Antioxidant systems of the avian embryo: Tissue-specific accumulation and distribution of Vitamin E in the turkey embryo during development

Surai PF, Sparks NH, Noble RC.

Br Poult Sci. 1999 Sep;40(4):458-66.

Tissue-specific accumulation of tocopherols and tocotrienols in turkey tissues during embryonic development and their susceptibility to lipid peroxidation were investigated. Fertile turkey eggs were incubated using standard commercial conditions. Embryonic tissues were collected at 16, 22, 25 d of incubation and from day-old poults (referred to as day 29) and alpha-; beta- + gamma- and delta-tocopherols and respective tocotrienolswere analysed by HPLC. A turkey diet provided to the parent hens contained the complete range of tocopherols and tocotrienols. Between days 16 and 22 of embryo development, the alpha-tocopherol concentration in the liver remained constant and then increased significantly (P<0.01) reaching a maximum just after hatching. Similar changes were observed for the other tocopherols and tocotrienols. The accumulation of alpha-tocopherol in the yolk sac membrane (YSM) started after day 20 of development and at hatching the alpha-tocopherol concentration in the YSM was twice that of beta- + gamma-tocopherols and 15 times greater than that of alpha-tocotrienol. In the kidney, heart, lung, muscle and adipose tissues a gradual increase in tocopherol and tocotrienol concentrations took place between days 20 and 25 of development with a sharp increase in particular of alpha-tocopherol between days 25 and 29. There was a discrimination between tocopherols and tocotrienols during their assimilation from the diet by the parent hen and during metabolism by the developing turkey embryo. Tissue-specific features in the susceptibility to lipid peroxidation were found with the brain being the most susceptible to lipid peroxidation at day 25 and in day-old poults.

Although scientific evidence is relatively limited, rice bran oil (RBO) is tenaciously believed to be a healthy vegetable oil in Asian countries. It exerts hypocholesterolemic activity in relation to more commonly used vegetable oils and is characterized by a relatively high content of non-fatty acid components, some of which are known to have beneficial health effects. Components specific for RBO such as gamma-oryzanol and tocotrienolscould participate in its hypocholesterolemic effects. In addition, blending RBO with safflower oil, but not with sunflower oil, may magnify the hypocholesterolemic efficacy. This observation is of particular interest with regard to dietary intervention with RBO. The possible mechanism underlying this effect may at least in part be related to the specific triglyceride structure of safflower oil, differing from that of sunflower oil.

Although vitamin E has been known as an essential nutrient for reproduction since 1922, we are far from understanding the mechanisms of its physiological functions. Vitamin E is the term for a group of tocopherols and tocotrienols, of which alpha-tocopherol has the highest biological activity. Due to the potent antioxidant properties of tocopherols, the impact of alpha-tocopherol in the prevention of chronic diseases believed to be associated with oxidative stress has often been studied, and beneficial effects have been demonstrated. Recent observations that the alpha-tocopherol transfer protein in the liver specifically sorts out RRR-alpha-tocopherol from all incoming tocopherols for incorporation into plasma lipoproteins, and that alpha-tocopherol has signaling functions in vascular smooth muscle cells that cannot be exerted by other forms of tocopherol with similar antioxidative properties, have raised interest in the roles of vitamin E beyond its antioxidative function. Also, gamma-tocopherol might have functions apart from being an antioxidant. It is a nucleophile able to trap electrophilic mutagens in lipophilic compartments and generates a metabolite that facilitates natriuresis. The metabolism of vitamin E is equally unclear. Excess alpha-tocopherol is converted into alpha-CEHC and excreted in the urine. Other tocopherols, like gamma- and delta-tocopherol, are almost quantitatively degraded and excreted in the urine as the corresponding CEHCs. All rac alpha-tocopherol compared to RRR-alpha-tocopherol is preferentially degraded to alpha-CEHC. Thus, there must be a specific, molecular role of RRR-alpha-tocopherol that is regulated by a system that sorts, distributes, and degrades the different forms of vitamin E, but has not yet been identified. In this article we try to summarize current knowledge on the function of vitamin E, with emphasis on its antioxidant vs. other properties, the preference of the organism for RRR-alpha-tocopherol, and its metabolism to CEHCs.

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.

Objectives: To summarize new knowledge surrounding the physiological activity of tocotrienol, a natural analogue of tocopherol.

Results: The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. In the vitamin E group, alpha-tocopherol is considered to be the most active form. However, recent research has suggested tocotrienol to be a better antioxidant. Moreover, tocotrienol has been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein B and lipoprotein(a) plasma levels. In addition, tocotrienol has been suggested to have an anti-thrombotic and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer.

Conclusion: The physiological activities of tocotrienol suggest it to be superior than alpha-tocopherol in many situations. Hence, the role of tocotrienol in the prevention of cardiovascular disease and cancer may have significant clinical implications. Additional studies on its mechanism of action, as well as, long-term intervention studies, are needed to clarify its function. From the pharmacological point-of-view, the current formulation of vitamin E supplements, which is comprised mainly of alpha-tocopherol, may be questionable

A crude palm-oil extract rich in vitamin E homologues was investigated by HPLC-MS and HPLC-NMR coupling. For mass spectrometry a newly introduced ionization technique called Coordination Ion Spray (CIS) was used. Through the addition of silver ions to the HPLC eluent, the ionization process of nonpolar substances is facilitated. Chromatography and all coupling experiments were conducted on a C(30) column which exhibited an extraordinary shape selectivity and overwhelming sample-loading capability. Experiments were performed with pure methanol as an eluent which proved to be ideal for NMR spectroscopy as well as mass spectrometry. All necessary information for unambiguous structural assignment was collected within 45 min of the LC-NMR experiment and 15 min of the LC-MS experiment. Six compounds were identified, i.e., α-, β-, γ-, and δ-tocotrienol, α-tocoenol, and α-tocopherol.

The effects of topical vitamin E on the cosmetic appearance of scars

Baumann LS, Spencer J.

Dermatol Surg. 1999 Apr;25(4):311-5.

BACKGROUND: Vitamin E is a generic term for a group of tocol and tocotrienol derivatives. Since the discovery that vitamin E is the major lipid soluble antioxidant in skin, this substance has been tried for the treatment of almost every type of skin lesion imaginable. Anecdotal reports claim that vitamin E speeds wound healing and improves the cosmetic outcome of burns and other wounds. Many lay people use vitamin E on a regular basis to improve the outcome of scars and several physicians recommend topical vitamin E after skin surgery or resurfacing.

OBJECTIVE: We attempted to determine whether topically applied vitamin E has any effect on the cosmetic appearance of scars as suggested by multiple anectodal reports.

METHODS: Fifteen patients who had undergone skin cancer removal surgery were enrolled in the study. All wounds were primarily closed in 2 layers. After the surgery, the patients were given two ointments each labeled A or B. A was Aquaphor, a regular emollient, and the B was Aquaphor mixed with vitamin E. The scars were randomly divided into parts A and B. Patients were asked to put the A ointment on part A and the B ointment on part B twice daily for 4 weeks. The study was double blinded. The physicians and the patients independently evaluated the scars for cosmetic appearance on Weeks 1, 4, and 12. The criteria was simply to recognize which side of the scar looked better if there was any difference. The patients’ and the physicians’ opinions were recorded. A third blinded investigator was shown photographs of the outcomes and their opinion was also noted.

RESULTS: The results of this study show that topically applied vitamin E does not help in improving the cosmetic appearance of scars and leads to a high incidence of contact dermatitis.

CONCLUSIONS: This study shows that there is no benefit to the cosmetic outcome of scars by applying vitamin E after skin surgery and that the application of topical vitamin E may actually be detrimental to the cosmetic appearance of a scar. In 90% of the cases in this study, topical vitamin E either had no effect on, or actually worsened, the cosmetic appearance of scars. Of the patients studied, 33% developed a contact dermatitis to the vitamin E. Therefore we conclude that use of topical vitamin E on surgical wounds should be discouraged.

Effect of dietary antioxidants on serum lipid contents and immunoglobulin productivity of lymphocytes in Sprague-Dawley rats

Kaku S, Yunoki S, Mori M, Ohkura K, Nonaka M, Sugano M, Yamada K

Biosci Biotechnol Biochem. 1999 Mar;63(3):575-6.

Sprague-Dawley rats were fed alpha-tocopherol, tocotrienol, or quercetin to examine their dietary effects on serum lipid contents and immunoglobulin productivity. In tocotrienol or quercetin groups, serum triglyceride was lower than in the none group. Moreover, in the alpha-tocopherol group, serum IgA level and IgA productivity of MLN lymphocytes were high, while in the tocotrienol group, IgM productivity of spleen lymphocytes and IgA, IgG, and IgM productivity of MLN lymphocytes were high. Thus, we suggested each antioxidant had different effects in rats.

Effects of gamma-tocotrienol on ApoB synthesis, degradation, and secretion in HepG2 cells

Theriault A, Wang Q, Gapor A, Adeli K.

Arterioscler Thromb Vasc Biol. 1999 Mar;19(3):704-12.

Gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen.

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BACKGROUND: Tocotrienols, lipid-soluble antioxidants with vitamin E activity, have been reported to lower LDL-cholesterol concentrations and platelet aggregation in men, but results are contradictory.

OBJECTIVE: To examine in detail the effects of a vitamin E concentrate rich in tocotrienols on serum lipoproteins and on platelet function in men at risk for cardiovascular disease.

DESIGN: In this randomized, double-blind, placebo-controlled parallel trial, 20 men received daily for 6 wk 4 capsules, each containing 35 mgtocotrienols and 20 mg alpha-tocopherol; 20 other men received 4 capsules daily, each providing 20 mg alpha-tocopherol. All men had concentrations of serum total cholesterol between 6.5 and 8.0 mmol/L or lipoprotein(a) concentrations > 150 mg/L.

RESULTS: Compliance was confirmed by changes in serum tocopherol and tocotrienol concentrations. Serum LDL cholesterol in the tocotrienolgroup was 4.80 mmol/L before and 4.79 mmol/L after intervention, and increased from 4.70 to 4.86 mmol/L in the placebo group (95% CI for the difference: -0.54, 0.19 mmol/L; P = 0.333). Also, changes in HDL cholesterol, triacylglycerol, lipoprotein(a), and lipid peroxide concentrations did not differ between the groups. After adjustment for differences in initial values, no effects were found on collagen-induced platelet aggregation velocity, maximum aggregation, or thromboxane B2 formation in citrated whole blood. ATP release, however, was lower in the tocotrienol group. Urinary thromboxane B2 and 11-keto-thromboxane B2 concentrations and coagulation and fibrinolytic measures did not change.

CONCLUSION: The tocotrienol supplements used had no marked favorable effects on the serum lipoprotein profile or on platelet function in men with slightly elevated lipid concentrations.

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