Effect of dietary vitamin E and Santoquin on regenerating rat liver

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.     

Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL)

Activation of polymorphonuclear neutrophils (PMNL) leads to the release of arachidonate from cellular phospholipids via a phospholipase A2, and conversion of products of the 5-lipoxygenase pathway. Evidence to date indicates the dietary vitamin E ((R,R,R)-alpha-tocopherol) can influence both cyclooxygenase and phospholipase A2 activities and that the effect of this vitamin is cell/tissue specific. The present study was undertaken in order to examine the effects of varying dietary tocopherol on PMNL tocopherol content and 5-lipoxygenase product profile using the ionophore A23187 as stimulant in the presence and absence of exogenous arachidonate. Feeding semi-purified diets containing 0, 30 or 3000 ppm of (R,R,R)-alpha-tocopherol acetate to weanling rats for 17 weeks resulted in a dose-related enrichment of PMNL tocopherol. Stimulation of PMNL elicited a significant and rapid loss of tocopherol. When PMNL were stimulated with A23187 alone, the synthesis of 5-HETE, LTB4 and 19-hydroxy-LTB4 was decreased in proportion to increasing dietary tocopherol concentrations. However, when exogenous arachidonate was provided with A23187, intermediate amounts of dietary tocopherol (30 ppm) still suppressed the formation of 5-lipoxygenase products, but high doses (3000 ppm) did not have any additional inhibitory effect. This differential response to high concentrations of vitamin E in the presence and absence of exogenous arachidonate highly suggest that at these concentrations, tocopherol may act principally at the level of substrate release whereas at lower concentrations, 5-lipoxygenase is inhibited. Data from this study demonstrated that attenuation of the formation of 5-lipoxygenase products in PMNL can be achieved by dietary vitamin E enrichment.     

Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity

The tocopherol transfer protein (TTP) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[1,2,5]oxadiazol-4-ylamino)nonyl]chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue shifted, and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analogue binds to TTP reversibly and with high affinity (K(d) = 8.5 +/- 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the ataxia with vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (K(d) = 71 +/- 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high-resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction.     

Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development

Diets specifically deficient in selenium (Se) and/or vitamin E or adequate in both nutrients were fed to chicks from the time of hatching. Lymphoid organs (bursa, thymus, and in some instances, spleen) were collected from chicks 7-35 days of age. Growth of the chicks fed these diets was monitored over the experimental period as was lymphoid organ growth. The development of the primary lymphoid organs was further assessed by histological techniques and the organ contents of vitamin E (alpha-tocopherol) and Se were determined. Specific deficiencies of either Se or vitamin E were found to significantly impair bursal growth as did a combined deficiency. Thymic growth was impaired only by the combined deficiency diet. Severe histopathological changes in the bursa resulted from the combined deficiency and these were detectable by 10-14 days after hatching. These changes were characterized by a gradual degeneration of the epithelium and an accompanying depletion of lymphocytes. Similar changes, although slower to develop and less severe, were observed in the thymus as a result of the combined deficiency. When both serum and tissue levels of vitamin E and Se were monitored, it was observed that these were rapidly and independently depleted by the specific deficiency diets. These data suggest that the primary lymphoid organs are major targets of Se and vitamin E dietary deficiencies and provide a possible mechanism by which immune function may be impaired.     

Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure

Human platelet 12-lipoxygenase (hp-12LOX, 662 residues + iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples. To understand the properties of hp-12LOX, we have expressed and studied the behavior, function and low-resolution structure of the hp-12LOX His-tagged recombinant enzyme and its mutants in solution. We have found that it is a dimer easily converted into bigger aggregates, which are soluble/covalent-noncovalent/reversible. The heavier oligomers show a higher activity at pH 8, in contrast to dimers with lower activity showing two maxima at pH 7 and pH 8, indicating the existence of two different conformers. In the seven-point C → S mutant, aggregation is diminished, activity has one broad peak at pH 8 and there is no change in specificity. Truncation of the N_t-β-barrel domain (PLAT, residues 1-116) reduces activity to ∼ 20% of that shown by the whole enzyme, does not affect regio- or stereospecificity and lowers membrane binding by a factor of ∼ 2. “NoPLAT” mutants show strong aggregation into oligomers containing six or more catalytic domains regardless of the status of the seven cysteine residues tested. Time-of-flight mass spectrometry suggests two arachidonic acid molecules bound to one molecule of enzyme. Small angle X-ray scattering studies (16 Å resolution, χ∼ 1) suggest that two hp-12LOX monomers are joined by the catalytic domains, with the PLAT domains floating on the flexible linkers away from the main body of the dimer.     

Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extra-hepatic vitamin E metabolism

The widely conserved preferential accumulation of α-tocopherol (α-TOH) in tissues occurs, in part, from selective postabsorptive catabolism of non-α-TOH forms via the vitamin E-ω-oxidation pathway. We previously showed that global disruption of CYP4F14, the major but not the only mouse TOH-ω-hydroxylase, resulted in hyper-accumulation of γ-TOH in mice fed a soybean oil diet. In the current study, supplementation of Cyp4f14^−/− mice with high levels of δ- and γ-TOH exacerbated tissue enrichment of these forms of vitamin E. However, at high dietary levels of TOH, mechanisms other than ω-hydroxylation dominate in resisting diet-induced accumulation of non-α-TOH. These include TOH metabolism via ω-1/ω-2 oxidation and fecal elimination of unmetabolized TOH. The ω-1 and ω-2 fecal metabolites of γ- and α-TOH were observed in human fecal material. Mice lacking all liver microsomal CYP activity due to disruption of cytochrome P450 reductase revealed the presence of extra-hepatic ω-, ω-1, and ω-2 TOH hydroxylase activities. TOH-ω-hydroxylase activity was exhibited by microsomes from mouse and human small intestine; murine activity was entirely due to CYP4F14. These findings shed new light on the role of TOH-ω-hydroxylase activity and other mechanisms in resisting diet-induced accumulation of tissue TOH and further characterize vitamin E metabolism in mice and humans.     

Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.     

Effect of cytochrome c on the linoleic acid-degrading activity of porcine leukocyte 12-lipoxygenase

Hemoproteins are known to have quasilipoxygenase activity that converts linoleic acid (LA) to its hydroperoxides. However, it is not still clear whether, like lipoxygenases, hemoproteins can produce LA hydroperoxides when the LA is part of a mixture containing many different saturated and unsaturated fatty acids. In this study, we found that such hemoprotein as cytochrome c (Cyt c) did not produce LA hydroperoxides from the phospholipase A(2) (PL-A(2)) hydrolysis products of egg yolk phosphatidylcholine (PC). We also found that traces of hydroperoxides and a high concentration of the target unsaturated fatty acid (LA) needs to be present in a fatty acid mixture before the quasi-lipoxygenase activity of Cyt c becomes apparent. We also attempted to elucidate how Cyt c interact with porcine leukocyte 12-lipoxygenase (12-LOX). Hemoproteins are known to possess pseudo-lipohydroperoxidase activity, and can remove the hydroperoxides of unsaturated fatty acids from a reaction mixture. However, we found that Cyt c catalyzed the reaction by which hydroperoxides degrade LA, and thus enhanced the LA-degrading activity of 12-LOX. This hemoprotein-induced promotion of the ability of 12-LOX to degrade LA was observed even when the reaction mixture contained many different saturated and unsaturated fatty acids.     

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce more TxA₂ than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI₂) than arterial tissue from vitamin E-supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Modulation of the platelet thromboxane A2 and aortic prostacyclin synthesis by dietary selenium and vitamin E

Vitamin E and selenium (Se) interact synergistically as an important antioxidant defense mechanism. Se, an essential component of glutathione peroxidase (GSH-Px) and vitamin E decompose fatty acid hydroperoxides and hydrogen peroxides generated by free radical reactions. Vitamin E and GSH-Px may modulate arachidonic acid metabolism and the activity of cyclooxygenase enzymes by affecting peroxide concentration. The balance between arterial wall prostacyclin (PGI₂) production and platelet thromboxane (TX)A₂ directly influences platelet activity. In order to elucidate the differential role of dietary vitamin E and Se in aortic PGI₂ and platelet TXA₂ synthesis, 1-mo-old F344 rats were fed semipurified diets containing different levels of vitamin E (0, 30, 200 ppm) and Se (0, 0.1, 0.2 ppm) for 2 mo. Thromboxane B₂ (TXB₂) and 6-keto-PGF₁α, were measured by radioimmunoassay (RIA) after incubation of whole blood and aortic rings at 37°C for 10 and 30 min, respectively. Vitamin E deficiency reduced plasma vitamin E to 5–17% of control-fed rats, and supplementation increased it to 53% of the control-fed rats. Se supplementation in vitamin E-supplemented animals increased plasma GSH-Px by 17%, compared to vitamin E-deficient rats. Se and vitamin E supplementation did not have a similar effect on TXB₂ and PGI₂ synthesis. Se deficiency did not alter platelet TXB₂ synthesis, but significantly decreased aortic PGI₂ synthesis. It was necessary to supplement with both antioxidants in order to increase, PGI₂ synthesis. Se and vitamin E deficient groups had a higher TXB₂/PGI₂ ratio (0.17±0.08) compared to Se- and vitamin E-supplemented groups (0.03±0.01). These results confirm previous reports in humans and animals and are in accordance with epidemiological data indicating an inverse relationship between plasma Se and platelet aggregation. Thus, further suggesting that vitamin E and Se may have a specific role in controlling TXA₂ and PGI₂ synthesis.     

Molecular basis of vitamin E action: tocotrienol modulates 12-lipoxygenase,a key mediator of glutamate-induced neurodegeneration

Vitamin E is a generic term for tocopherols and tocotrienols. This work is based on our striking evidence that, in neuronal cells, nanomolar concentrations of alpha-tocotrienol, but not alpha-tocopherol, block glutamate-induced death by suppressing early activation of c-Src kinase (Sen, C. K., Khanna, S., Roy, S., and Packer, L. (2000) J. Biol. Chem. 275, 13049-13055). This study on HT4 and immature primary cortical neurons suggests a central role of 12-lipoxygenase (12-LOX) in executing glutamate-induced neurodegeneration. BL15, an inhibitor of 12-LOX, prevented glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOX-deficient mice were observed to be resistant to glutamate-induced death. In the presence of nanomolar alpha-tocotrienol, neurons were resistant to glutamate-, homocysteine-, and l-buthionine sulfoximine-induced toxicity. Long-term time-lapse imaging studies revealed that neurons and their axo-dendritic network are fairly motile under standard culture conditions. Such motility was arrested in response to glutamate challenge. Tocotrienol-treated primary neurons maintained healthy growth and motility even in the presence of excess glutamate. The study of 12-LOX activity and metabolism revealed that this key mediator of glutamate-induced neurodegeneration is subject to control by the nutrient alpha-tocotrienol. In silico docking studies indicated that alpha-tocotrienol may hinder the access of arachidonic acid to the catalytic site of 12-LOX by binding to the opening of a solvent cavity close to the active site. These findings lend further support to alpha-tocotrienol as a potent neuroprotective form of vitamin E.     

Inhibition of platelet arachidonic acid 12-lipoxygenase by acetylenic acid compounds

Eighteen acetylenic fatty acids were tested as inhibitors of human platelet arachidonic acid 12-lipoxygenase. 4,7,10,13-Eicosatetraynoic (4,7,10,13-ETYA) acid emerged as the most potent compound. Additional experiments have shown that 4,7,10,13-ETYA selectively blocked the 12-lipoxygenase in washed human platelets with lesser activity against the cyclooxygenase. The ID50 value for lipoxygenase was 7.8 microM in comparison with an ID50 of 100 microM for the cyclooxygenase. The commonly used inhibitor 5,8,11,14-eicosatetraynoic acid inhibited both enzymes with equal potency. It appears that 4,7,10,13-ETYA may be a valuable lead for selective modulation of the 12-lipoxygenase pathway in platelet or other target tissues.     

Conditions for the formation of the oxo derivatives of arachidonic acid from platelet 12-lipoxygenase and soybean 15-lipoxygenase

Three carbonyl compounds derived from arachidonic acid have recently been characterized in human platelets, namely, 12-ketoeicosatetraenoic acid and two isomeric 12-oxododecatrienoic acids. The conditions for the synthesis of these compounds and for the synthesis of analogous products from soybean lipoxygenase, i.e., 15-ketoeicosatetraenoic acid and 15-oxopentadecatetraenoic acids, were compared with regard to the role of oxygen and fatty acid availability, and heme catalysis. Using platelet homogenates or soybean lipoxygenase and arachidonic acid as a substrate, it was found that the establishment of anaerobic conditions during the incubation was mandatory only for the synthesis of 15-oxopentadecatetraenoic acids. Anaerobic conditions, however, greatly increased the formation of 15-ketoeicosatetraenoic acid and, to a lesser extent, of 12-oxododecatrienoic acids. On the other hand, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was transformed into 12-ketoeicosatetraenoic acid and 12-oxododecatrienoic acids by platelet homogenates or soybean lipoxygenase. This transformation was increased when the incubation was performed in anaerobic conditions and in the presence of a fatty acid substrate of the enzyme. These data suggest that oxygen deprivation and excess fatty acid could play a stimulatory role in the synthesis of 12-oxo compounds by platelets. Finally, we have compared the heme-catalyzed generation of the 12-oxo and 15-oxo derivatives from their hydroperoxide precursors: whereas 12-oxododecatrienoic acids and 12-ketoeicosatetraenoic acid were formed in the proportion of 8.5: 1.5 from 12-HPETE incubated with hematin (150 nM), 15-ketoeicosatetraenoic acid was the only carbonyl compound generated from 15-HPETE in the same conditions, emphasizing the unique reactivity of the 12-HPETE.     

Effect of tocopherol and tocopherol quinone complexes with proteins on activity of leukotriene B4 lipoxygenase

While estimating the effect of tocopherol, tocopherylquinone and their complexes with the tocopherol-binding proteins from the rat liver cytosole on arachidonate 5-lipoxygenase from peritoneal-lymphocytes and soybean linoleate-5-lipoxygenase alpha-tocopherol and especially its complex with tocopherol-binding protein was defined to inhibit the activity of both vegetative- and animal nature-lipoxygenase.