David O. Foster

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.

Biokinetics of dietaryRRR-α-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not “spare” vitamin Ein vivo

The net rates of uptake of “new” and loss of “old”2R,4′ R,8′ R-α-tocopherol (RRR-α-TOH, which is natural vitamin E) have been measured in the blood and in nine tissues of male guinea pigs over an eight week period by feeding diets containing deuterium-labelled α-tocopheryl acetate (d6-RRR-α-TOAc). There was an initial two week “lead-in” period during which 24 animals [the “high” vitamin E (HE) group] received diets containing 36 mg of unlabelled (d0)RRR-α-TOAc and 250 mg of ascorbic acid per kg diet, while another 24 animals [the “low” vitamin E (LE) group] received diets containing 5 mgd0-RRR-α-TOAc and 250 mg ascorbic acid per kg diet. The HE group was then tivided into three equal subgroups, which were fed diets containing 36 mgd6-RRR-α-TOAc and 5000 mg [the “high” vitamin C (HEHC) subgroup], 250 mg [the “normal” vitamin C (HENC) subgroup] and 50 mg [the “low” vitamin C (HELC) subgroup] ascorbic acid per kg diet. One animal from each group was sacrificed each week and the blood and tissues were analyzed ford0- andd6-RRR-α-TOH by gas chromatography-mass spectrometry. The LE group was similarly divided into three equal subgroups with animals receiving diets containing 5 mgd6-RRR-α-TOAc and 5,000 mg (LEHC), 250 mg (LENC) and 50 mg (LELC) ascorbic acid per kg diet with a similar protocol being followed for sacrifice and analyses. In the HE group the totald0-+d6-)RRR-α-TOH concentrations in blood and tissues remained essentially constant over the eight week experiment, whereas in the LE group the totalRRR-α-TOH concentrations declined noticeably (except in the brain, an organ with a particularly slow turnover of vitamin E). There were no significant differences in the concentrations of “old”d0-RRR-α-TOH nor in the concentrations of “new”d6-RRR-α-TOH found in any tissue at a particular time between the HEHC, HENC and HELC subgroups, nor between the LEHC, LENC and LELC subgroups. We conclude that the long-postulated “spring” action of vitamin C on vitamin E, which is well documentedin vitro, is of negligible importancein vivo in guinea pigs that are not oxidatively stressed in comparison with the normal metabolic processes which consume vitamin E (e.g., by oxidizing it irreversibly) or elminate it from the body. This is true both for guinea pigs with an adequate, well-maintained vitamin E status and for guinea pigs which are receiving insufficient vitamin E to maintain their body stores.The biokinetics of vitamin E uptake and loss in the HE guinea pigs are compared with analogous data for rats reported previously (Lipids 22, 163–172, 1987). For most guinea pig tissues the uptake of vitamin E under “steadystate” conditions was faster than for the comparable rat tissues. However, the brain was an exception with the turnover of vitamin E occurring at only one-third of the rate for the rat.

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.

Comparison of microspheres and 86Rb+ as tracers of the distribution of cardiac output in rats indicates invalidity of 86Rb+-based measurements

The technique of using gamma-labeled plastic microspheres (15 +/- 5 micrometer) to measure cardiac output (CO) and its fractional distribution (FD) to individual tissues and organs was judged by various criteria to give valid data when applied to barbital-sedated warm-acclimated or cold-acclimated (CA) white rats, which were either resting or responding calorigenically to infused noradrenaline (NA). The FD of CO to each of 16 tissues or organs of CA rats at rest or responding to NA was then estimated both with 86Rb+ and with microspheres, the two tracers being injected simultaneously. For only seven of the tissues examined in resting rats and only one in NA-infused rats was the FD of CO estimated with 86Rb+ not significantly different from that estimated with microspheres. 86Rb+ to microsphere ratios of the FD of CO to individual tissues ranged from 3.5 and 3.0 for liver and skeletal muscle, respectively, down to 0.09 and 0.07 for brown adipose tissue (BAT) and brain. Since microsphere-based estimates of blood flow to the interscapular BAT of CA rats responding to NA were corroborated by direct measurements of venous efflux from the tissue, it is unequivocal that the 86Rb+-based estimate of the fraction of CO directed to interscapular BAT was highly erroneous. When considered along with data from the literature, the present findings support a conclusion that the uptake of 86Rb+ by a tissue frequently does not provide a valid measure of the FD of CO to the tissue. Some of the factors that are likely responsible for this situation are discussed, and it is suggested that only by a fortuitous combination of circumstances does the uptake of 86Rb+ by a tissue sometimes match the FD of CO to the tissue.

Evidence for pore-like opening of the blood-brain barrier following forebrain ischemia in rats

The nature of the delayed blood-brain barrier (BBB) opening that occurs in rats subjected to forebrain ischemia by the technique of two-vessel (carotid) occlusion plus hypovolemic hypotension (2VO ischemia) was probed by examining the simultaneous, trans-barrier movement of two hydrophilic, normally poorly permeative solutes of markedly different molecular size: sucrose (MW = 342) and inulin (MW approximately 5000). Pentobarbital-anesthetized, male, Sprague-Dawley rats (342-374 g) were subjected to 10 min of 2VO ischemia (tympanic temperature, 37.5-38.0 degrees C); 6 h later they were reanesthetized and, along with non-ischemic controls, injected i.v. with [14C]sucrose and [3H]inulin. Transfer constants (Kis) for blood-to-brain movement of the tracers and Vis (apparent initial volumes of tracer distribution) were determined for six brain regions by the multiple-time, graphical method (tracer circulation times from 3 to 30 min). Vis differed little or insignificantly between the two tracers, or between control and post-ischemic rats; the values did not suggest appreciable endothelial binding of either tracer that might lead to its uptake by adsorptive-phase endocytosis. In the controls, regional Kis +/- S.E.M. (nl g(-1) s(-1)) for inulin ranged from 0.18 +/- 0.04 to 0.31 +/- 0.09 and were significantly lower (P < 0.01) than Kis for sucrose (1.53 +/- 0.16-1.91 +/- 0.29). The Ki ratio (sucrose/inulin) across brain regions (mean, 6.6; S.E.M., 0.6) was much lower than would be expected according to the concept that movement of most organic non-electrolytes across the intact BBB occurs by dissolution in and diffusion through endothelial cell plasma membranes, at a rate proportional to the lipid solubility and diffusivity of the solute. This finding is interpreted as indicating that a portion of the transfer of sucrose and inulin occurred by a mechanism other than dissolution-diffusion (e.g., via pores or vesicles). In the post-ischemic rats, Kis for both tracers were elevated significantly (P < 0.01) in parietal cortex, striatum, hippocampus, and midbrain. The post-ischemic increases (delta Kis) in these regions were greater for sucrose (1.90-3.31 nl g(-1) s(-1)) than for inulin (0.80-1.33). Across brain regions the ratio between sucrose delta Ki and inulin delta Ki averaged 2.9 (S.E.M., 0.2), a value significantly greater than the ratio of 1 that would be expected were the BBB opening due to an enhancement of micropinocytosis and vesicular transport. The correspondence of the mean delta Ki ratio with the ratio of the free diffusion coefficients of the tracers (D(f, suc)/D(f, inu) = 2.9; water, 38 degrees C) suggests that the delayed opening of the BBB following 2VO ischemia involves the formation of trans- or paracellular, aqueous pores or channels.

Chiral discrimination in the exchange of α-tocopherol stereoisomers between plasma and red blood cells

The transport of 2R,4′R, 8′R-α-tocopherol and 2S,4′R,8′R-α-tocopherol from plasma into rat red blood cell membranes occurs with essentially no chiral discrimination. The previously demonstrated (10) preference of red blood cell membranes favoring 2R,4′R,8′R-α-tocopherol over the 2S,4′R, 8′R stereoisomer is shown to be due to better retention of the former compound, i.e., to preferential retention of natural vitamin E.

Brown adipose tissue: the dominant site of nonshivering thermogenesis in the rat.

Measurements with tracer microspheres of changes in tissue blood flow associated with noradrenaline (NA)-induced calorigenesis in warm-acclimated and in cold-acclimated (CA) rats revealed very large increases in flow to brown adipose tissue (BAT). The data on flow together with measurements of the arteriovenous difference in blood O2 across interscapular BAT indicate that BAT accounts for at least 60% of the NA-induced nonshivering thermogenesis (NST) of CA rats. Skeletal muscle was found to be only minimally, if at all, involved in this NST.

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...