Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

To evaluate vitamin E metabolism, a method was developed to quantitate liver alpha- and gamma-tocopherol metabolites, alpha-carboxyethyl hydroxychroman [alpha-CEHC; 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman] and gamma-CEHC [2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman], respectively. Vitamin E supraenriched livers were obtained from rats that were injected with vitamin E daily for 18 days. Liver samples (approximately 50 mg) were homogenized, homogenate CEHC-conjugates were hydrolyzed, CEHCs were extracted with ethyl ether, and then CEHCs were quantitated using liquid chromatography-mass spectrometry (LC-MS). Precision, based on intersample variability, ranged from 1% to 3%. Recovery of alpha- and gamma-CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of alpha- and gamma-CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver alpha-tocopherol, the MS peak for liver alpha-CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 +/- 0.01 to 15 +/- 2 nmol/g). Liver alpha-CEHC concentrations were correlated with serum alpha-CEHC, liver alpha-tocopherol, and serum alpha-tocopherol (P < 0.001 for each comparison). alpha-CEHC represented 0.5-1% of the liver alpha-tocopherol concentration. Thus, LC-MS can be successfully used to quantitate alpha- and gamma-CEHC in liver samples. These data suggest that in times of excess liver alpha-tocopherol, increased metabolism of alpha-tocopherol to alpha-CEHC occurs.     

Urinary alpha-tocopherol metabolites in alpha-tocopherol transfer protein-deficient patients

Patients with alpha-tocopherol transfer protein (alpha-TTP) defects experience neurological symptoms characteristic of vitamin E deficiency and depend on continuous high alpha-tocopherol supplements. We investigated the excretion of 2,5,7, 8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a urinary metabolite of alpha-tocopherol, as a putative marker for the alpha-tocopherol status of alpha-TTP-deficient patients and control subjects. In three patients vitamin E supplementation was stopped for short periods of time, during which plasma alpha-tocopherol concentrations and urinary alpha-CEHC excretion were measured. In the patients, plasma alpha-tocopherol decreased below normal (<5 micromol/l) but alpha-CEHC excretion remained above the range of unsupplemented control subjects (0.118-0.306 mg/day, n = 6). In healthy subjects, however, alpha-CEHC excretion was increased only after surpassing a plasma alpha-tocopherol threshold of 30-40 micromol/l. Such a threshold did not exist in patients. The general mechanism of alpha-tocopherol degradation did not appear to differ between patients and control subjects. The presumed mechanism of omega- and subsequent beta-oxidation was supported by the detection of alpha- CPHC, an alpha -CEHC homolog with a side chain longer by 3 carbon atoms, both in supplemented patients and in control subjects.     

Synthesis and analysis of conjugates of the major vitamin E metabolite,alpha-CEHC

Glucuronide and sulphate conjugates of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the major metabolite of alpha-tocopherol (vitamin E), have been synthesized and used for the first direct analysis of conjugated urinary vitamin E metabolites. The metabolites of vitamin E (alpha-tocopherol) could be useful as markers of the function(s) of vitamin E in vivo. A number of methods have been described for the analysis of urinary vitamin E metabolites but these have relied on either acid or enzymatic deconjugation of the metabolites prior to analysis by high performance liquid chromatography or gas chromatography/mass spectrometry. These methods have provided useful information about the amount and types of metabolites excreted in the urine but suffer from a number of disadvantages. Deconjugation has been shown to produce artifacts as a result of the conversion of alpha-CEHC to alpha-tocopheronolactone and the efficiency of deconjugation is also difficult to assess. Methods that allow the direct measurement of the conjugated metabolites would overcome these problems and would also substantially reduce the preparation and analysis time. Here we describe the use of conjugated standards to characterize alpha-CEHC conjugates in human urine by tandem mass spectrometry (MS-MS). The future use of MS-MS to measure urinary vitamin E metabolites is also discussed.     

Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in human serum.

alpha- and gamma-tocopherol are the major vitamin E compounds found in human blood and tissues. The metabolites are 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC, LLU-alpha), respectively. alpha-CEHC is excreted mainly as glucuronide or sulfate conjugates in the urine. Here we describe a sensitive and reliable method to analyze alpha- and gamma-CEHC in human serum. The concentration of alpha-CEHC in human serum is in the range of 5-10 pmol/ml but increases significantly up to 200 pmol/ml upon supplementation with RRR-alpha-tocopherol. About one-third of the alpha-CEHC circulating in the blood is present as a glucuronide conjugate. Baseline levels of gamma-CEHC are about 50 to 85 pmol/ml.     

Alpha-tocopherol modulates genes involved in hepatic xenobiotic pathways in mice

Hepatic proteins involved in xenobiotic pathways (Phases I, II and III) are responsible for the metabolism and disposition of endogenous and exogenous compounds including dietary phytochemicals. To test the hypothesis that elevated alpha-tocopherol intakes alter gene expression of hepatic xenobiotic pathways, mice were fed diets supplemented with either 1000 IU (+E) or 35 IU (E) all-rac-alpha-tocopheryl acetate for 4 months; liver RNA was isolated, and gene expression was determined using both whole genome microarray and real-time quantitative polymerase chain reaction analyses. Hepatic alpha-tocopherol (173+/-18 vs. 21+/-1 nmol/g, mean+/-S.E.) and its metabolite (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman, 0.232+/-0.046 vs. 0.031+/-0.019 nmol/g) concentrations were approximately eightfold higher following the +E dietary treatment. In +E relative to E mice, gene expression of Phase I enzymes, P450 oxidoreductase and cytochrome P450 3a11 increased 1.6- and 4.0-fold, respectively; two Phase II genes, sulfotransferase 2a and glutathione S-transferase mu 3, increased 10.8- and 1.9-fold respectively, and a Phase III biliary transporter, Abcb1a, doubled. Thus, consumption of high-level dietary alpha-tocopherol simultaneously coordinated Phase I, II and III gene expression. These data demonstrate that increased hepatic alpha-tocopherol modulates its own concentrations through increasing xenobiotic metabolism, a process that may alter metabolism of other foreign compounds, such as therapeutic drugs and phytochemicals, in humans.     

In vitro antioxidant activity of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a vitamin E metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of alpha-CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS.+) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, alpha-tocopherol, ascorbic acid, and (-)-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, alpha-CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that alpha-CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.     

Cell calcium, vitamin E, and the thiol redox system in cytotoxicity

The controversial role of extracellular Ca2+ in toxicity to in vitro hepatocyte systems is reviewed. Recent reports demonstrate that extracellular Ca2+-related cytotoxicity is dependent on Ca2+-influenced vitamin E (alpha-tocopherol) content of isolated hepatocytes. Based on a Ca2+-omission model of in vitro oxidative stress, the role of vitamin E in cytotoxicity is further explored. This model demonstrates the interdependence of the GSH redox system and vitamin E as protective agents during oxidative stress. Following chemical oxidant-induced depletion of intracellular GSH, cell morphology and viability are maintained by the continuous presence of cellular alpha-tocopherol above a threshold level of 0.6-1.0 nmol/10(6) cells. alpha-Tocopherol threshold-dependent cell viability is directly correlated with the prevention of the loss of cellular protein thiols in the absence of intracellular GSH. Potential mechanisms for this phenomenon are explored and include a direct reductive action of alpha-tocopherol on protein thiyl radicals, and the prevention of oxidation of protein thiols by scavenging of lipid peroxyl radicals by alpha-tocopherol. It is suggested that in light of the threshold phenomenon of vitamin E prevention of potentially severe oxidative stress-induced cytotoxicity, its use as a protective agent against an oxidative challenge in vivo should be reassessed.     

A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds

There is currently interest in measuring urinary metabolites of vitamin E. It has been suggested that alpha-to-copheronolactone (alphaTL), with an oxidized chroman ring, may be an indicator of in vivo oxidative stress and 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), with a shortened side chain but intact hydroxychroman ring, may provide a measure of adequate or excess vitamin E status. To date, methods in the literature have tended to concentrate on the estimation of single metabolites. We describe the establishment and validation of a relatively simple and reproducible method to extract and quantitate a range of vitamin E metabolites using 0.5 ml of human urine. The vitamin E metabolites were extracted from urine using solid phase extraction cartridges, deconjugated enzymatically, and analyzed using gas chromatography-mass spectrometry. Using this method we have identified alphaTL and the CEHC metabolites derived from alpha-, delta-, and gamma-tocopherol. In addition we have tentatively identified a novel group of vitamin E metabolites, which are related to the CEHCs but have three extra carbons in the side chain. The possibility of the artifactual oxidation of alpha-CEHC to alphaTL during the assay procedure is also discussed.     

Vitamin E protection against chemical-induced cell injury. II. Evidence for a threshold effect of cellular alpha-tocopherol in prevention of adriamycin toxicity

The cardiomyopathy produced by the widely used anticancer drug adriamycin (ADR) is believed to be related to the production of reaction oxygen species and consumption of reduced glutathione (GSH) during redox cycling of the drug. Protection by vitamin E against the toxicity of ADR was studied in a model of compromised isolated hepatocytes, generated by physiological alterations in the concentration of cell calcium. A decrease in cell calcium concentration leads to a greater loss of endogenous alpha-tocopherol and enhances the intracellular hydrolysis of exogenous alpha-tocopheryl esters. With this model, vitamin E (alpha-tocopheryl succinate) at 25 microM protected the calcium-depleted hepatocytes against the toxicity of ADR, in association with greater cellular alpha-tocopherol content as compared to calcium-adequate cells. The incubation of calcium-adequate hepatocytes with increasing concentrations of alpha-tocopheryl succinate up to 200 microM demonstrated that maximal protection by vitamin E was directly dependent on the alpha-tocopherol content of the cells, regardless of the concentration of cell calcium. The viability of the cells was closely associated with the alpha-tocopherol-mediated maintenance of cellular protein thiols. Viability and protein thiol content of the cells were maximal at cellular alpha-tocopherol levels in the range 0.6-1.0 nmol/10(6) cells in both calcium-depleted and -adequate cells. It is suggested that the potential use of vitamin E as a protective agent against ADR toxicity in vivo be reevaluated with an emphasis placed on the threshold level of intracellular alpha-tocopherol in the critical target tissue.     

Alpha-tocopherol modulates Cyp3a expression, increases gamma-CEHC production, and limits tissue gamma-tocopherol accumulation in mice fed high gamma-tocopherol diets

Although all forms of vitamin E are absorbed, the liver preferentially secretes alpha-, but not gamma-tocopherol, into plasma. Liver alpha-tocopherol secretion is under the control of the alpha-tocopherol transfer protein (TTP). Therefore, to assess gamma-tocopherol bioactivities Ttpa-/-, +/- and +/+ mice were fed for 5 weeks diets containing gamma-tocopherol 550 (gamma-T550), gamma-tocopherol 60 (gamma-T60) mg/kg that also contained trace amounts of alpha-tocopherol, a vitamin E-deficient diet, or a control diet. Plasma and tissues from mice fed gamma-T550 diets were found to contain similar gamma- and alpha-tocopherol concentrations despite the high dietary gamma-tocopherol content; nervous tissues contained almost no gamma-tocopherol. Liver vitamin E metabolites (carboxyethyl hydroxychromans, CEHCs) were also measured. In mice with widely ranging liver alpha- (from 0.7 to 16 nmol/g) and gamma-tocopherol concentrations (0 to 13 nmol/g), hepatic alpha-CEHC was undetectable, but gamma-CEHC concentrations (0.1 to 0.8 nmol/g) were correlated with both alpha- and gamma-tocopherol concentrations (P < 0.004). Hepatic cytochrome P450s (CYPs) involved in vitamin E metabolism, Cyp4f and Cyp3a, were also measured. There were no variations in Cyp4f protein expression as related to diet or mouse genotype. However, Cyp3a was correlated (P < 0.0001) with liver alpha-, but not gamma-tocopherol concentrations. These data support the hypothesis that alpha-tocopherol modulates xenobiotic metabolism by increasing Cyp3a expression, gamma-CEHC formation, and the excretion of both gamma-tocopherol and gamma-CEHC.     

Intracellular Transport of Fat‐Soluble Vitamins A and E

Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat‐soluble vitamins. Because of their lipophilicity, fat‐soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid‐binding proteins such as cellular retinol‐binding protein (CRBP), cellular retinoic acid‐binding protein (CRABP) and cellular retinal‐binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP‐mediated intracellular retinoid transport is essential for vision in human. α‐Tocopherol, the main form of vitamin E found in the body, is transported by α‐tocopherol transfer protein (α‐TTP) in hepatic cells. Defects of α‐TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α‐TTP with phosphoinositides plays a critical role in the intracellular transport of α‐tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.      

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Identification of a novel cytosolic tocopherol-binding protein: structure, specificity, and tissue distribution

Alpha-tocopherol plays an important role as a lipid-soluble antioxidant. It is present in all major mammalian cell types and shows tissue-specific distribution. This suggests the presence of specific proteins involved in intracellular distribution or metabolism of alpha-tocopherol. A diminution of tocopherol plasma concentrations contributes to the development of diseases such as vitamin E deficiency (AVED), atherosclerosis, and prostate cancer. Further evidence has been obtained for the existence of sites in cellular metabolism and signal transduction where alpha-tocopherol potentially plays a regulatory role. A signal transduction modulation specific for alpha-tocopherol has been described in several model systems. Using radioactively labeled alpha-tocopherol as tracer, we have isolated a new alpha-tocopherol-associated protein (TAP) from bovine liver. This protein has a molecular mass of 46 kDa and an isoelectric point of 8.1. From its partial amino acid sequence, a human gene has been identified with high homology to the newly described protein. Sequence analysis has established that the new TAP has structural motifs suggesting its belonging to a family of hydrophobic ligand-binding proteins (RALBP, CRALBP, alpha-TTP, SEC 14, PTN 9, RSEC 45). Human TAP has been cloned into Escherichia coli, and its tissue-specific expression has been assessed by Northern blot analysis.     

Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice

Metabolism of vitamin E is initiated by cytochrome P450 (CYP) enzymes usually involved in the metabolism of xenobiotics. Like other CYP substrates, vitamin E induced a reporter gene under the control of the pregnane X receptor (PXR) which regulates the expression of CYPs including CYP3A4. gamma-Tocotrienol, the most effective PXR activator, also induced endogenous CYP3A4 mRNA in HepG2 cells. Since these findings imply an interference of vitamin E with drug metabolism it was deemed necessary to investigate their in vivo relevance. Therefore, mice were grown for 3 months with alpha-tocopherol-deficient, -adequate, and -supranutritional diet, i.e. 2, 20 and 200 mg RRR-alpha-tocopheryl acetate/kg diet, respectively. Half of them received 250 microg gamma-tocotrienol/day for the last 7 days. After 3 months, hepatic levels of Cyp3a11 mRNA, the murine homolog to human CYP3A4, were about 2.5-fold higher in the 20 and 200 mg alpha-tocopherol groups than in the 2 mg group. After feeding 200 mg alpha-tocopherol for 9 months, Cyp3a11 mRNA was 1.7-fold higher than after 3 months. In contrast, gamma-tocotrienol did not induce Cyp3a11 mRNA. This could be explained by its high metabolism as demonstrated by the 20- to 25-fold increase in the urinary excretion of gamma-CEHC, the final metabolite of gamma-tocotrienol degradation. In conclusion, alpha-tocopherol maintains an adequate level of xenobiotic-metabolizing enzymes. If fed in supranutritional dosages, especially for longer times, alpha-tocopherol induces Cyp3a11 to levels which might interfere with drug metabolism.     

New synthesis of (+/-)-alpha-CMBHC and its confirmation as a metabolite of alpha-tocopherol (vitamin E)

There is currently interest in the metabolism of the various compounds which make up the vitamin E family, especially with regards to the possible use of vitamin E metabolites as markers of oxidative stress and adequate vitamin E supply. A number of vitamin E metabolites have been described to date and we have recently developed a method to extract and quantitate a range of vitamin E metabolites in human urine. During the development of this method a new metabolite of alpha-tocopherol was identified, which we tentatively characterised as 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid (alpha-CMBHC).(1) Here we describe the synthesis of alpha-CMBHC as a standard and confirm that it is a metabolite of alpha-tocopherol.     

Vitamin E: underestimated as an antioxidant

Some 80 years after its discovery, vitamin E has experienced a renaissance which is as surprising as it is trivial. Although vitamin E is essential for reproduction, in rats at least, and deficiency causes neurological disorders in humans, the main interest in the last decades has concentrated on its antioxidant functions. This focus has highly underestimated the biological importance of vitamin E, which by far exceeds the need for acting as a radical scavenger. Only recently has it become clear that vitamin E can regulate cellular signaling and gene expression. Out of the eight different tocols included in the term vitamin E, alpha-tocopherol often exerts specific functions, which is also reflected in its selective recognition by proteins such as the alpha-tocopherol transfer protein and alpha-tocopherol-associated proteins. Vitamin E forms other than alpha-tocopherol are very actively metabolised, which explains their low biopotency. In vivo, metabolism may also attenuate the novel functions of gamma-tocopherol and tocotrienols observed in vitro. On the other hand, metabolites derived from individual forms of vitamin E have been shown to exert effects by themselves. This article focuses on the metabolism and novel functions of vitamin E with special emphasis on differential biological activities of individual vitamin E forms.     

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.     

Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma

Alpha- and gamma-tocopherol (alpha- and gamma-T, respectively) metabolite analysis is of key relevance in the study of vitamin E metabolism. Whilst there is information on urinary excretion of the two major metabolites of these vitamin E homologues, namely the 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), their concentration and response to supplements in plasma remains poorly investigated. In this study we describe a gas chromatography-mass spectrometry (GC/MS)-based assay to measure both alpha- and gamma-T and their corresponding CEHC metabolites in human plasma. As an example of the application of this method we report data obtained following the supplemention of two healthy volunteers with 100 mg of deuterium-labeled gamma-T acetate (d(2)-gamma-TAC). Under routine analytical conditions a good linearity in the range 0.0025--1 microM was observed for both the alpha- and gamma-CEHC deuterated standards. In plasma samples, the detection limit for alpha- and gamma-CEHC was 2.5 and 5 nmol/l, respectively. The minimum amount of plasma required for the assay was 500 microl. The plasma concentrations of alpha-CEHC and gamma-CEHC in unsupplemented healthy subjects were 12.6 +/-7.5 and 160.7 +/- 44.9 nmol/l, respectively. In the two volunteers supplemented with 100 mg of d(2)-gamma-TAC, plasma d(2)-gamma-T concentrations increased 250 to 450-fold 6 h postsupplementation. Plasma and urinary d(2)-gamma-CEHC concentrations increased 20 to 40-fold 9--12 h postsupplementation. Interestingly, the acute increase in d(2) gamma-T did not significantly affect the baseline plasma concentrations of d(0)-gamma-T and only slight lowered alpha-T concentrations. Likewise, plasma alpha-CEHC levels were not influenced and urinary excretion of alpha-CEHC were unaltered. This GC/MS method provides a versatile and accurate mean for assessing carboxyethyl-hydroxychroman metabolites of vitamin E in plasma.