This paper describes a simple method for the analysis of tocopherols in tissues by which frozen tissues-70 degrees C were pulverized at dry ice temperatures (-70 degrees C) and immediately extracted with hexane. There was no need to remove the coeluting lipids from tissues by saponification, since at that level of neutral lipids in the sample, there was no reduction in fluorescence response. For the analysis of oil, in which large amounts of neutral lipids were coextracted, a 20% reduction of fluorescence response was observed, but the response was equal for all tocopherol forms, and was appropriately corrected. Saponification was used only when tocopherol esters were present, and only after an initial hexane extraction to remove the free tocopherols in order to avoid their loss by saponification, particularly non alpha-tocopherol and tocotrienols. All the tocopherols and tocotrienols were separated on a normal-phase diol (epoxide) column that gave consistent and reproducible results, without the disadvantages of nonreproducibility with silica columns, or the lack of separation with reversed-phase columns. The tocopherols were quantitated by using a tocopherol form not present in the sample as an internal tocopherol standard, or using an external tocopherol standard if all forms were present, or when the sample was saponified. Piglet heart and liver samples showed the presence of mainly alpha-tocopherol, with minor amounts of beta- and gamma-tocopherol and alpha-tocotrienol, but no delta-tocopherol. Only small amounts of tocopherol esters were present in the liver but not in the heart.
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Interactions between vitamin E homologues and ascorbate free radicals in murine skin homogenates irradiated with ultraviolet light
Kitazawa M, Podda M, Thiele J, Traber MG, Iwasaki K, Sakamoto K, Packer L.
Photochem Photobiol. 1997 Feb;65(2):355-65.
The mechanism of oxidation of ascorbic acid in mouse skin homogenates by UV light was investigated by measuring ascorbate free radical formation using electron spin resonance signal formation. Addition of vitamin E (alpha-tocopherol or alpha-tocotrienol) had no effect, whereas short-chain homologues (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid [Trolox] and 2,2,5,7,8-pentamethyl-6-hydroxychromane [PMC]) accelerated ascorbate oxidation. The similar hydrophilicity of ascorbate, Trolox and PMC increased their interaction, thus rapidly depleting ascorbate. When dihydrolipoic acid was added simultaneously with the vitamin E homologues, the accelerated ascorbate oxidation was prevented. This was due to the regeneration of ascorbate and PMC from their free radicals by a recycling mechanism between ascorbate, vitamin E homologues and dihydrolipoic acid. Potentiation of antioxidant recycling may be protective against UV irradiation-induced damage. The rate of ascorbate oxidation in the presence of vitamin E homologues was enhanced by a photosensitizer (riboflavin) but was not influenced by reactive oxygen radical quenchers, superoxide dismutase or 5,5-dimethyl-1-pyrroline-N-oxide. These experimental results suggest that the UV irradiation-induced ascorbate oxidation in murine skin homogenates is caused by photoactivated reactions rather than reactive oxygen radical reactions.
Ozone depletes tocopherols and tocotrienols topically applied to murine skin
Thiele JJ, Traber MG, Podda M, Tsang K, Cross CE, Packer L.
FEBS Lett. 1997 Jan 20;401(2-3):167-70.
To evaluate ozone damage to hairless mouse skin, two parameters of oxidative damage, vitamin E depletion and malondialdehyde (MDA) production, were measured in vitamin E-enriched and in control skin from mice exposed to ozone (10 ppm). A 5% vitamin E solution (tocotrienol-rich fraction, TRF) in polyethylene glycol (PEG) was applied to 2 sites on the back of hairless mice, PEG to 2 sites. After 2 h, the sites were washed, one of each pair of sites covered and the mice exposed ozone for 2 h. Ozone exposure (compared with covered sites) increased epidermal MDA in PEG-treated sites, while vitamin E was unchanged. In contrast, ozone exposure significantly depleted vitamin E in TRF-treated sites, while significant MDA accumulation was prevented. This is the first demonstration that ozone exposure causes damage to cutaneous lipids, an effect which can be attenuated by vitamin E application.
Nitrofurantoin-induced hepatic and pulmonary biochemical changes in mice fed different vitamin E doses
Adam A, Marzuki A, Ngah WZ, Top GM.
Pharmacol Toxicol. 1996 Dec;79(6):334-9.
The hepatic and pulmonary effects of nitrofurantoin (40 mg/kg, intraperitoneally) were determined at 4 and 24 hr following its administration in mice fed for 10 weeks with a vitamin E sufficient, deficient or enriched diet. Liver glutathione (GSH) was reduced by nitrofurantoin at 4 hr but was unchanged 20 hr later. Nitrofurantoin did not affect liver glutathione peroxidase, glutathione reductase or superoxide dismutase activities. Liver catalase activities were decreased by nitrofurantoin at 4 hr. Lung GSH levels were increased whilst glutathione peroxidase activity was decreased at 4 and 24 hr. Lung glutathione reductase activity was reduced in certain groups. Nitrofurantoin did not affect lung superoxide dismutase, but catalase was decreased at 24 hr. Liver malondialdehyde levels were increased by nitrofurantoin in the vitamin E deficient group whilst lung malondialdehyde levels remained unchanged. Both liver and lung malondialdehyde levels were unaffected by vitamin E supplementation when compared to the vitamin E-sufficient group. These results suggest that nitrofurantoin (40 mg/kg) was deleterious to the liver and lung. Nitrofurantoin-induced lipid peroxidation was seen in vitamin E deficiency but an increase in dietary vitamin E content did not provide additional protection compared to the recommended daily allowance. The antioxidant activities of alpha-tocopherol and gamma-enriched tocotrienol were similar.
Lymphatic transport of alpha-, gamma- and delta-tocotrienols and alpha-tocopherol was measured in thoracic duct-cannulated rats. Animals were administered 3 ml of a test emulsion containing 200 mg sodium taurocholate, 50 mg fatty acid free-albumin, 200 mg fat and 100 mg of a mixture oftocotrienols and alpha-tocopherol (Exp. 1) or 10 mg of purified alpha-, gamma- or delta-tocotrienol or alpha-tocopherol (Exp. 2) through a gastric tube. Quantitative lymphatic recovery of oleic acid given as triolein was obtained in these experimental conditions. The 24-hours recovery of tocotrienols and alpha-tocopherol were 10-20% of the administered dose in Exp. 1. The recovery of alpha-tocotrienol was about 2-times higher than that of alpha-tocopherol, while that of gamma- and delta-tocotrienols was intermediate between these two alpha-forms. In Exp. 2, where these compounds were administered individually, the 24 hours recovery ranged from 22 to 37% of the administered dose. Again, the recovery of alpha-tocotrienol was significantly higher than that of the other tocotrienols and alpha-tocopherol, while that of gamma- and delta-tocotrienols and alpha-tocopherol was comparable. Thus, the results show the preferential absorption of alpha-tocotrienol compared to gamma- and delta-tocotrienols and alpha-tocopherol.
The available data in humans suggest that rice bran oil (RBO) is an edible oil of preference for improving plasma lipid and lipoprotein profiles similar to more commonly used vegetable oils. The observation that blending RBO with safflower oil at a specific proportion magnifies the hypocholesterolemic efficacy is of particular interest with regard to utilization of this oil. The occurrence of peculiar components such as gamma-oryzanol and tocotrienolsin RBO might be responsible for its hypocholesterolemic effect.
A liquid chromatographic (LC) method was developed for determination of tocopherols and tocotrienols in foods. Tocopherols and tocotrienols were released after saponification of test portions for 40 min at 80 degrees C, followed by extraction with hexane-ethyl acetate. After evaporation of the organic solvents, the extract was injected in the LC system. Separation of individual tocopherols and tocotrienols was satisfactory. Some interferences were encountered from tocomoneols, tocodienols, 2-tert-butyl-4-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-methylphenol, (BHT), and plastochromanol-8. The response of the LC system was linear over a range of 0-10 micrograms/mL for tocopherols and tocotrienols. The detection limit for these compounds was 0.1 micrograms/mL. Repeatabilty relative standard deviation values for tocopherols and tocotrienols in margarine, infant formula, and broccoli (concentration range, 0.11-22 mg/100 g) varied from 1.3 to 6.4%. Recoveries ranged from 91 to 105%.
The chemistry and antioxidant properties of tocopherols and tocotrienols
Kamal-Eldin A, Appelqvist LA.
Lipids. 1996 Jul;31(7):671-701.
This article is a review of the fundamental chemistry of the tocopherols and tocotrienols relevant to their antioxidant action. Despite the general agreement that alpha-tocopherol is the most efficient antioxidant and vitamin E homologue in vivo, there was always a considerable discrepancy in its “absolute” and “relative” antioxidant effectiveness in vitro, especially when compared to gamma-tocopherol. Many chemical, physical, biochemical, physicochemical, and other factors seem responsible for the observed discrepancy between the relative antioxidant potencies of the tocopherols in vivo and in vitro. This paper aims at highlighting some possible reasons for the observed differences between the tocopherols (alpha-, beta-, gamma-, and delta-) in relation to their interactions with the important chemical species involved in lipid peroxidation, specifically trace metal ions, singlet oxygen, nitrogen oxides, and antioxidant synergists. Although literature reports related to the chemistry of the tocotrienols are quite meager, they also were included in the discussion in virtue of their structural and functional resemblance to the tocopherols.
A tissue-specific distribution of the various vitamin E forms, tocotrienols and tocopherols, has been found, suggesting that these forms have unique roles in cellular functions. A sensitive procedure is described for the simultaneous determination of individual tocopherols, tocotrienols, ubiquinols, and ubiquinones using gradient high pressure liquid chromatography (HPLC) and electrochemical detection for vitamin E homologues and ubiquinols, and in-line UV detection for ubiquinones. Using this method, the lipophilic antioxidant complement of a variety of hairless mouse tissues was analyzed. Of the vitamin E forms, brain contained virtually only alpha-tocopherol (5.4 +/- 0.1 nmol/g; 99.8%) and no detectable tocotrienols were found. By contrast, skin contained nearly 15% tocotrienols and 1% gamma-tocopherol. In other tissues, the alpha-tocopherol content was higher (20 nmol/g), while each of the other forms represented about 1% of the total (gamma-tocopherol 0.2 to 0.4 nmol/g, alpha-tocotrienol 0.1, gamma-tocotrienol 0.2). Ubiquinol-9 concentrations were highest in kidney (81 nmol/g) and in liver (42 nmol/g), while the highest ubiquinone-9 concentrations were found in kidney (301 +/- 123 nmol/g) and heart (244 +/- 22 nmol/g). Liver contained nearly identical concentrations of each of the redox couple (ubiquinol-9 (41 +/- 16 nmol/g) and ubiquinone-9 (46 +/- 18 nmol/g). The unique distribution of these various antioxidants in the tissues measured suggests their distribution may be dependent upon selective mechanisms for maintaining antioxidant defenses in each tissue.
The concentration-dependent impact of gamma-tocotrienol on serum cholesterol can be traced to the posttranscriptional down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. gamma-Tocotrienol also suppresses tumor growth. Palmvitee, the tocopherol and tocotrienol-rich fraction of palm oil, is the sole commercial source of gamma-tocotrienol. Contrary to the universal findings of the efficacy of gamma-tocotrienol there are conflicting reports of the impact of Palmvitee on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, serum cholesterol concentrations and tumor development. These conflicting reports led us to examine the impact of alpha-tocopherol on the cholesterol-suppressive action of gamma-tocotrienol. Control and experimental diets were fed to groups of White Leghorn chickens (n = 10) for 26 d. The control diet was supplemented with 21 nmol alpha-tocopherol/g. All experimental diets provided 141 nmol of blended tocols/g diet. The alpha-tocopherol and gamma-tocotrienol concentrations of the experimental diets ranged from 21 to 141 and 0 to 120 nmol/g, respectively. We now report that including alpha-tocopherol in tocol blends containing adequate gamma-tocotrienol to suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase activity results in an attenuation of the tocotrienol action (P < 0.001). A summary of results from studies utilizing different Palmvitee preparations shows that effective preparations consist of 15-20% alpha-tocopherol and approximately 60% gamma- (and delta-) tocotrienol, whereas less effective preparations consist of > or = 30% alpha-tocopherol and 45% gamma- (and delta-) tocotrienol.