The effect of α tocopherol,all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation of rod outer segments

The effect of tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation induced by ascorbate-Fe²⁺ of rod outer segment membranes isolated from bovine retina was examined. The inhibition of light emission (maximal induced chemiluminescence) by tocopherol, all-trans retinol and retinyl palmitate was concentration dependent. All trans retinol showed a substantial degree of inhibition against ascorbate-Fe²⁺ induced lipid peroxidation in rod outer segment membranes that was 10 times higher than the observed in the presence of either tocopherol or retinyl palmitate. Inhibition of lipid peroxidation of rod outer segment membranes by tocopherol and retinyl palmitate was almost linear for up to 0,5 mol vitamin/mg membrane protein, whereas all-trans retinol showed linearity up to 0,1 mol vitamin/mg membrane protein. Incubation of rod outer segments with increasing amounts of low molecular weight cytosolic proteins carrying 1-[¹⁴C] linoleic acid, [³H] retinyl palmitate or [³H] all-trans retinol during the lipid peroxidation process produced a net transfer of ligand from soluble protein to membranes. Linoleic acid was 4 times more effectively transferred to rod outer segment membranes than all-trans retinol or retinyl palmitate. Incubation of rod outer segments with delipidated low molecular weight cytosolic proteins produced inhibition of lipid peroxidation. The inhibitory effect was increased when the soluble retinal protein fraction containing a tocopherol was used. These data provide strong support for the role of all-trans retinol as the major retinal antioxidant and open the way for many fruitful studies on the interaction and precise roles of low molecular weight cytosolic retinal proteins involved in the binding of antioxidant hydrophobic compounds with rod outer segments.     

Relative efficacies of α-tocopherol, N-acetyl-serotonin, and melatonin in reducing non-enzymatic lipid peroxidation of rat testicular microsomes and mitochondria

In this study, we examined the relative efficacies of alpha-tocopherol, N-acetyl-serotonin, and melatonin in reducing ascorbate-Fe(2+) lipid peroxidation (LPO) of rat testicular microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids (PUFAs) C20:4 n6 and C22:5 n6. The LPO of testicular microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. Both long-chain PUFAs were protected when the antioxidants were incorporated either in microsomes or mitochondria. By comparison of the IC50 values obtained between alpha-tocopherol and both indolamines, it was observed that alpha-tocopherol was the most efficient antioxidant against the LPO induced by ascorbate-Fe(2+) under experimental conditions in vitro, IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.14 mM) than in mitochondria (0.08 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6] + [C22:5 n6] in microsomes than that in mitochondria. Melatonin and N-acetyl-serotonin were more effective in inhibiting the LPO in mitochondria than that in microsomes. Thus, a concentration of 1 mM of both indolamines was sufficient to inhibit in approximately 70% of the light emission in mitochondria, whereas a greater dosage of 10 times (10 mM) was necessary to produce the same effect in microsomes. It is proposed that the vulnerability to LPO of rat testicular microsomes and mitochondria in the presence of both indolamines is different because of the different proportion of PUFAs in these organelles.     

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μM) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe²⁺. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μs after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl⁺. Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.     

Effects of Schisandrin B and α-tocopherol on lipid peroxidation, in vitro and in vivo

Effects of Schisandrin B (Sch B) and -tocopherol (-TOC) on ferric chloride (Fe³⁺) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl₄) induced lipid peroxidation in vivo were examined. While -TOC could produce prooxidant and antioxidant effect on Fe³⁺-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with -TOC (3 mmol/kg/day × 3) did not protect against CCl₄-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day × 3) produced a dose-dependent protective effect on the CCl₄-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.Abbreviations ALT alanine aminotransferase - CCl₄ carbon tetrachloride - Fe³⁺ ferric chloride - MDA malondialdehyde - Sch B Schisandrin B - TBA 2-thiobarbituric acid - TBARS thiobarbituric acid reactive substances - -TOC dl--tocopherol     

Enhanced lipid peroxidation and inflammation during heat exposure in rats of different ages: Role of α-tocopherol

To investigate early events possibly related to the development of heat shock, we examined whether inflammatory-(interleukin-6, tumor necrosis factor α and 15-keto-13,14-dihydro-PGF_2α) and peroxidative-(8-iso-PGF_2α and malondialdehyde) markers are altered during acute heat exposure and aging. We also studied the relationships between inflammatory and peroxidative markers in these settings. In order to prevent these reactions developed as a consequence of the conditions mentioned above, we tested the effects of α-tocopherol. Our results demonstrated that 15-keto-13,14-dihydro-PGF_2α and malondialdehyde in the liver were altered during acute heat exposure in the young and middle-aged rats and could be predicted by changes in the levels of circulatory cytokines. Regardless of age, the supplementation with α-tocopherol prevented changes in the plasma cytokine levels and 15-keto-13,14-dihydro-PGF_2α and malondialdehyde levels in the liver, during acute heat exposure. This study notably emphasized the ability of α-tocopherol to prevent different heat induced mechanisms, involved in induction of inflammatory or peroxidative reactions.     

The influence of α-tocopherol and pyritinol on oxidative DNA damage and lipid peroxidation in human lymphocytes

Unscheduled DNA synthesis (UDS) and lipid peroxidation (LPO) were measured in human peripheral lymphocytes from healthy volunteers. These processes were induced by the catalytic system Fe²⁺-sodium ascorbate. The degree of induced LPO was measured spectrophotometrically by the thiobarbituric acid assay. UDS was detected by scintillometric measurement of the incorporation of ³H-thymidine into DNA. The protective action by fat-soluble vitamin E (d,l-α-tocopherol) and the artificial antioxidant pyritinol on UDS and LPO was also investigated.The system Fe²⁺ (2 μmole/1)-sodium ascorbate (30 μmole/1) increased the LPO level in healthy volunteers approximately 2.5 times and the incorporation of ³H-thymidine by 60–70%. α-Tocopherol (0.2 mmole/1) very efficiently suppressed LPO processes (p < 0.01) and the oxidative damage of DNA measured as UDS was also significantly diminished (p < 0.05). Pyritinol had no effect on LPO and UDS under our experimental conditions.     

Transmembrane distribution of α-tocopherol in single-lamellar mixed lipid vesicles

Summary A study of the molar ratio dependence of the incorporation of -tocopherol into single-lamellar vesicles showed that the number of molecules which the bilayers can accommodate increased linearly with increasing -tocopherol/phosphatidylcholine initial molar ratios till about 0.05, then approached a saturation limit. At 5 mol%, one -tocopherol molecule per 60 phospholipids can be incorporated into the membranes. Up to this limit the distribution of -tocopherol in the bilayers is uniform, while at initial molar ratios higher than 0.05 a disproportionation toward the inner monolayer of the vesicles is observed. The average outer/total ratio is found to be 0.27±0.03 at -tocopherol/phosphatidylcholine molar ratios above 0.07 and is similar to asymmetrical distributions that have been reported in vesicles containing other one-chain amphiphiles (e.g., cholesterol). This large disproportionation is in contrast with the packing distribution of certain twochain amphiphiles, and indicates that one of the driving forces for asymmetry formation in lipid bilayers might be dependent on the number of hydrocarbon chains per amphiphile molecule. A possible reason for the disproportionation effect observed in our experiments is the displacement of unsaturated phospholipids to the outer monolayer of the single-lamellar vesicles, by the more rigid isoprene units of -tocopherol.     

Comparative effect of N-substituted dehydroamino acids and α-tocopherol on rat liver lipid peroxidation activities

Free radical damage has been associated with a growing number of diseases and conditions, such as autoimmune diseases, neurodegenerative disorders and multiple types of cancer. Some dehydroamino acids and corresponding peptides can function as radical scavengers. In this study the in vitro effects on rat liver lipid peroxidation levels of fourteen N-substituted dehydroamino acid derivatives and α-tocopherol were investigated. α-Tocopherol is a powerful antioxidant that is beneficial in the treatment of many free radical related diseases. The results indicated that all the compounds showed very good inhibitory effect on the lipid peroxidation compound with α-tocopherol at 1 mM concentrations and the inhibition rate was in the range of 70–79 % with the exception of compound 5. At 0.1 mM concentrations compounds 1, 2 and 9 were found more active than α-tocopherol. The results confirmed that molecules such as dehydroamino acids which have reactive double bonds can act as a guard in vitro against oxidants.     

Ascorbic acid spares α-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells

Ascorbic acid, or vitamin C, can recycle -tocopherol in lipid bilayers, but even sparing of -tocopherol has not been a consistent finding in intact cells. Therefore, we tested the ability of ascorbate loading to spare -tocopherol and to prevent lipid peroxidation of cultured H4IIE rat liver cells. Although -tocopherol was undetectable in H4IIE cells, its cell content was increased by overnight incubation with -tocopherol in culture. Cells incubated with ascorbate 2-phosphate accumulated ascorbate to concentrations as high as 0.6 mM after overnight loading, but also released ascorbate into the medium. Ascorbate loading of -tocopherol-treated cells spared -tocopherol in a concentration-dependent manner during overnight incubation. Lipid peroxidative damage, measured as a decrease in fluorescence of cell-bound cis-parinaric acid, was decreased in cells loaded with either -tocopherol or ascorbate 2-phosphate, and showed an additive effect. These results suggest that ascorbate loading of H4IIE cells spares cellular -tocopherol and either directly or through recycling of -tocopherol prevents lipid peroxidative damage due to oxidant stress in culture.     

Inhibition of peroxidation in linoleic acid membranes by nitroxide radicals,butylated hydroxytoluene,and α-tocopherol

The thermal oxidation of the membranes of linoleic acid vesicles was preceded by a lag period, as long as the membranes contained low levels of preformed peroxides. Incorporation of 0.034 to 0.170 mol% of nitroxide spin label increased the length of this lag between 4.8 and 10.1 times. At the same time, the intensity of the ESR signal fell. The inclusion of as little as 0.04 mol% of butylated hydroxytoluene in the membranes also lengthened the lag period by a factor of 2.5. However, a similar molar proportion of α-tocopherol was without effect. When the linoleic acid from which vesicle membranes were formed contained between 0.45 and 1.43 mol% of peroxide, α-tocopherol produced a significant increase in the lag period, during which the antioxidant was gradually oxidized.     

Modification of the structure of plasmatic membranes of the liver by the action of α-tocopherol in vitro

Biophysics - The effect of the natural antioxidant α-tocopherol in a broad concentration range (10?4–10?25 M) on the viscosity characteristics and thermally induced...     

Model of singlet oxygen scavenging by α-tocopherol in biomembranes

Quenching of singlet molecular oxygen (${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$) by α-tocopherol (I) involves the hydroxy function of the chromanol ring of I. In phosphatidylcholine (PC) uni- and multilamellar vesicles this structural element of I is localized at the interface polar headgroup/hydrophobic core. A dielectric constant of ? ～ 25 was determined for this special region of the PC bilayer. The ratio k_Q/k_R of rate constants of quenching processes (k_Q) and irreversible reactions (k_R) of I with ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ increases with decreasing polarity of the solvent. In ethanolic solutions where ? = 25.5, k_Q/k_R is about 40. Extrapolation of these results to phospholipid bilayers suggests that at the nearness of the ester carbonyl oxygen of the PC fatty acid moieties, α-tocopherol can deactivate approximately 40 ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ molecules before being destroyed. It is concluded that in vivo, one may expect to find a higher k_Q/k_R ratio if the chromanol ring of I hides within the more hydrophobic interiors of the membrane surface peptides.     

Purification of a complex of transducin βγ subunits from isotonic extracts of bovine retinal rod outer segments

A method is proposed for obtaining a free complex of transducin βγ subunits from bovine retinal rod outer segments in a highly purified state.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Involvement of binding lipoproteins in the absorption and transport of α-tocopherol in the rat

1. Specific lipoproteins binding alpha-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered alpha-[(3)H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of alpha-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of alpha-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of alpha-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.     

Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of γ-glutamyltranspeptidase activity

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of γ-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20–25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H₂O₂ plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent path-way of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.     

On the inhibition of α-oxobutyrate utilization by fatty acids in rat liver mitochondria

It has been found that free fatty acids and acylcarnitine inhibit α-oxobutyrate utilization in rat liver mitochondria. It has been recognized that the intramitochondrial accumulation of acetyl-CoA, produced by the β-oxidation of activated fatty acids, is responsible for such inhibition. In fact acetyl-CoA is shown to inhibit α-oxobutyrate dehydrogenase (α-oxoglutarate: lipoate oxidoreductase (acceptor acylating) EC 1.2.4.2)