Lise Hughes

Biokinetics of and discrimination between dietaryRRR- andSRR-α-tocopherols in the male rat

The net rates of uptake of the natural (2R,4′R,8′R) diastereoisomer of α-tocopherol (α-T) and the biodiscrimination relative to its 2S-epimer (2S,4′R,8′R) have been measured, in two experiments, for the blood and 21 tissues of male Sprague-Dawley rats fed over a period of several months diets containing deuterium-substituted forms of the α-T acetates. Gas chromatography-mass spectrometry was used to measure the amount of deuterated tocopherols taken up relative to the amount of nondeuterated tocopherol remaining. The measurements were performed at different times after the rats, placed for one month on a basal diet containing nondeuterated, natural α-T acetate, were switched to a diet containing the same total quantity of deuterated forms of either natural α-T acetate or a mixture of the acetates of the 2R- and 2S-epimers (i.e.,ambo-α-T acetate). In experiment 1 the source of vitamin E in the replacement diet was trideuterio-2R,4′R,8′R-α-T acetate. The data obtained provide the first direct measure of the rate at which natural vitamin E is replaced and augmented in the tissues of growing animals under normal laboratory dietary conditions. There are dramatic differences in the tissue kinetics; for example, the apparent half-life of vitamin E, i.e., the time at which the total amount of ingested trideuterio-α-T taken up is the same as the amount of nondeuterated α-T remaining, varies from ca. 1 wk for the lung to ca. 11 wk for the spinal cord. In experiment 2 the vitamin E in the replacement diet was an equimolar mixture of trideuterio-2S,4′R,8′R- and hexadeuterio-2R,4′R,8′R-α-T acetates. The results show that there is a preferential uptake of the natural diastereoisomer of α-T by all tissues (except the liver during the first month). Examination of fecal material reveals that the biodiscrimination begins in the gut; the incomplete hydrolysis of the acetates shows clearly that this reaction proceeds to a greater extent with the natural diastereoisomer. The greatest discrimination of all the tissues examined was found to occur in the brain. After five months, the level of the deuterated natural diastereoisomer was more than five times that of the deuterated 2S-epimer. These results have potential implications for human nutrition.

Oxidative degradation of β-carotene and β-apo-8′-carotenal

Abstract In the self-initiated oxidation of β-carotene with molecular oxygen the rate of oxygen uptake was shown to depend on the oxygen partial pressure. Epoxides, dihydrofurans, carbonyl compounds, carbon dioxide, oligomeric material, traces of alcohols, and probably carboxylic acids were formed. The main products in the early stage of the oxidation were shown to be 5,6-epoxy-β-carotene. 15,15′-epoxy-β-carotene, diepoxides, and a series of β-apo-carotenals and -carotenones. As the oxidation proceeded uncharacterised oligomeric material and the carbonyl compounds became more important and the epoxides degraded. In the final phase of the oxidation the longer chain β-apo-carotenals were themselves oxidized to shorter chain carbonyl compounds, particularly β-apo-13-carotenone, β-ionone, 5,6-epoxy-gb-ionone, dihydroactinidiolide and probably carboxylic acids. The effect of iron, copper and zinc stearates on the product composition and proportions was studied, as was the effect of light. The oxidation was inhibited by 2,6-di- t -butyl-4-methylphenol and α-tocopherol. The oxidations of β-apo-8′-carotenal and retinal under similar conditions were studied briefly, and the main products from the former compound were characterized. The initiation, the formation of the epoxides, the β-apo-carotenals and -carotenones, the successive chain shortening of the aldehydes to the ketones, and the formation of dihydroactinidiolide are explained in terms of free radical peroxidation chemistry.

Biokinetics in humans of RRR-alpha-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E.

The bioavailability of RRR-alpha-tocopherol from the oral administration of RRR-alpha-tocopherol itself and its acetate and succinate esters was determined in healthy human subjects. Venous blood samples were withdrawn periodically over a 51-h period following oral administration of a gelatin capsule containing an equimolar mixture of RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate. In a second study, subjects received a capsule containing an equimolar mixture of RRR-alpha-tocopheryl acetate and RRR-alpha-tocopheryl succinate. In Study 1, RRR-alpha-tocopherol was absorbed at similar rates from both the free phenol, and the acetate ester and maximum plasma levels occurred at 12 h in most subjects. The extent of absorption of RRR-alpha-tocopherol varied considerably between subjects in absolute terms, but the relative absorption from the two forms was remarkably consistent, and a ratio of 1.0 was found for parameters of relative bioavailability in plasma. The concentration of RRR-alpha-tocopherol from each form was maximal at approximately 27 h in red blood cells and, as seen with the plasma data, there was a large interindividual variability. In Study 2, there was no significant difference in the extent of absorption of RRR-alpha-tocopherol from the acetate ester and the succinate ester, although there was an apparently higher initial rate of absorption from the acetate ester.

Estimation of the location of natural α-tocopherol in lipid bilayers by 13C-NMR spectroscopy☆

Natural, 2R,4′R,8′R-α-tocopherol (vitamin E), labelled selectively with 13C in the methyl group at position 5, was incorporated into unilamellar vesicles of egg phosphatidylcholine. The vesicles are impermeable to the shift reagent Pr3+ and, in the presence of this reagent, separate 13C resonances due to labelled α-tocopherol in the outer and inner monolayers could be observed with relative intensities, 2:1. Subsequent addition of the relaxation reagent Gd2+ causes broadening and greatly shortened spin-lattice relaxation times for the resonance due to α-tocopherol in the outer monolayer only. These data confirm that α-tocopherol is located in both halves of the bilayers with its more hydrophilic chroman moiety very near the lipid-water interface, and indicate that the methyl group at position 5 of the α-tocopherol in the inner monolayer must be at least 40 A from the aqueous interface of the outer monolayer.

Comparison of free α-tocopherol and α-tocopheryl acetate as sources of vitamin E in rats and humans

The uptake of α-tocopherol from 2R,4′R,8′R-α-tocopherol and 2R,4′R,8′R-α-tocopheryl acetate has been compared in rats and humans. The two forms of vitamin E were compared simultaneously in each subject (rat and human) by using a combination of deuterium-substitution and gas chromatography-mass spectrometry (GC-MS) to distinguish and measure the competitive uptake of α-tocopherol from an orally ingested mixture of the acetate and the free phenol forms. When rats were dosed in a manner analogous to that used in traditional bioassays, i.e., providing the two forms of vitamin E one daily in tocopherol-stripped corn oil for four successive days immediately prior to sacrifice, the net uptake of α-tocopherol from the free phenol form was only half that from the acetate. This result is consistent with the greater activity of the acetate that had been observed previously in bioassays. However, when the two forms of tocopherol were intubated into rats as a single dose mixed in with an aqueous bolus of standard laboratory diet, the amount of α-tocopherol taken up from the free form after 24 hr was very similar to that derived from the acetate. In five adult humans, competitive uptake studies of the two forms after a single dose taken with a meal showed that the amount of α-tocopherol from the free phenol form was equal to that from the acetate in plasma and red blood cells. These findings illustrate the value and potential of using deuterium-substituted α-tocopherol and GC-MS in evaluating the effectiveness of different forms of vitamin E in human studies. The results also stress the need for caution in using data obtained from animal bioassays when considering comparative human nutritional standards.

Exploratory study of β-carotene autoxidation

Abstract The main products in the early stages of β-carotene autoxidation were epoxides, β-ionone, β-apo-13-carotenone, retinal, and related carbonyl compounds; in the final mixture short chain carbonyl compounds predominated.

Antioxidant Mechanisms of Vitamin E and β-Carotene

Vitamin E is an excellent trap for peroxyl radicals (ROO•) and it is the major lipid soluble antioxidant present in mammalian cells. It therefore occupies a unique position in the arsenal of natural antioxidants providing protection against various diseases. Product studies of the reaction of α-tocopherol with peroxyl radicals suggest that the existence of an α-tocopherol regeneration mechanism is essential for maintaining the antioxidant viability of the vitamin. Evidence now exists that vitamin C may regenerate vitamin E in some tissues. Studies carried out with deuterium-labeled α-tocopherol have confirmed that turnover of vitamin E is very slow in neural tissue, the tissue most susceptible to the effects of a deficiency of vitamin E in humans.

Vitamin E Activity of 1-Thio-α-Tocophero as Measured by the Rat Curative Myopathy Bioassay

The bioactivity of the acetate of the all-racemic, 1-thio analog of a-tocopherol (all-rac-]-thio-α-tocopheryl acetate) has been determined by measuring its ability to decrease plasma levels of pyruvate kinase in vitamin E deficient rats using the curative myopathy bioassay. The thio analog is only 0.22 times as active as RRR-α-tocopheryl acetate and is therefore approximately 0.33 times as active as all-rac-α-tocopheryl acetate, since the latter has been shown to be 1.47 times less active than RRR-α-tocopheryl acetate in the same bioassay (H. Weiser, M. Vecchi and M. Schlachter, Internal. J. Vit. Nutr. Res. 55 149-158 (1985)). The 0.33:1.0 ratio is similar to the ratio of 0.41:1.0 measured for the in vitro antioxidant activities of the corresponding free phenols. This finding lends further support to our view that the vitamin E activity in the curative myopathy bioassay of close structural analogs of α-tocopherol is determined primarily by the in vitro antioxidant activity of the analog relative to α-toco...