Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.     

Role of ��-tocopherol in cellular signaling: ��-tocopherol inhibits stress-induced mitogen-activated protein kinase activation

Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.     

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T₂ seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.     

Acute toxicity study of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol, and γ-tocopherol in rats

Liposomes have been used for the delivery of antioxidants to different tissues and organs for the treatment of oxidative stress-induced injuries. In this study, the acute toxicity of a single dose of intravenously (i.v.) administered liposomal antioxidant formulation, containing N-acetylcysteine (NAC) with or without α-tocopherol (α-T) or γ-tocopherol (γ-T), in rats was examined. Each group consisted of 5 male and 5 female Sprague-Dawley rats, with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (660 mg/kg) and test groups receiving DPPC liposomes (660 mg/kg) entrapped with 1) NAC (200 mg/kg), 2) NAC (200 mg/kg) and α-T (83.3 mg/kg), and 3) NAC (200 mg/kg) and γ-T (71.4 mg/kg). These dose levels were determined from the dose-range-finding study and were considered to be the maximum feasible dose (MFD) levels, based on the volume of 10 mL/kg and physical properties and viscosity of the test articles that could be safely administered to rats by an i.v. injection. Two weeks after treatment (day 15), rats in the control group and three test groups exhibited no clinical signs of toxicity during the dosing period or during the 14-day post-treatment period. Weight gain and food consumption in all animals was appropriate for the age and sex of animals. Clinical pathology findings (e.g., hematology, coagulation, clinical chemistry, and urinalysis) were unremarkable in all rats and in all groups. In conclusion, the results of this study showed no treatment-related toxicity in rats at the MFD level by a single bolus i.v. administration.     

Serum α-tocopherol and selenium in belgian infants and children

Previous studies showed low selenium (Se) concentrations in Belgian children. Serum α-tocopherol, retinol, total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, selenium (Se), and thiobarbituric acid-reactive substances were examined. In order to obtain further information on the Se status in Belgian children, Se, α-tocopherol, retinol, and lipid concentrations were examined and signs of peroxidative lipid damage were evaluated in a subgroup. The study was performed in 524 children (0–14 yr old) during vaccination campaigns. Three age groups were analyzed: 0–1, 1–4, and 4–14 yr. In 87 of them, where sufficient amounts of serum were available, analysis of thiobarbituric acid-reactive substances was done. Infants have high serum α-tocopherol concentrations: (23.2 μmol/L [median and interquartile range: 18.6–30.2]) and low Se concentrations (0.37 mol/L [0.27–0.47]). Se concentrations rise significantly during the first 4 yr (p < 0.0001) (Mann-Whitney U-test, tied p-values): 0.70 μmol/L (0.59–0.82); in the 4–14 yr olds, it was 0.75 μmol/L (0.67–0.86). These values remain low compared to results coming from other parts of the world. α-Tocopherol concentrations decrease significantly after infancy (p < 0.0001). The ratio α-tocopherol/total cholesterol is higher in infants. This is induced by the high vitamin E content of infant formulas. Signs of serum lipid peroxidation could not be detected by analysis of serum malondialdehyde concentrations. High α-tocopherol concentrations, as those observed in infant serum lipids, could be one of the protective mechanisms from the peroxidative lipid damages, sometimes observed in a low-Se status.     

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.     

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.     

Solubilization and interaction of α-tocopherol in water-aerosol OT-isooctane systems

Solubilization and interaction of α-tocopherol into bis(2-ethylhexyl)sulphosuc cinate sodium salt microemulsion systems have been studied by temperature dependent phase transition, viscosity and nuclear magnetic resonance studies. Tocopherol being an amphiphilic molecule dissolves into the interfacial surfactant monolayer of the microemul sion droplets. The dissolution leads to an enhancement of the rigidity of the surfactant monolayer as studied by the increase in mixing and phase transition temperatures of the microemulsion droplets. Solubilization of tocopherol into microemulsion droplets causes an increase in the effective size of the droplet and as a consequence, the inter-droplet interactions are also increased. The water binding capacity of the surfactant (bis(2-ethylhexyl)sulphosuccinate sodium salt) is reduced due to solubilization of tocopherol as is evidenced from the downfield shifts of water proton magnetic resonances. In the presence of the dissolved electrolytes into the aqueous core, tocopherol is squeezed out of the microemulsion droplets increasing the membrane fluidity and permeability.     

The role of α-tocopherol in motor hypofunction with aging in α-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network — including α-tocopherol (αT) and αT transfer protein — in aging in vivo, α-tocopherol transfer protein knockout (αTTP^?/?) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% αT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F₂α, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of αTTP^?/? mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in αTTP^?/? mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the αTTP^?/? mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels.In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that αT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.     

Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

Alpha-tocopherol (vitamin E) is synthesized from gamma-tocopherol in chloroplasts by gamma-tocopherol methyltransferase (gamma-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of alpha-tocopherol, but are low in gamma-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene gamma-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allele (vte4-2) carrying a T-DNA insertion in the coding sequence of gamma-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of gamma-tocopherol accumulated, whereas alpha-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the gamma-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of alpha-tocopherol and gamma-tocopherol increased in wild type, and gamma-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that alpha-tocopherol can be replaced by gamma-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.     

Plasma concentration of α-tocopherol in different free-ranging birds of prey

In this study, we investigated the α-tocopherol plasma concentrations in healthy free-ranging nestlings of the white-tailed sea eagle (Haliaeetus albicilla) (n = 32), osprey (Pandion haliaetus) (n = 39), northern goshawk (Accipiter gentilis) (n = 25), common buzzard (Buteo buteo) (n = 31), and honey buzzard (Pernis apivorus) (n = 18) as well as of free-ranging adults of the white-tailed sea eagle (n = 10), osprey (n = 31), and northern goshawk (n = 45). α-Tocopherol plasma concentrations were determined by reverse-phase high-performance liquid chromatography. α-Tocopherol plasma concentrations in nestlings of osprey, white-tailed sea eagle, and northern goshawk did not differ significantly amongst the species, but the common buzzard and honey buzzard nestlings had significantly lower α-tocopherol plasma concentrations than nestlings of the other species (both P < 0.001). Adult male ospreys and white-tailed sea eagles had significantly higher α-tocopherol concentrations compared to adult females (both P < 0.005). Adult ospreys and northern goshawks had significantly higher α-tocopherol plasma concentrations compared to their nestlings (both P < 0.001). In adult female northern goshawks, plasma concentrations of α-tocopherol increased significantly before egg laying (P < 0.001). These results demonstrate α-tocopherol plasma concentrations in birds of prey to be species specific and influenced by age and reproductive status.     

Pulse radiolytic study of α-tocopherol radical mechanisms in ethanolic solution

Pulse radiolytic studies of α-tocopherol (αTH) oxidation-reduction processes were carried out with low doses (5 Gy) of high-energy electrons in O₂₋, N₂₋, and air-saturated ethanolic solutions. Depending on the concentration of oxygen in solution, two different radicals, A· and B·, were observed. The first, A·, was obtained under N₂ and results from aTH reaction with solvated electron (k_aTH+csolv⁻ = 3.4 × 10⁸ mol⁻¹ liter s⁻¹) and with H₃C-ĊH-OH, (R·) (k_{aTH + R·} = 5 × 10⁵ mol⁻¹ liter s⁻¹). B·, observed under O₂, is produced by αTH reaction with RO₂ peroxyl radicals (k_aTH + RO₂^. = 9.5 × 10⁴ mol⁻¹ liter s⁻¹).     

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.     

Serum α-tocopherol and γ-tocopherol concentrations and prostate cancer risk in the PLCO Screening Trial: a nested case-control study

Background

Vitamin E compounds exhibit prostate cancer preventive properties experimentally, but serologic investigations of tocopherols, and randomized controlled trials of supplementation in particular, have been inconsistent. Many studies suggest protective effects among smokers and for aggressive prostate cancer, however.

Methods

We conducted a nested case-control study of serum α-tocopherol and γ-tocopherol and prostate cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, with 680 prostate cancer cases and 824 frequency-matched controls. Multivariate-adjusted, conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CIs) for tocopherol quintiles.

Results

Serum α-tocopherol and γ-tocopherol were inversely correlated (r = −0.24, p<0.0001). Higher serum α-tocopherol was associated with significantly lower prostate cancer risk (OR for the highest vs. lowest quintile = 0.63, 95% CI 0.44–0.92, p-trend 0.05). By contrast, risk was non-significantly elevated among men with higher γ-tocopherol concentrations (OR for the highest vs. lowest quintile = 1.35, 95% CI 0.92–1.97, p-trend 0.41). The inverse association between prostate cancer and α-tocopherol was restricted to current and recently former smokers, but was only slightly stronger for aggressive disease. By contrast, the increased risk for higher γ-tocopherol was more pronounced for less aggressive cancers.

Conclusions

Our findings indicate higher α-tocopherol status is associated with decreased risk of developing prostate cancer, particularly among smokers. Although two recent controlled trials did not substantiate an earlier finding of lower prostate cancer incidence and mortality in response to supplementation with a relatively low dose of α-tocopherol, higher α-tocopherol status may be beneficial with respect to prostate cancer risk among smokers. Determining what stage of prostate cancer development is impacted by vitamin E, the underlying mechanisms, and how smoking modifies the association, is needed for a more complete understanding of the vitamin E-prostate cancer relation.     

Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.     

ATP-Binding cassette transporter A1 is involved in hepatic α-tocopherol secretion

Vitamin E (α-tocopherol) is an essential fat-soluble nutrient with antioxidant properties. α-Tocopherol transfer protein (α-TTP), the product of the gene responsible for familial isolated vitamin E deficiency, plays an important role in maintaining the plasma α-tocopherol level by mediating the secretion of α-tocopherol by the liver. However, the mechanisms underlying hepatic α-tocopherol secretion are not fully understood. This study was undertaken to elucidate the mechanism of α-tocopherol re-efflux from hepatocytes, the cells that have the most important role in regulating plasma-α-tocopherol concentrations. From in vitro experiments using [³H]α-tocopheryl acetate and McARH7777 cells that stably express α-tocopherol transfer protein (α-TTP), the following results were obtained. First, addition of apolipoprotein A-I (apoA-I), a direct acceptor of the ATP-binding cassette transporter A1 (ABCA1)-secreted lipids, increased α-tocopherol secretion in a dose-dependent manner. Second, probucol, an antiatherogenic compound reported to be an inactivator of ABCA1 reduced hepatic α-tocopherol secretion. Third, ABCA1-RNAi suppressed hepatic α-tocopherol secretion. In a mouse in vivo experiment, addition of 1% probucol to the diet decreased plasma α-tocopherol concentrations. These results strongly suggest that ABCA1 is substantially involved in hepatic α-tocopherol secretion.     

Spectroscopic studies of D-α-tocopherol concentration-induced transformation in egg phosphatidylcholne vesicles

The effects of embedding up to 60 mol% of α-tocopherol (α-Toc) on the morphology and structure of the egg phosphatidylcholine (PC) membrane were studied using spectroscopic techniques. The resulting vesicles were subjected to turbidometric and dynamic light scattering measurements to evaluate their size distribution. The α-Toc intrinsic fluorescence and its quenching was used to estimate the tocopherol position in the membrane. Optical microscopy was used to visualize morphological changes in the vesicles during the inclusion of tocopherol into the 2 mg/ml PC membrane. The incorporation of up to 15 mol% of tocopherol molecules into PC vesicles is accompanied by a linear increase in the fluorescence intensity and the simultaneous formation of larger, multilamellar vesicles. Increasing the tocopherol concentration above 20 mol% induced structural and morphological changes leading to the disappearance of micrometer-sized vesicles and the formation of small unilamellar vesicles of size ranging from 30 to 120 nm, mixed micelles and non-lamellar structures.     

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.     

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