Cyclooxygenase (COX-1/COX-2)-catalyzed eicosanoid formation plays a key role in inflammation-associated diseases. Natural forms of vitamin E are recently shown to be metabolized to long-chain carboxychromanols and their sulfated counterparts. Here we find that vitamin E forms differentially inhibit COX-2-catalyzed prostaglandin E 2 in IL-1␤-stimulated A549 cells without affecting COX-2 expression, showing the relative potency of ␥-tocotrienol Ϸ ␦-tocopherol > ␥-tocopherol Ͼ Ͼ ␣-or ␤-tocopherol. The cellular inhibition is partially diminished by sesamin, which blocks the metabolism of vitamin E, suggesting that their metabolites may be inhibitory. Consistently, conditioned media enriched with long-chain carboxychromanols, but not their sulfated counterparts or vitamin E, reduce COX-2 activity in COX-preinduced cells with 5 M arachidonic acid as substrate. Under this condition, 9-or 13-carboxychromanol, the vitamin E metabolites that contain a chromanol linked with a 9-or 13-carbon-length carboxylated side chain, inhibits COX-2 with an IC 50 of 6 or 4 M, respectively. But 13-carboxychromanol inhibits purified COX-1 and COX-2 much more potently than shorter side-chain analogs or vitamin E forms by competitively inhibiting their cyclooxygenase activity with K i of 3.9 and 10.7 M, respectively, without affecting the peroxidase activity. Computer simulation consistently indicates that 13-carboxychromanol binds more strongly than 9-carboxychromanol to the substrate-binding site of COX-1. Therefore, long-chain carboxychromanols, including 13-carboxychromanol, are novel cyclooxygenase inhibitors, may serve as anti-inflammation and anticancer agents, and may contribute to the beneficial effects of certain forms of vitamin E.cancer ͉ inflammation ͉ PGE2 ͉ tocopherol ͉ tocotrienol C yclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ), the common precursor to prostaglandins and thromboxanes that are important lipid mediators for regulation of many physiological and pathophysiological responses (1). COXs are bifunctional enzymes that carry out two sequential activities-i.e., the cyclooxygenase activity, which leads to the formation of prostaglandin G 2 (PGG 2 ), and the peroxidase activity, which reduces PGG 2 to PGH 2 (2). COX-1 is constitutively expressed in many tissues, including platelets where thromboxanes are generated by this enzyme to promote platelet aggregation. COX-2 is often induced under acute/chronic inflammatory conditions and is mainly responsible for the generation of proinflammatory eicosanoids, including prostaglandin E 2 (PGE 2 ) (3). COX inhibitors, which are nonsteroidal anti-inflammatory drugs (NSAIDs), have been used for the relief of fever, pain, and inflammation (4). Chronic inflammation has been identified as a significant factor in the development of cancer (5). It is well established that NSAIDs are effective chemoprevention agents for cancer (6), although their long-term use has been questioned due to the associated gastrointestinal sid...

Although cell-based studies have shown that c-tocotrienol (cTE) exhibits stronger anticancer activities than other forms of vitamin E including c-tocopherol (cT), the molecular bases underlying cTE-exerted effects remains to be elucidated. Here we showed that cTE treatment promoted apoptosis, necrosis and autophagy in human prostate PC-3 and LNCaP cancer cells. In search of potential mechanisms of cTE-provoked effects, we found that cTE treatment led to marked increase of intracellular dihydroceramide and dihydrosphingosine, the sphingolipid intermediates in de novo sphingolipid synthesis pathway but had no effects on ceramide or sphingosine. The elevation of these sphingolipids by cTE preceded or coincided with biochemical and morphological signs of cell death and was much more pronounced than that induced by cT, which accompanied with much higher cellular uptake of cTE than cT. The importance of sphingolipid accumulation in cTE-caused fatality was underscored by the observation that dihydrosphingosine and dihydroceramide potently reduced the viability of both prostate cell lines and LNCaP cells, respectively. In addition, myriosin, a specific inhibitor of de novo sphingolipid synthesis, counteracted cTE-induced cell death. In agreement with these cell-based studies, cTE inhibited LNCaP xenograft growth by 53% (p < 0.05), compared to 33% (p 5 0.07) by cT, in nude mice. These findings provide a molecular basis of cTE-stimulated cancer cell death and support the notion that elevation of intracellular dihydroceramide and dihydrosphingosine is likely a novel anticancer mechanism.Vitamin E has eight lipophilic antioxidants, that is, a-, b-, cand d-tocopherol (aT, bT, cT and dT) and a-, b-, c-and dtocotrienol (aTE, bTE, cTE and dTE). Specific forms of vitamin E have been proposed to be promising anticancer agents.

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(b-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13 ¶-hydroxychromanol and longchain 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 g-tocopherol (g-T) and d-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, ?45% long-chain carboxychromanols from g-T but only 10% from d-T were sulfated. Upon supplementation with g-T, rats had increased tissue levels of 9 ¶S, 11 ¶S, and 13 ¶S, 13 ¶-hydroxychromanol, 13 ¶-carboxychromanol, and g-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 g-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with b-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.-Jiang, Q., H. Freiser, K. V. Wood, and X. Yin. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats. J. Lipid Res. or y-T) and four corresponding tocotrienols] (Scheme 1). All of these molecules have a chromanol ring and a 16 carbon phytyl chain, which are responsible for the potent antioxidant activity and lipophilic property, respectively. Despite structural similarity, different vitamin E forms appear to have distinct bioactivity and to be distinctively metabolized (1, 2). a-T is the predominant vitamin E form in tissues and is the least catabolized. On the other hand, some portions of non-a forms are likely to be readily metabolized and/or excreted directly via the bile (3), because they do not appear to be accumulated to the same extent as a-T in most tissues. To elucidate the potential biological functions of each vitamin E form, it is important to understand their metabolism and to identify the major metabolites in the body.The understanding of how vitamin E is metabolized was initiated by the identification of their urine-excreted metabolites. The water-soluble metabolite of y-T, y-7,8-dimethyl-2-(b-carboxyethyl)-6-hydroxychroman (CEHC), was first found in rat urine (4) (Scheme 1). Subsequently, g-CEHC and a-CEHC were identified to be the major urine-excreted metabolites of g-T and a-T, respectively (5-9). g-CEHC has been shown to have natriuretic activity (9) and to inhibit t...

The metabolism of gamma-tocotrienol (gamma-TE) and gamma-tocopherol (gamma-T) was investigated in human A549 cells and in rats. Similar to gamma-T, A549 cells metabolized gamma-TE to sulfated 9'-, 11'-, and 13'-carboxychromanol and their unconjugated counterparts. After 72-h incubation with the cells, 90% of long-chain carboxychromanols in the culture media from gamma-TE, but <45% from gamma-T, were in the sulfated form. The formation of these metabolites was further investigated in rats gavaged by gamma-TE at 10 or 50 mg/kg, gamma-T at 10 mg/kg, or tocopherol-stripped corn oil in controls. Six hours after a single dosing, the supplemented rats had increased plasma concentrations of 13'-carboxychromanol and sulfated 9'-, 11'-, 13'-carboxychromanol, whereas none of these metabolites were detectable in the controls. Sulfated 11'-carboxychromanol was the most abundant long-chain metabolite in gamma-TE-supplemented rats. Sulfatase/glucuronidase hydrolysis revealed for the first time that >88% 2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), the terminal beta-oxidation metabolite, was in the conjugated form in the plasma. In all groups, conjugated gamma-CEHC accounted for >75% of total metabolites, whereas free CEHC was a minor metabolite. At 10 mg/kg, the plasma concentrations of total metabolites from gamma-TE-supplemented rats were higher (P < 0.05) than those from gamma-T-fed rats. These results demonstrate that in rats, conjugation such as sulfation occurs parallel to beta-oxidation in the liver and is quantitatively important to vitamin E metabolism. Conjugated long-chain carboxychromanols may be novel excreted metabolites during supplementation. Our data also provide in vivo evidence that gamma-TE is more extensively metabolized than gamma-T.

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 have recently shown 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 un-conjugated 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 de-conjugation 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 (2ml/5mL) 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, three fold. This protocol may be useful for the analysis of other conjugated phenolic compounds in complicated biological matrixes like plasma.