BACKGROUND & AIMS: Selective removal of activated pancreatic stellate cells (PSCs) through induction of their own programmed death is a goal of therapeutic interest in patients with chronic pancreatitis. Here, we investigated the effects of tocotrienols on PSC death outcomes.

METHODS: Activated and quiescent PSCs and acinar cells from rat pancreas were treated with vitamin E derivatives alpha-tocopherol; individual alpha-, beta-, gamma-, and delta-tocotrienols; and a tocotrienol rich fraction (TRF) from palm oil.

RESULTS: TRF, but not alpha-tocopherol, reduced viability of activated PSC by setting up a full death program, independent of cell cycle regulation. Activated PSCs died both through apoptosis, as indicated by increased DNA fragmentation and caspase activation, and through autophagy, as denoted by the formation of autophagic vacuoles and LC3-II accumulation. In contrast to alpha-tocopherol, TRF caused an intense and sustained mitochondrial membrane depolarization and extensive cytochrome c release. Caspase inhibition with zVAD-fmk suppressed TRF-induced apoptosis but enhanced autophagy. However, mitochondrial permeability transition pore blockade with cyclosporin A completely abolished the deadly effects of TRF. beta-, gamma-, and delta-tocotrienol, but not alpha-tocotrienol nor alpha-tocopherol, reproduced TRF actions on activated PSCs. TRF death induction was restricted to activated PSCs because it did not cause apoptosis either in quiescent PSCs or in acinar cells.

CONCLUSIONS: Tocotrienols selectively trigger activated pancreatic stellate cell death by targeting the mitochondrial permeability transition pore. Our findings unveil a novel potential for tocotrienols to ameliorate the fibrogenesis associated with chronic pancreatitis.

Natural vitamin E comprises 8 different analogues, the alpha-, beta-, gamma-, and delta-tocopherols and the alpha-, beta-, gamma-, and delta-tocotrienols. However, only alpha-tocopherol is selectively enriched by the liver; the other vitamin E analogues and also excess alpha-tocopherol are converted to several metabolites and eliminated. Recently, a novel phosphorylated form of tocopherol, alpha-tocopheryl phosphate, was shown to occur naturally in animal and human tissues as well as in foods. Several synthetic vitamin E derivatives have been synthesized that are either converted by esterases to the natural form, or exert novel or vitamin E related biological activities. During the last years, specific cellular effects for each individual vitamin E analogue have been described that are the consequence of modulating signal transduction and gene expression. These effects possibly reflect specific interactions of each of the vitamin E analogues with enzymes, structural proteins, lipids and transcription factors. In this review, the different natural vitamin E analogues and synthetic derivatives are compiled in relation to their major molecular and cellular activities.

Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts. Side chain stereochemistry had no effect on omega-hydroxylation of alpha-tocopherol (alpha-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, alpha-TOH was a positive effector of omega-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.

Tocotrienols, a group of Vitamin E stereoisomers, offer many health benefits including their ability to lower cholesterol levels, and provide anticancer and tumor-suppressive activities. Several recent studies determined the cardioprotective abilities of tocotrienols, although the number is only 1% compared to the study with tocopherols. Both in acute perfusion experiments and in chronic models, tocotrienols attenuate myocardial ischemia-reperfusion injury, artherosclerosis, and reduced ventricular arrythmias. Apart from the antioxidative role of tocotrienols, it appears that tocotrienols mediated cardioprotection is also achieved through the preconditioning-like effect, the best yet devised method of cardioprotection. Hence, tocotrienols likely fulfills the definition of a pharmacological preconditioning agent and give a tremendous opportunity to place tocotrienols as an important therapeutic option in cardiovascular system.

Naturally occurring vitamin E, comprised of four forms each of tocopherols and tocotrienols, are synthesized solely by photosynthetic organisms and function primarily as antioxidants. These different forms vary in their biological availability and in their physiological and chemical activities. Tocopherols and tocotrienols play important roles in the oxidative stability of vegetable oils and in the nutritional quality of crop plants for human and livestock diets. The isolation of genes for nearly all the steps in tocopherol and tocotrienol biosynthesis has facilitated efforts to alter metabolic flux through these pathways in plant cells. Herein we review the recent work done in the field, focusing on branch points and metabolic engineering to enhance and alter vitamin E content and composition in oilseed crops.

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of alpha-tocopherol and gamma-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of gamma-tocotrienol was also larger than that of alpha-tocopherol. To investigate the intestinal uptake of alpha-tocopherol and gamma-tocotrienol in vivo, the mice were fed single doses of alpha-tocopherol or gamma-tocotrienol with triolein. The gamma-tocotrienol responded faster in plasma than alpha-tocopherol, although the maximal level of gamma-tocotrienol was lower than that of alpha-tocopherol. This suggested that the intestinal uptake properties of administered alpha-tocopherol and gamma-tocotrienol would characterize their plasma level transitions in mice.

A rapid analytical method including pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) has been developed for the determination of tocopherols and tocotrienols in cereals. The pressurized liquid extraction parameters were optimized in order to maximize the extraction efficiency. The use of methanol as extraction solvent at a temperature of 50 degrees C and a pressure of 110 bar, using one cycle of extraction with a static time of 5 min, provided the best results. A good LC separation was achieved using a C(18) column and a solution of 6.0 mM ammonia in methanol/water (97:3, v/v) as the mobile phase at a flow rate of 0.2 mL min(-1). MS coupling with an ESI interface in the negative ion mode was used as the detection technique. In the present work, it is shown that the addition of a base to the mobile phase is required to enhance the ionization of tocopherols and tocotrienols in negative ion mode electrospray ionization. The applicability of the method to cereal samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the 6-10% range. The recoveries of added tocopherols from cereal samples ranged from 91 to 109%.