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Nutrition is undergoing a revolution owing to the recognition that some foods contain trophic, health-promoting factors distinct from essential nutrients. In this revolution, whey is increasingly being viewed as more than a source of proteins with a particularly nutritious composition of essential amino acids. Milk evolved under continuous Darwinian selection pressure to nourish mammalian neonates. Evolutionary pressure appears to have led to the elaboration of a complex food that contains proteins, peptides, complex lipids, and oligosaccharides that act as growth factors, toxin-binding factors, antimicrobial peptides, prebiotics, and immune regulatory factors within the mammalian intestine. Importantly, these trophic macromolecules are not essential, although the health benefits that their biological activities within the intestine provide likely contributed to neonatal survival. Human and bovine milks contain many homologous components, and bovine whey may prove to be a source for molecules capable of providing biological activities to humans when consumed as food ingredients. To approach this potential, food and nutrition research must move beyond the description of food ingredients as delivering only essential nutrients and develop a mechanistic understanding of the interactions between dietary components and the metabolic and physiological properties of the intestine.

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The intent of this symposium is to assemble current knowledge of the role of arachidonic acid (AA) in the diet to provide a conceptual and mechanistic framework for future research. The principal focus is on the varied biological effects of dietary AA, including opposing effects of n-3 and n-6 polyunsaturated fatty acids (PUFA); regulation of n-6 PUFA metabolism, eicosanoid synthesis and gene expression; the importance of AA in infant nutrition and the contemporary Western diet in general; and the effects of AA on tumor promotion. Through its myriad actions and remarkably ubiquitous presence in cells, AA can be argued to affect every cell of the body. Although the varied molecular events associated with the metabolism of AA have been subjects of intense investigation, the ability of AA in the diet to alter AA levels in cellular membranes is poorly described and is thus the focus of this symposium.

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In vivo interactions of vitamin E with diethylmaleate (DEM) and bromotrichloromethane (CBrCl3) were examined in rats fed a diet either without vitamin E or supplemented with 30 IU dl-alpha-tocopheryl acetate/kg. Groups of rats within each dietary group were given two injections 30 min apart. One group received two injections of the mineral oil carrier. The other groups were injected with either DEM and mineral oil, mineral oil and CBrCl3, or DEM and CBrCl3. The rats were killed 10 min after the second injection. Measurements were made of hepatic GSH, thiobarbituric acid-reactive substances (TBARS) as a lipid peroxidation index, and 11 enzymes as potential markers of oxidant damage. Special focus was placed on reactive cysteine-containing aldehyde dehydrogenase (ALDH). Although dietary vitamin E protected ALDH, the enzyme was highly susceptible to oxidant damage. ALDH activity was correlated with GSH (r = 0.83, p less than 0.001) and there was an inverse relationship between the logarithmic values of ALDH activity and TBARS (r = 0.78, p less than 0.001). Similar results were observed for a number of other enzymes when GSH depletion preceded oxidant treatment. Two-way analysis of variance revealed significant effects of vitamin E and of injection treatments on hepatic GSH. There was a significant interaction between vitamin E and the injection treatments on the activities of five enzymes. The results suggested that vitamin E and GSH functioned together to protect sensitive enzymes against oxidant stress. The sensitive enzymes may be useful markers of hepatic damage in vivo.

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Bilirubin has been suggested as a physiological antioxidant, and recent studies suggest that its synthesis is induced in response to oxidative stress. Numerous reports in the literature show increases in serum bilirubin when using halogenated hydrocarbons as oxidative stress inducers. Analogously, these increases should also be expected for other inducers. On the other hand, bilirubin is destroyed by the same molecules that induce its production. The measurement of bilirubin may be a useful index of in vivo oxidative stress, although no big differences in bilirubin levels should be expected.

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Over the past twenty-years of lipid peroxidation research in this laboratory, considerable effort has gone into development of new methods, with emphasis on measurement of lipid-soluble fluorophores and the volatile hydrocarbons ethane and pentane. Application of these and other methods has been made to biological materials and living animals. Although the various methodologies used in lipid peroxidation research do not necessarily measure the same class of products, and although agreement of results is not always 100%, there is substantial documentation of good correlations between measurements; for example, of trace volatile hydrocarbons with thiobarbituric acid-reacting substances, of pentane production with dietary and/or tissue vitamin E content, and of pentane production with lipid-soluble fluorophores accumulated in spleen as a function of oxidant stress. Individual methodologies do have their inherent limitations. However, measurements of multiple products and their correlations have added significantly to the base of information on biological damage and protection by dietary antioxidants against nutritional and toxicological insults to tissues, cells, and macromolecules as a result of peroxidative and oxidative reactions.

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The active component of aurothioglucose (ATG) in effecting changes in plasma sulfhydryl (SH) levels and plasma SH reactivity with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was determined. These two measurements are applied clinically to rheumatoid arthritis patients undergoing chrysotherapy. Normal rats were injected intramuscularly daily for seven days with 30 mumol of either ATG or sodium thioglucose (STG)/kg body wt or with an equivalent volume of the carrier, 0.05% benzyl alcohol. ATG but not STG significantly increased total SH levels in plasma, liver, and kidney. The seven-day treatment with ATG significantly increased glutathione levels in kidney but not in liver or plasma. Thus, gold(I) rather than thioglucose was the active moiety that affected SH levels in ATG-injected rats. In vivo, gold(I) was also the active moiety that stimulated plasma SH reactions with DTNB at pH 7.4, since injection of ATG but not STG stimulated the SH reactivity in fresh plasma. In vitro, ATG increased the rate of plasma reaction with DTNB at pH 7.4, thus, gold(I) ions acted as a catalyst in the SH-disulfide exchange reaction. This study demonstrates that gold(I) but not its thiol ligand strongly interacts with protein SH groups in the rat tissues. Such an interaction may play an important role in the biological actions of gold.

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Catalase activity and cytochrome content were measured in kidneys of Fisher 344 rats injected with aurothioglucose (ATG) either daily for 3 days or 5 days a week for up to 8 wk. Catalase activity was decreased 39%, 59%, and 48% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. Microsomal cytochrome P-450 levels decreased 71%, 86%, and 80% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. In contrast, cytochrome b5 was significantly increased at 3 days and 2 wk, but not at 8 wk. Microsomal heme contents decreased 44% (p less than 0.001), 34% (p less than 0.001), and 22% (p greater than 0.05) at 3 days, 2 wk, and 8 wk, respectively. The content of mitochondrial cytochromes aa3, b, c1, and c were not affected after 8 wk of ATG treatment. In vitro inhibition of the heme-containing enzyme delta-aminolevulinic acid dehydratase by ATG was reversible in the presence of physiological concentrations of small thiols. Although the activity of this enzyme in kidneys of ATG-treated rats was not measured, its significant inhibition in vivo by ATG appears unlikely. This study demonstrates that there were differential effects of gold on the various cytochromes and that changes in catalase activity paralleled changes in cytochrome P-450 and heme contents in the kidneys of ATG-treated rats. The findings are relevant to nephrotoxicity during chrysotherapy.

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Fisher 344 rats injected with a total of 20.8 +/- 1.5 mg of gold (Au) as aurothioglucose over an 8-wk period were used to study the effect of long-term Au treatment on selenium-dependent glutathione peroxidase (SeGSHPx), other enzymes related to GSH metabolism, GSH, nonprotein sulfhydryls, and total sulfhydryls (SH) in various tissues. The indirect coupled assay for SeGSHPx revealed decreased activity in platelets of Au-treated rats but not in other tissues. Inhibition of SeGSHPx by Au is reversible upon dilution. A direct assay of GSH consumption by concentrated tissue cytosols that was developed to minimize enzyme dilution provided evidence of in vivo inhibition of SeGSHPx in kidney and liver from Au-injected rats. Kidneys of these rats had decreased (P less than 0.05) activities of GSSG reductase (36%), gamma-glutamylcysteine synthetase (19%), and gamma-glutamyl transpeptidase (26%), and increased (P less than 0.05) activities of glucose 6-phosphate dehydrogenase (90%) and GSH S-transferase (130%). The reactivity of fresh plasma SH groups with 5,5'-dithiobis-(2-nitrobenzoic acid) increased as a function of injection time. Enhanced SH reactivity suggests that Au may react with protein GSH-disulfides to release GSH. New findings were (i) decreased platelet SeGSHPx and kidney GSSG reductase in aurothioglucose-injected rats, (ii) direct in vivo inhibition of kidney and liver SeGSHPx in aurothioglucose-injected rats, and (iii) no significant correlation between the activity of GSH-metabolizing enzymes and levels of tissue GSH.

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The antirheumatic drug aurothioglucose is an inhibitor of the selenoenzyme GSH peroxidase. During chrysotherapy, the decreased levels of erythrocyte GSH and serum sulfhydryls of rheumatoid arthritis patients are normalized concomitant with clinical efficacy. This investigation examined the in vivo and in vitro effect of gold(I) as aurothioglucose on enzymes related to the GSH redox cycle or metabolism. The enzymes measured were GSH peroxidase, GSSG reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase, GSH S-transferase, GSH thiotransferase, glucose-6-phosphate dehydrogenase, superoxide dismutase and catalase. Rats were injected with 30 mumol aurothioglucose/kg body wt. daily for 7 days by intramuscular injection. GSH levels in aurothioglucose-treated rats were 68% higher in erythrocytes (P less than 0.005) and 45% higher in kidney (P less than 0.001) than in control rats. Treatment with aurothioglucose did not elevate plasma or liver GSH. The enzyme activities that were changed by aurothioglucose treatment were GSH peroxidase in kidney (41% decreased, P = 0.005) and liver (13% decreased, P less than 0.05), gamma-glutamyl transpeptidase in kidney (15% decreased, P less than 0.05), and catalase in kidney (58% decreased, P less than 0.001). Kidney glucose-6-phosphate dehydrogenase activity was increased 50% (P less than 0.005) and GSH S-transferase was increased 72% (P less than 0.001). In vitro the only liver enzymes inhibited more than 50% by concentrations of less than 50 microM aurothioglucose were GSH peroxidase (50% inhibited by 25 microM aurothioglucose) and GSH thiotransferase (50% inhibited by 5 microM aurothioglucose). Studies of in vitro enzyme inhibition by aurothioglucose could not be used to predict decreased enzyme activities in vivo. Although decreased activities of two major enzymes that utilize GSH, GSH peroxidase and gamma-glutamyl transpeptidase, coincided with elevated GSH in kidneys of aurothioglucose-treated rats, a direct cause and effect relationship remains speculative.

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In vivo, cysteine in proteins or glutathione is the major amino acid involved in sulfhydryl oxidation-reduction reactions. An in vitro model of cysteine oxidation accelerated by selenium compounds was used to study the interaction of selenocystine and sodium selenite with metal ions. The interaction of metal ions with selenium compounds inhibited cysteine oxidation. The ionic forms of three toxic soft-acid metals, mercury, silver, and gold, were the most effective inhibitors. The antiarthritic gold drugs, aurothiomalate and aurothioglucose, were of particular interest as they inhibit the activity of selenium-glutathione peroxidase. The effect of gold ligands on gold(I) inhibition of selenocystine-accelerated cysteine oxidation was tested. Sodium cyanide partially reversed inhibition and potassium iodide had no effect. Inhibition of selenium-accelerated oxidation-reduction reactions by soft-acid metal ions may be of biological relevance during toxicities or during antiarthritic gold therapy.

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Gold interacts with selenium in vivo, and the normal distribution of selenium among tissues and subcellular compartments changes. Literature evidence shows that many of the effects of gold compounds on the polymorphonuclear neutrophil, macrophage, and lymphocyte cellular components of the immune system are similar to effects observed in these cellular components in selenium-deficient animals. Affected by these two metals are immune functions related to phagocytic cell migration, phagocytosis, microbial killing, lymphocyte mitogenesis/DNA synthesis, arachidonic acid metabolism/prostaglandin synthesis, and immunoglobulin production. The interaction of gold with selenium in vivo may be responsible for some of the multiparameter-based actions of gold compounds used in the treatment of inflammatory diseases such as rheumatoid arthritis. One mechanism by which gold exerts its clinical effects may be related to its interaction with selenium to produce, in specific microenvironments, decreased levels of this essential trace element.

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The influence of dietary vitamin E and Santoquin on lipid peroxidation and liver regeneration in partially-hepatectomized rats was studied. Rats were fed either a basal 10% tocopherol-stripped corn oil diet, the basal diet plus 40 mg dl-alpha-tocopheryl acetate/kg, or the basal diet plus 2 g Santoquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline)/kg. After 6 weeks, rats fed the antioxidant-deficient diet produced more of the lipid peroxidation product, pentane, than did the rats fed antioxidants. Partial hepatectomy was performed after six and one-half weeks or ten weeks of feeding the diets. At 3 and 6 days after surgery, pentane production was significantly elevated over pre-surgery levels in rats fed the antioxidant-deficient or vitamin E-supplemented diets, but not in rats fed the Santoquin-supplemented diet. Six days after surgery, there were fewer thiobarbituric acid reactants in regenerating liver of Santoquin-fed rats than of vitamin-E fed rats or antioxidant-deficient rats. There was no increase in the 6-day level of thiobarbituric acid reactants over the 3-day level in livers of rats fed Santoquin, while there was an increase in livers of the antioxidant-deficient and vitamin E-supplemented rats. Liver sulfhydryl levels were higher at 3 and 6 days post surgery in the Santoquin-fed rats than in the antioxidant-deficient or vitamin E-supplemented rats. Plasma gamma-glutamyl-transpeptidase activity was not different among the groups of rats. Between the third and sixth day following surgery, liver regeneration was significantly stimulated in Santoquin-fed, but not vitamin E-fed rats. After 11 days, a stimulatory, but not statistically significant, effect of vitamin E was found. Although DNA content of liver was higher at 6 days than at 3 days post surgery, it was not different among the dietary groups, indicating that cell proliferation rather than hypertrophy had occurred. Partial hepatectomy could have altered the ability of the liver to metabolize pentane, thus explaining part of the increased production of pentane. However, the results obtained support the interpretation that elevated levels of dietary antioxidants can be beneficial in terms of reduced lipid peroxidation and increased rates of liver regeneration following liver surgery.

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The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.

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Gold (Au) thioglucose, which has been used in the treatment of rheumatoid arthritis, inhibits selenium (Se)-glutathione peroxidase. Since Au and Se play roles in inflammation, the effects of dietary Se (0, 0.2, and 2.0 ppm for 10 weeks) and injected gold thioglucose (5 mg Au/day/kg body weight for 28 days) in adjuvant-treated rats were investigated. Au toxicity was evidenced by lower body weights and higher tissue weight/body weight ratios for kidneys and spleens of Au-treated rats. Adjuvant-induced inflammation, measured by paw thickness, was not influenced by dietary Se, although Au decreased inflammation in Se-deficient rats. Liver glutathione peroxidase activity was depressed by Se deficiency and by Au. Sulfhydryl levels in liver soluble fraction and plasma were highest for Se-deficient rats. Among liver, kidney, spleen, and plasma, thiobarbituric acid reactants were highest in kidneys of Au-treated rats and lowest in plasma of rats fed 2 ppm Se. gamma-Glutamyltranspeptidase activity in plasma indicated liver damage in Se-deficient rats. Kidney PGE2 output in 24-hour urine samples was unaffected by Au, Se, or adjuvant. Au-Se interactions in vivo are complex, but decreased glutathione peroxidase activity in Au-injected rats suggests that Se nutrition of Au-treated rheumatoid arthritis patients may be a practical concern.

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Gold (Au) thioglucose, used to treat inflammatory diseases such as rheumatoid arthritis, inhibits the selenium (Se)-dependent glutathione peroxidase. The present study examines the ability of Au to act either as a Se antagonist or as a GSH peroxidase inhibitor in vivo. The effects of gold thioglucose loading on Se distribution, and on Se-dependent GSH peroxidase and GSH S-transferase, were examined in rats fed three dietary levels of Se (0, 0.2, and 2.0 ppm), and with or without adjuvant-induced inflammation. Kidney, liver, spleen, testes, and erythrocytes were selected for study based upon their high Se content and their ability to concentrate Au. Au loading increased kidney, liver, and spleen Se concentrations, and this effect was dependent upon dietary Se levels. Rats fed Se-supplemented diets had higher levels of Au in kidney, liver, and spleens than did rats fed a Se-deficient diet. Au loading decreased GSH peroxidase activity in kidney, liver, and erythrocytes. The decrease in GSH peroxidase in kidney and liver, and the increase in Se concentration in these tissues, suggest that Au-Se complexes may have limited the biosynthesis of the enzyme. Au affects Se distribution, and Se concentrates Au in tissues with a high lysosomal content.

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The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.

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Indirect evidence has suggested that lipid peroxidation is associated with iron overload in vivo. As a measure of lipid peroxidation, pentane expired in the breath of rats loaded with an accumulated dose of either 100 mg or 186-200 mg of iron injected intraperitoneally as iron dextran was measured over a 7 to 8 week period, and the effect on pentane production of feeding antioxidant-supplemented diets was determined. By the seventh week of feeding the diets, rats fed 0.3% L-ascorbic acid produced 17% less (P = 0.03) pentane than did rats fed the basal antioxidant-deficient diet, whereas rats fed 0.004% dl-alpha-tocopherol acetate produced 92% less (P less than 0.001). After being fed the basal diet for 7 weeks, iron-loaded rats produced 76 +/- 9 pmol pentane/100 g body wt/min. When synthetic antioxidants were added to the diet at a concentration of 0.25%, the order of effectiveness in decreasing pentane production after 1 week was: N,N'-diphenyl-p-phenylenediamine greater than ethoxyquin greater than butylated hydroxyanisole greater than butylated hydroxytoluene greater than propyl gallate approximately equal to no antioxidant. After removal of either ethoxyquin or N,N'-diphenyl-p-phenylenediamine from the diets for 1 week, pentane production increased to a high level. The total amount of lipid soluble fluorophores in individual spleens of rats fed N,N'-diphenyl-p-phenylenediamine, ethoxyquin, dl-alpha- tocopherol acetate, ascorbic acid and no antioxidant were correlated significantly with the corresponding total integrated amount of pentane produced by the individual rats over the 7 to 8 week period. This study has provided some of the most direct evidence to date that lipid peroxidation is associated with iron overload in vivo.

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Pentane production was used as an index of lipid peroxidation in male rats fed either 0 or 1000 ppm copper in diets with and without vitamin E. Pentane production by vitamin E-deficient rats not fed copper was greater than that by vitamin E-supplemented rats not fed copper. Pentane production was low by all groups of rats. Copper-fed, vitamin E-deficient and vitamin E-supplemented rats produced more pentane than did respective controls not fed copper. After 9 weeks of feeding the diets, more pentane was produced by vitamin E-deficient than by vitamin E-supplemented rats following intraperitoneal injection of 5 mg of copper/kg of body weight, and vitamin E-deficient rats fed copper produced 5-fold more pentane than did those not fed copper. Thiobarbituric acid-reactants were highest in blood, kidney and liver from copper-fed rats. Lipid-soluble fluorophores in spleen were lowest in vitamin E-supplemented rats not fed copper and highest in copper-fed, vitamin E-deficient rats.

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The relative antioxidant effectiveness of RRR-alpha-tocopherol and d-gamma-tocopherol against in vivo lipid peroxidation in vitamin E-depleted, iron-loaded rats was assessed by measurement of expired pentane. Rats fed a vitamin E-deficient diet were each administered 103 +/- 2 mg of iron as iron dextran over a 4-week period. After 3 weeks, their erythrocytes were 96.9 +/- 0.6% hemolyzed by dialuric acid. After 6 weeks, the rats exhaled 22.4 +/- 3.4 pmol pentane/(100 g body weight . minute). Groups of 4 rats each were then fed varying levels of RRR-alpha- and d-gamma-tocopherol for 2 weeks, after which the pentane levels were directly related to the dietary tocopherol content. Covariance analysis of the log of pentane production versus the log of dietary tocopherol showed the relative antioxidant effectiveness of 1:0.31 for alpha-tocopherol:gamma-tocopherol. In an independent estimation of relative antioxidant effectiveness, covariance analysis of the log of lipid soluble fluorophores in the spleens of the rats versus the log of dietary tocopherol showed a ratio of 1:0.37 for alpha-tocopherol:gamma-tocopherol. Regression analysis showed the fluorophores also to be correlated with the integrated amount of pentane produced over the 7-week experiment (r = 0.84, P less than 0.001). gamma-Tocopherol was more effective as an in vivo antioxidant than has been reported for its inhibition of vitamin E-deficiency syndromes.

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