Antioxidant action of vitamin E in vivo as assessed from its reaction products with multiple biological oxidants.

Vitamin E acts as essential antioxidant against detrimental oxidation of biological molecules induced by multiple reactive species. To gain more insight into the physiological role of vitamin E, the levels of its oxidation products in humans under normal and pathological conditions were compared. α-Tocopherol quinone (α-TQ) and 5-nitro-γ-tocopherol (5-NgT) were focused. α-TQ is produced by multiple oxidants including oxygen radicals, peroxynitrite, hypochlorite, singlet oxygen, and ozone, while 5-NgT is produced by nitrogen dioxide radical derived from peroxynitrite and the reaction of nitrite and hypochlorite. The reported concentrations of α-TQ and 5-NgT in healthy human plasma are highly variable ranging from 15 to 360 and 4 to 170 nM, respectively. In general, the molar ratio 5-NgT/γ-tocopherol was higher than the ratio α-TQ/α-tocopherol. Both absolute concentrations of α-TQ and 5-NgT and the molar ratios to the parent tocopherols were elevated significantly in the plasma of patients with various diseases compared with healthy subjects except neurological diseases. The molar ratios of the products to the respective parent compounds decreased in the order of 5-NgT/γ-tocopherol > α-TQ/α-tocopherol > hydroxyoctadecadienoate/linoleate > 3-nitrotyrosine/tyrosine > isoprostane/arachidonate. The molar ratios of nitrated products to the respective parent compounds in human plasma are approximately 10-2 for 5-NgT and 10-5 for 3-nitrotyrosine, nitro-oleic acid, and 8-nitroguaine. These data indicate that vitamin E acts as an important physiological antioxidant and that α-TQ and 5-NgT represent biomarker for oxidative stress and nitrative stress respectively.

α-Tocopherol Stereoisomers in Human Plasma Are Affected by the Level and Form of the Vitamin E Supplement Used.

BACKGROUND: Studies examining vitamin E intake and the percentage of the population meeting dietary guidelines do not distinguish between natural (RRR-α-tocopherol) and synthetic (all-rac-α-tocopherol) intake, even though these different isomeric forms differ in bioactivity. OBJECTIVE: This study aimed to determine the effect of RRR-α-tocopherol vs. all-rac-α-tocopherol intake on the percentage of the population meeting the vitamin E recommendation and on plasma α-tocopherol stereoisomer distribution. METHODS: With the use of data from the Irish National Adult Nutrition Survey (NANS), this study examined the percentage of the Irish population meeting the European Union (EU) RDA for vitamin E of 12 mg/d, correcting for a bioactivity difference in all-rac- vs. RRR-α-tocopherol, where 1 mg of all-rac-α-tocopherol is considered to be equivalent to 1:1.36 (0.74) mg in the EU RDA. In a subcohort of supplement users and nonusers, plasma α- and γ-tocopherol concentrations and α-tocopherol stereoisomer distribution were measured. Receiver operating characteristic (ROC) curve analysis was conducted to determine ability to discriminate supplement user types. RESULTS: Analysis of the NANS showed that 100% of participants still met the recommended intake of 12 mg/d, after all-rac-α-tocopherol intake was corrected for α-tocopherol equivalent bioactivity. In the subcohort analysis, the percentage of plasma RRR-α-tocopherol was significantly lower in high all-rac-α-tocopherol supplement (>11 mg/d) users (82%) compared with nonusers and with high RRR-α-tocopherol supplement (>35 mg/d) users (91% and 93% respectively, P < 0.01). High RRR-α-tocopherol supplement users had a significantly higher plasma α-tocopherol than low all-rac-α-tocopherol supplement (<2.5 mg/d) users (34 vs. 25 µmol/L, P = 0.01). ROC analysis demonstrated an ability to distinguish between RRR- and all-rac-α-tocopherol consumers, which may be useful in investigating the potential effect of RRR- and all-rac-α-tocopherol intake on health. CONCLUSIONS: This study demonstrated that the percentage of the population meeting the vitamin E recommendation was unaffected when all-rac-α-tocopherol intake was corrected for α-tocopherol equivalent bioactivity. all-rac-α-Tocopherol intake led to a decrease in the percentage of plasma RRR-α-tocopherol relative to RRR-α-tocopherol intake.

α-Tocopherol supplementation reduces 5-nitro-γ-tocopherol accumulation by decreasing γ-tocopherol in young adult smokers.

γ-Tocopherol (γ-T) scavenges reactive nitrogen species (RNS) to form 5-NO2-γ-T (NGT). However, α-T supplementation decreases circulating γ-T, which could limit its RNS scavenging activities. We hypothesized that α-T supplementation would mitigate NGT accumulation by impairing γ-T status. Healthy smokers (21 ± 1 y, n = 11) and non-smokers (21 ± 2 y, n = 10) ingested 75 mg/d each of RRR- and all-rac-α-tocopheryl acetate for 6 d. Plasma α-T, γ-T, γ-carboxyethyl hydroxychromanol (CEHC), NGT, and nitrate/nitrite were measured prior to supplementation (Pre), the morning after 6 consecutive evenings of supplementation (Post 1), and on the mornings of d 6 (Post 6) and d 14 (Post 14) during the post-supplementation period. α-T supplementation increased plasma α-T, and decreased γ-T, in both groups and these returned to Pre concentrations on Post 6 regardless of smoking status. Plasma γ-CEHC increased after the first dose of supplementation in both groups, suggesting that α-T supplementation decreased plasma γ-T in part by increasing its metabolism. Plasma NGT and nitrate/nitrite concentrations at Pre were greater in smokers, indicating greater nitrative stress due to cigarette smoking. Plasma NGT concentration was lowered only in smokers on Post 1 and Post 6 and was restored to Pre levels on Post 14. Plasma nitrate/nitrite tended (P = 0.07) to increase post-supplementation only in smokers, supporting decreases in RNS scavenging by γ-T. Plasma NGT concentration was more strongly correlated (P < 0.05) with γ-T in smokers (R = 0.83) compared with non-smokers (R = 0.50), supporting that α-T-mediated decreases in γ-T reduces NGT formation. These data indicate that α-T supplementation limits γ-T scavenging of RNS in smokers by decreasing γ-T availability.

Gamma-tocopherol-N,N-dimethylglycine ester as a potent post-irradiation mitigator against whole body X-irradiation-induced bone marrow death in mice.

We examined the radioprotective and mitigative effects of gamma-tocopherol-N,N-dimethylglycine ester (GTDMG), a novel water-soluble gamma-tocopherol derivative, against X-irradiation-induced bone marrow death in mice. Mice (C3H, 10 weeks, male) were injected intraperitoneally with GTDMG suspended in a 0.5% methyl cellulose solution before or after receiving of 7.5-Gy whole body X-irradiation. GTDMG significantly enhanced the 30-day survival rate when given 30 min before or immediately after the irradiation. Its mitigative activity (administered after exposure) was examined further in detail. The optimal concentration of GTDMG given immediately after irradiation was around 100 mg/kg body weight (bw) and the 30-day survival rate was 97.6 ± 2.4%. When GTDMG was administered 1, 10 and 24 h post-irradiation, the survival rate was 85.7 ± 7.6, 75.0 ± 9.7 and 36.7 ± 8.8%, respectively, showing significant mitigation even at 24 h after irradiation (P < 0.05). The value of the dose reduction factor (100 mg/kg bw, given intraperitoneally (i.p.) immediately after irradiation) was 1.25. GTDMG enhanced the recovery of red blood cell-, white blood cell-, and platelet-counts after irradiation and significantly increased the number of endogenous spleen colonies (P < 0.05). Subcutaneous (s.c.) administration also had mitigative effects. In conclusion, GTDMG is a potent radiation mitigator.

Gamma-tocopherol dominates in young leaves of runner bean (Phaseolus coccineus) under a variety of growing conditions: the possible functions of gamma-tocopherol.

It has been shown that young leaves of runner bean (Phaseolus coccineus) plants grown under natural conditions have an unusually high content of gamma-tocopherol, accounting for up to 90% of all tocopherols and 50% of the chlorophyll content. The level of gamma-tocopherol gradually decreased during the first two weeks of leaf development. The high content of gamma-tocopherol in young leaves was not significantly influenced by growth conditions. In contrast to seeds, gamma-tocopherol was also the main tocopherol found in light-grown and etiolated primary leaves of runner bean. The obtained results suggest that gamma-tocopherol decline during leaf development is not only due to conversion of gamma- to alpha-tocopherol but probably also due to degradation of gamma-tocopherol to non-tocochromanol compounds. We have also shown that gamma-tocopherol found in young leaves is mainly localized in thylakoid membranes within chloroplast. In the primary leaves subjected to different abiotic stresses, only during simultaneous drought and light stress, gamma-tocopherolquinone, an oxidation product of gamma-tocopherol, was preferentially accumulated. Since one of the other possible functions of gamma-tocopherol could be its action as a nitric oxide scavenger, young leaves were analyzed for the presence of nitro-gamma-tocopherol. However, despite the use of a sensitive detection method, it was not found. The possible physiological function of the increased level of gamma-tocopherol in the young leaves was discussed.

[UVB-induced skin damage and the protection/treatment--effects of a novel, hydrophilic gamma-tocopherol derivative].

Ultraviolet radiation is the major environmental cause of skin damage. Although only 0.5% of ultraviolet B (UVB) radiation reaches the earth, it is the main cause of sunburn and inflammation and the most carcinogenic constituent of sunlight. We investigated whether the topical application of a novel, water-soluble gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG), could protect against UV-induced skin damage. Topical pre- or postapplication of gamma-TDMG solution significantly prevented sunburn cell formation, lipid peroxidation, and edema/inflammation that were induced by exposure to a single dose of UV irradiation. Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E(2) (PGE(2)) levels seen after UV exposure were significantly suppressed by pre- or posttreatment with gamma-TDMG. The increase in COX-2 activity was significantly inhibited by gamma-TDMG, suggesting that the reduction in PGE(2) concentration was due to the direct inhibition of COX-2 activity by gamma-TDMG. The derivative strongly inhibited inducible nitric oxide synthase mRNA expression and nitric oxide production. With the application of gamma-TDMG, the pigmentation in melanocytes was lightened and the increase melanin concentration was suppressed. Gamma-TDMG is converted to gamma-Toc in the skin and has higher bioavailability than gamma-Toc itself. These results suggest that gamma-TDMG-derived gamma-Toc acts as an antioxidant, antiinflammatory and antipigmentation agent. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing and reducing UV-induced skin damage in humans.

Topical application of gamma-tocopherol derivative prevents UV-induced skin pigmentation.

We previously reported that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopheryl-N,N-dimethylglycinate hydrochloride (gamma-TDMG) gets converted to the antioxidant gamma-Toc in skin. We also found that this derivative displayed greater bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce UV-induced skin pigmentation in brownish guinea pigs. gamma-TDMG (0.1 or 0.5%) was topically applied to the skin before and after it was exposed to UVB plus UVA (3 times/week for 1 week), and then 10 times/week for 4 weeks thereafter. Treatment with 0.5% gamma-TDMG resulted in significant skin lightening (70% of the pigmentation of irradiated controls). We also found that melanin synthesis was dose-dependently inhibited by gamma-TDMG in murine B16 melanoma cells. When gamma-TDMG or kojic acid (250 microM) were added to homogenates of B16 melanoma cells, their tyrosinase activity was significantly inhibited by approximately 40% and 75%, respectively. Mushroom tyrosinase activity was significantly inhibited by 200 microM gamma-Toc and kojic acid, but not gamma-TDMG. When B16 cells were incubated with 250 microM gamma-TDMG for 24 or 48 h, their intracellular gamma-Toc concentrations rose over 100 fold to 10.5 and 11.2 nmol/10(6) cells, respectively, suggesting that gamma-TDMG was rapidly converted to gamma-Toc in these cells and that their reduced melanin synthesis may have been due to the activity of gamma-Toc. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing photo-induced skin pigmentation in humans.

Topical application of a novel, hydrophilic gamma-tocopherol derivative reduces photo-inflammation in mice skin.

We previously demonstrated that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG) converts to gamma-Toc in the mouse skin and has a higher bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce photo-inflammation in mouse skin, and compared its effectiveness to that of alpha-Toc acetate (alpha-TA). Topical pre- or post-application of 5% gamma-TDMG significantly reduced the formation of edema and tempered the increase in cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E2 (PGE2) that were induced by a single dose of UV irradiation of 2 kJ/m2 (290-380 nm, maximum 312 nm). The pre-treatment of mouse skin with 10% alpha-TA had the same anti-inflammatory effect as did gamma-TDMG. In spite of same having the ability to reduce PGE2 levels, the effect of gamma-TDMG pre-treatment on the inhibition of COX-2 mRNA/protein expression was less than that seen with 10% alpha-TA. In contrast, the increase in COX-2 activity seen after UV exposure was reduced more by gamma-TDMG than by alpha-TA, suggesting that the reduction in PGE2 levels might have been due to the direct inhibition of COX-2 activity by gamma-TDMG-derived gamma-Toc. Both Toc derivatives strongly suppressed inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production, both of which play important roles in UV-induced inflammation. Both derivatives also significantly reduced lipid peroxidation in response to UV exposure, though gamma-TDMG's ability in this regard was less than that seen with alpha-TA, which correlated with their abilities to suppress COX-2 expression. Thus, the gamma-TDMG-derived gamma-Toc acts as an antioxidant, suppresses iNOS expression and directly inhibits COX-2 activity, all of which likely play a role in mediating its suppressive effects on photo-inflammation. Our data further suggest that the topical application of gamma-TDMG, a novel hydrophilic gamma-Toc derivative, may be efficacious in preventing and reducing UV-induced inflammation in humans.

Nitration of gamma-tocopherol prevents its oxidative metabolism by HepG2 cells.

Gamma-tocopherol (gammaT) is one of the major forms of vitamin E consumed in the diet. Previous reports have suggested increased levels of nitrated gamma-tocopherol (5-NO2-gammaT) in smokers and individuals with conditions associated with elevated nitrative stress. The monitoring of 5-NO2-gammaT and its possible metabolite(s) may be a useful marker of reactive nitrogen species generation in vivo. The major pathway for the metabolism of gammaT is the cytochrome P450 dependent oxidation to its water-soluble metabolite gamma-CEHC, which is excreted in urine. In order to determine if 5-NO2-gammaT could be metabolised via the same route and detected in urine we developed a sensitive gas chromatography-mass spectrometry assay for 5-NO2-gamma-CEHC. 5-NO2-gamma-CEHC was synthesised and its structure confirmed by proton nuclear magnetic resonance and mass spectrometry. While gamma-CEHC was abundant in urine from healthy volunteers, as well as patients with coronary heart disease and type 2 diabetes, 5-NO2-gamma-CEHC was undetectable (limit of detection of 5 nM). To understand this observation we examined the uptake and metabolism of gammaT and 5-NO2-gammaT by HepG2 cells. gammaT was readily incorporated into cells and metabolised to gamma-CEHC over a period of 48 hours. In contrast, 5-NO2-gammaT was poorly incorporated into HepG2 cells and not metabolised to 5-NO2-gamma-CEHC over the same time period. We conclude that nitration of gammaT prevents its incorporation into liver cells and therefore its metabolism to the water-soluble metabolite. Whether 5-NO2-gammaT could be metabolised via other pathways in vivo requires further investigation.

5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo.

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-gamma-tocopherol (NGT). In order to assess gamma-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted approximately 75% of alpha-T, approximately 60% of gamma-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for approximately 20% of the gamma-T decrease. NGT also correlated (R2 = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or gamma-T; plasma alpha-T and gamma-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.

Novel d-gamma-tocopherol derivative as a prodrug for d-gamma-tocopherol and a two-step prodrug for S-gamma-CEHC.

d-gamma-Tocopherol (gamma-Toc) and its major metabolite, 2, 7, 8-trimethyl-2S-(beta-carboxyethyl)-6-hydroxychroman (S-gamma-CEHC), are currently receiving attention concerning their unique pharmacological activities. In order to achieve the efficient delivery of gamma-Toc and S-gamma-CEHC in vivo, we synthesized d-gamma-tocopheryl N,N-dimethylglycinate hydrochloride (gamma-TDMG) as a water-soluble prodrug of gamma-Toc and a two-step prodrug of S-gamma-CEHC. gamma-TDMG is a solid (mp 161-163 degrees C) and is quite soluble in water over 50 mM. The hydrolysis of gamma-TDMG was effectively catalyzed by esterases in rat and human liver microsomes. The disposition of gamma-TDMG after iv administration in rats was compared with that of gamma-Toc solubilized with the surfactant, polyoxyethylene hydrogenated castor oil. The plasma and liver levels of gamma-Toc rapidly increased after the iv administration of the gamma-TDMG. The liver availability of gamma-Toc after the administration of gamma-TDMG was two times higher than that of the gamma-Toc administration. The relative systemic availability of S-gamma-CEHC after the gamma-TDMG administration was an equivalent value (102%), and the mean residence time of S-gamma-CEHC was eight times longer than the racemic gamma-CEHC administration. Based on these results, gamma-TDMG was identified as the most promising water-soluble prodrug of gamma-Toc and the two-step prodrug of S-gamma-CEHC.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection.

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

Evidence for the nitration of gamma-tocopherol in vivo: 5-nitro-gamma-tocopherol is elevated in the plasma of subjects with coronary heart disease.

This study investigated the hypothesis that nitration of gamma-tocopherol may be an important mechanism for the detoxification of reactive nitrogen oxide species in vivo. Using liquid chromatography-tandem MS we have shown that gamma-tocopherol can be nitrated in vivo to form 5-nitro-gamma-tocopherol and that concentrations of this compound are elevated in the plasma of subjects with coronary heart disease. In addition, we demonstrate in carotid-artery atherosclerotic plaque that nitration of gamma-tocopherol is also evident at levels similar to that seen in the plasma of subjects with coronary heart disease.

The nitration product 5-nitro-gamma-tocopherol is increased in the Alzheimer brain.

Oxidative stress and quasi-inflammatory processes recently have been recognized as contributing factors in the pathogenesis of Alzheimer's disease (AD). Reactive nitrating species have specifically been implicated in AD based on immunochemical and instrumental detection of nitrotyrosine in AD brain protein. The significance of lipid-phase nitration has not been investigated in AD. This study documents a significant two- to threefold increase in the lipid nitration product 5-nitro-gamma-tocopherol in affected regions of the AD brain as determined by high-performance liquid chromatography with electrochemical detection. In a bioassay to compare the relative potency of alpha-tocopherol and gamma-tocopherol against nitrative stress, rat brain mitochondria were exposed to the peroxynitrite-generating compound SIN-1. The oxidation-sensitive Kreb's cycle enzyme alpha-ketoglutarate dehydrogenase was inactivated by SIN-1, in a manner that could be significantly attenuated by gamma-tocopherol (at <10 microM) but not by alpha-tocopherol. These data indicate that nitric oxide-derived species are significant contributors to lipid oxidation in the AD brain. The findings are discussed in reference to the neuroinflammatory hypothesis of AD and the possible role of gamma-tocopherol as a major lipid-phase scavenger of reactive nitrogen species.

Measurement of 3-nitrotyrosine and 5-nitro-gamma-tocopherol by high-performance liquid chromatography with electrochemical detection.

Nitric oxide (NO) is a lipophilic gaseous molecule synthesized by the enzymatic oxidation of L-arginine. During periods of inflammation, phagocytic cells generate copious quantities of NO and other reactive oxygen species. The combination of NO with other reactive oxygen species promotes nitration of ambient biomolecules, including protein tyrosine residues and membrane-localized gamma-tocopherol. The oxidative chemistry of NO and derived redox congeners is reviewed. Techniques are described for the determination of 3-nitro-tyrosine and 5-nitro-gamma-tocopherol in biological samples using high-performance liquid chromatography with electrochemical detection.

Plastid enzymes of terpenoid biosynthesis. Purification and characterization of gamma-tocopherol methyltransferase from Capsicum chromoplasts.

gamma-Tocopherol methyltransferase was solubilized and purified from Capsicum chromoplast membranes by a combination of standard fractionation techniques. The purified enzyme was electrophoretically homogeneous, and its molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 33,000. In the absence of detergent, the enzyme formed high molecular weight aggregates. Several properties of the enzyme have been determined. The Km values were 2.5 and 13.7 microM for S-adenosylmethionine and gamma-tocopherol, respectively. The enzyme was able to transfer the methyl group S-adenosylmethionine to N-4-azido-2-nitrophenyl-beta-alanyl-gamma-tocopherol. The rate of transfer was less efficient compared to gamma-tocopherol. In the presence of ultraviolet light, this analog inhibited the gamma-tocopherol methyltransferase activity.