) in the gut is rare, some studies show that this ester is hydrolyzed in the intestine and the released tocopherol (Tol) is absorbed. An HPLC method is published for the simultaneous analysis of Tol and Tac in mixed micelles. This study shows how this method is used for the measurement of esterase activity on Tac using commercial cholesterol esterase. The rate of hydrolysis is determined in less than 20 min on the basis of both Tac disappearance and Tol appearance. The present method is useful for investigating esterase activities on Tac in model systems that mimick the absorption environment or in any biological or nonbiological medium.
Subject(s)
Chromatography, High Pressure Liquid/methods , Sterol Esterase/metabolism , alpha-Tocopherol/analogs & derivatives , Intestinal Absorption , Tocopherols/analysis , alpha-Tocopherol/analysis , alpha-Tocopherol/metabolismABSTRACT
This report improves analytical procedures to investigate the behaviour of the two Vitamin E forms, alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac), in model systems mimicking the intestinal medium. We describe how to prepare mixed micelles as vehicle for Tac and Tol and the HPLC method for their quantification in the micelles. Tac and Tol were extracted using ethanol-hexane-drying procedure, whereas the separation and detection were performed in methanol and by UV method, respectively. Both compounds were eluted in less than 4 min. In the range between 1.7 microM and 54 microM of Tac or Tol in the micelles, their recovery were 89% and 81%, respectively, with correlation coefficient over 0.99 and R.S.D. of less than 7.2% in all cases. Limits of detection and quantification for Tac and Tol in mixed micelles ranged between 1 microM and 2 microM and between 3 microM and 5 microM, respectively. The behaviours of Tac and Tol were quite different during the extraction procedure and both were influenced by the vitamin concentration and the relative volume of organic solvents.
