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.     

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.     

Vitamin E: function and metabolism.

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

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.     

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.     

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.     

A rapid method for the extraction and determination of vitamin E metabolites in human urine

A method for the direct extraction and routine analysis of the vitamin E metabolites gamma- and alpha-carboxyethyl hydroxychroman (gamma- and alpha-CEHC) from human urine has been developed. A relatively small sample volume (5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms and acidification, the metabolites are extracted with diethyl ether. Recovery of alpha- and gamma-CEHC was compared to that of trolox, used as an internal standard, added to 24-h urine collections from vitamin E-unsupplemented volunteers. Various solvent conditions were initially tested; acidification and ether extraction gave the highest recovery. It was found that after addition and extraction from urine, trolox, alpha- and gamma-CEHC are recovered to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (ECD). HPLC-ECD was found to give higher selectivity and higher sensitivity compared to GC or HPLC with UV detection at 290 nm. The HPLC-ECD detection limit was 10 fmol, linearity (r(2) > 0.98) was achieved in the range of 40 to 200 fmol, which was found to be optimal for 24-h urines from unsupplemented subjects. Inter-sample variability was typically 2-5%. This greater sensitivity and selectivity means that vitamin E metabolites can be analyzed even in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent approximately 10% of the total alpha- and gamma-CEHC. This method can be used routinely for the determination of vitamin E metabolites in urine. The new extraction and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods.     

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.     

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.     

Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status

Postabsorptive elimination of the various forms of vitamin E appears to play a key role in regulation of tissue tocopherol concentrations, but mechanisms of tocopherol metabolism have not been elucidated. Here we describe a pathway involving cytochrome P450-mediated omega-hydroxylation of the tocopherol phytyl side chain followed by stepwise removal of two- or three-carbon moieties, ultimately yielding the 3'-carboxychromanol metabolite that is excreted in urine. All key intermediates of gamma-tocopherol metabolism via this pathway were identified in hepatocyte cultures using gas chromatography-mass spectrometry. NADPH-dependent synthesis of the initial gamma- and alpha-tocopherol 13'-hydroxy and -carboxy metabolites was demonstrated in rat and human liver microsomes. Functional analysis of several recombinant human liver P450 enzymes revealed that tocopherol-omega-hydroxylase activity was associated only with CYP4F2, which also catalyzes omega-hydroxylation of leukotriene B(4) and arachidonic acid. Tocopherol-omega-hydroxylase exhibited similar binding affinities but markedly higher catalytic activities for gamma-tocopherol than alpha-tocopherol, suggesting a role for this pathway in the preferential physiological retention of alpha-tocopherol and elimination of gamma-tocopherol. Sesamin potently inhibited tocopherol-omega-hydroxylase activity exhibited by CYP4F2 and rat or human liver microsomes. Since dietary sesamin also results in elevated tocopherol levels in vivo, this pathway appears to represent a functionally significant means of regulating vitamin E status.     

Pregnenolone and dexamethasone, modulators of cytochrome P450-3A, not increase but reduce urinary alpha-CEHC excretion in rats

In this study, the CYP3A inducer pregnenolone-16alpha-carbonitrile (PCN) and the CYP3A inhibitor ketoconazole (KCZ) were used to investigate whether the metabolism of alpha-tocopherol to its metabolite, alpha-carboxyethyl hydroxychroman (alpha-CEHC), is CYP3A-dependent in rats. In experiment 1, two groups of Wistar rats were fed for 3 wk with either a basal diet (containing 50 ppm of alpha-tocopherol) or the same diet containing 10-fold more alpha-tocopherol. In the last 3 days, each group was divided into 2 subgroups which were given a single i.p. injection of either PCN at 75 mg/kg/d (P50 & P500 groups) or DMSO (D50 & D500 groups). The liver TBARS concentration was highest in the P50 group. Two-way ANOVA analysis showed that alpha-tocopherol levels in the plasma and liver were both significantly decreased by PCN (p < 0.0001), as were alpha-CEHC levels in the urine (p = 0.0004). In experiment 2, alpha-tocopherol levels in the liver were increased and alpha-CEHC excretion in the urine decreased in the Wistar rats fed with KCZ containing diet. In experiment 3, Wistar rats administered with dexamethasone (DEX) significantly decreased alpha-tocopherol levels in the plasma and liver and alpha-CEHC levels in the urine. These data showed CYP3A is not a major contributor of the metabolism of alpha-tocopherol to alpha-CEHC. Nevertheless, vitamin E status was markedly reduced by CYP3A inducers due to increased lipid peroxidation and this would increase the consumption of alpha-tocopherol in the liver.     

Pregnenolone and dexamethasone, modulators of cytochrome P450-3A, not increase but reduce urinary alpha-CEHC excretion in rats

In this study, the CYP3A inducer pregnenolone-16alpha-carbonitrile (PCN) and the CYP3A inhibitor ketoconazole (KCZ) were used to investigate whether the metabolism of alpha-tocopherol to its metabolite, alpha-carboxyethyl hydroxychroman (alpha-CEHC), is CYP3A-dependent in rats. In experiment 1, two groups of Wistar rats were fed for 3 wk with either a basal diet (containing 50~ppm of alpha-tocopherol) or the same diet containing 10-fold more alpha-tocopherol. In the last 3 days, each group was divided into 2 subgroups which were given a single i.p. injection of either PCN at 75 mg/kg/d (P50 & P500 groups) or DMSO (D50 & D500 groups). The liver TBARS concentration was highest in the P50 group. Two-way ANOVA analysis showed that alpha-tocopherol levels in the plasma and liver were both significantly decreased by PCN (p < 0.0001), as were alpha-CEHC levels in the urine (p = 0.0004). In experiment 2, alpha-tocopherol levels in the liver were increased and alpha-CEHC excretion in the urine decreased in the Wistar rats fed with KCZ containing diet. In experiment 3, Wistar rats administered with dexamethasone (DEX) significantly decreased alpha-tocopherol levels in the plasma and liver and alpha-CEHC levels in the urine. These data showed CYP3A is not a major contributor of the metabolism of alpha-tocopherol to alpha-CEHC. Nevertheless, vitamin E status was markedly reduced by CYP3A inducers due to increased lipid peroxidation and this would increase the consumption of alpha-tocopherol in the liver.     

Complexity of vitamin E metabolism

Bioavailability of vitamin E is influenced by several factors, most are highlighted in this review. While gender, age and genetic constitution influence vitamin E bioavailability but cannot be modified, life-style and intake of vitamin E can be. Numerous factors must be taken into account however, i.e., when vitamin E is orally administrated, the food matrix may contain competing nutrients. The complex metabolic processes comprise intestinal absorption, vascular transport, hepatic sorting by intracellular binding proteins, such as the significant α-tocopherol-transfer protein, and hepatic metabolism. The coordinated changes involved in the hepatic metabolism of vitamin E provide an effective physiological pathway to protect tissues against the excessive accumulation of, in particular, non-α-tocopherol forms. Metabolism of vitamin E begins with one cycle of CYP4F2/CYP3A4-dependent ω-hydroxylation followed by five cycles of subsequent β-oxidation, and forms the water-soluble end-product carboxyethylhydroxychroman. All known hepatic metabolites can be conjugated and are excreted, depending on the length of their sidechain, either via urine or feces. The physiological handling of vitamin E underlies kinetics which vary between the different vitamin E forms. Here, saturation of the side-chain and also substitution of the chromanol ring system are important. Most of the metabolic reactions and processes that are involved with vitamin E are also shared by other fat soluble vitamins. Influencing interactions with other nutrients such as vitamin K or pharmaceuticals are also covered by this review. All these processes modulate the formation of vitamin E metabolites and their concentrations in tissues and body fluids. Differences in metabolism might be responsible for the discrepancies that have been observed in studies performed in vivo and in vitro using vitamin E as a supplement or nutrient. To evaluate individual vitamin E status, the analytical procedures used for detecting and quantifying vitamin E and its metabolites are crucial. The latest methods in analytics are presented.     

Inhibition of monosodium urate monohydrate-mediated hemolysis by vitamin E

Microcrystals of monosodium urate monohydrate(MSUM)induce cytolysis and hemolysis inerythrocytes.In this report,we studied the effect of vitamin E on MSUM-mediated hemolysis in humanerythrocytes.Vitamin E significantly inhibited hemolysis induced by MSUM.The hydroxyl group in thechromanol ring of vitamin E is dispensable for protecting erythrocytes against hemolysis induced by MSUM,indicating that the inhibitory effect of vitamin E is not due to its antioxidant properties.However,both thechromanol ring and the isoprenoid side chain are important for vitamin E to suppress MSUM-induced hemolysis.Our current study suggests that vitamin E inhibits hemolysis induced by MSUM as a membrane stabilizer.     

Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 microM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of gamma- and alpha-tocopherol in rat primary hepatocytes, and metabolism of gamma- and delta-tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 microM. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.     

Optimization of the enzymatic hydrolysis and analysis of plasma conjugated γ-CEHC and sulfated long-chain carboxychromanols, metabolites of vitamin E

Natural forms of vitamin E are metabolized by ω-hydroxylation and β-oxidation of the hydrophobic side chain to generate urinary-excreted 2-(β-carboxyethyl)-6-hydroxychroman (CEHC) and CEHC conjugates (sulfate, glucuronide, or glucoside). We recently showed that sulfated long-chain carboxychromanols, the conjugated intermediate β-oxidation products, are formed from tocopherols and tocotrienols in human cells and in rats. CEHC conjugates have been quantified after being converted to its unconjugated counterpart by sulfatase/glucuronidase. Although the enzymatic hydrolysis is critical for appropriate quantification of conjugated CEHC, it is not clear whether brief incubation of the plasma with sulfatases/glucuronidases results in complete deconjugation of conjugated CEHC. Here we show that quantitative hydrolysis of the conjugated vitamin E metabolites in the plasma requires an extraction procedure using methanol/hexane (2 ml/5 ml) and an overnight sulfatase/glucuronidase hydrolysis. Using this procedure, we demonstrate that conjugated γ-CEHC and some sulfated long-chain carboxychromanols are fully deconjugated. In contrast, direct enzymatic hydrolysis of the whole plasma underestimates the conjugated metabolites by at least threefold. This protocol may be also useful for the analysis of other conjugated phenolic compounds in complicated biological matrices such as plasma.     

Anti-inflammatory effects of tocopherol metabolites

Our objective was to assess the anti-inflammatory effects of alpha-tocopherol, gamma-tocopherol, and their metabolites 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC) in defined cell culture systems. Rat aortic endothelial cells and mouse microglial cultures were treated with tumor necrosis factor TNFalpha or bacterial lipopolysaccharide (LPS) and nitrite and prostaglandin E(2) (PGE(2)) were measured. alpha-CEHC suppressed TNFalpha-stimulated nitrite production in both cell types, whereas both CEHC derivatives inhibited LPS-stimulated microglial nitrite efflux. Both alpha-CEHC and gamma-CEHC inhibited microglial PGE(2) production, but neither alpha- nor gamma-tocopherol was effective at inhibiting cytokine-stimulated inflammatory processes. These results show that the anti-inflammatory effects of tocopherols are highly cell type-, stimulus-, and endpoint-dependent.     

Synthesis of putative metabolites of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is under phase III clinical trials in Japan for the treatment of osteoporosis and bone fracture prevention. Since ED-71 has a substituent at the 2beta-position of the A-ring, it is recognized that the metabolic pathway of ED-71 might be more complicated than 1,25(OH)(2)D(3) because of metabolism at the 2beta-position substituent in addition to the inherent metabolism of the side chain. To clarify the metabolism of hydroxypropoxy substituent of the 2beta-positon and a combination of metabolism between side chain and 2beta-positon, four putative metabolites of ED-71 have been prepared as authentic samples. The metabolites at the 2beta-positon, the methyl ester derivative considered as an ester standard of the oxidized metabolite and the tetraol derivative as the truncated metabolite were synthesized from alpha-epoxide, a key intermediate of ED-71 synthesis. The combination metabolites between side chain and 2beta-positon, the 24(S)- and 24(R)-pentaols were synthesized using Trost's convergent method.     

Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13'-hydroxychromanol and long-chain carboxychromanols. Conjugated (including sulfated) metabolites were reported previously but were limited to CEHCs. Here, using electrospray and inductively coupled plasma mass spectrometry, we discovered that gamma-tocopherol (gamma-T) and delta-T were metabolized to sulfated 9'-, 11'-, and 13'-carboxychromanol (9'S, 11'S, and 13'S) in human A549 cells. To further study the metabolites, we developed a HPLC assay with fluorescence detection that simultaneously analyzes sulfated and nonconjugated intermediate metabolites. Using this assay, we found that sulfated metabolites were converted to nonconjugated carboxychromanols by sulfatase digestion. In cultured cells, approximately 45% long-chain carboxychromanols from gamma-T but only 10% from delta-T were sulfated. Upon supplementation with gamma-T, rats had increased tissue levels of 9'S, 11'S, and 13'S, 13'-hydroxychromanol, 13'-carboxychromanol, and gamma-CEHC. The plasma concentrations of combined sulfated long-chain metabolites were comparable to or exceeded those of CEHCs and increased proportionally with the supplement dosages of gamma-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with beta-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.