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...

Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13'-COOH and δTE-13'-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygnease (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anti-cancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosin and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOHinduced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define “optimal” vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid–liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9–111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 molecules in biological samples.

the corresponding tocotrienols ( ␣ TE, ␤ TE, ␥ TE, and ␦ TE) ( Fig. 1 ). Among the members of the vitamin E family, ␣ T is the predominant form in tissues and is preferentially bound to a tocopherol transport protein that prevents ␣ T from being extensively metabolized ( 1 ). In contrast, other vitamin E forms are largely metabolized in the liver via -hydroxylation and oxidation to generate 13 ′ -hydroxychromanol (13 ′ -OH) and 13 ′ -carboxychromanol (13 ′ -COOH), the latter of which is subsequently metabolized via ␤ -oxidation to various shorter-chain carboxychromanols including the terminal metabolite observed in urine, 3 ′ -COOH [or 2-( ␤ -carboxyethyl)-6-hydroxychroman (CEHC)] ( Fig. 2 ) ( 1-3 ). In parallel with ␤ -oxidation, long-chain carboxychromanols and CEHCs appear to be conjugated via sulfation to form conjugated carboxychromanols ( Fig. 2 ) ( 4,5 ). The terminal metabolite CEHCs and their conjugated forms are found in the urine ( 6-11 ). On the other hand, unconjugated short-, mid-, and long-chain carboxychromanols have recently been detected in feces after supplementation of ␥ T or ␦ T ( 12-14 ).Although research on vitamin E has predominantly focused on tocopherols and tocotrienols, their metabolites have been shown to have biological activities that are stronger than or differ from the unmetabolized vitamins. For instance, ␥ -CEHC has been reported to have natriuratic activity in vitro and in animals ( 15 ). Recent studies including ours have demonstrated that long-chain carboxychromanols appear to have unique anti-infl ammatory effects by inhibition of cyclooxygenases and 5-lipoxygenase and induced apoptosis in cancer cells (16)(17)(18). For these activities, long-chain carboxychromanols are much stronger than tocopherols or shorter-chain carboxychromanols. Despite these interesting fi ndings of vitamin E metabolites, Abstract Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13 ′ -hydroxychromanols (13 ′ -OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13 ′ -carboxychromanol (13 ′ -COOH), appear to be more bioactive than tocopherols in anti-infl ammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with effi cacy of Ն 89% and interday/intraday variation of 3-11% for major metabolites. To ensure accurate quantifi cation, short-chain, long-chain, and sulfated carboxychromanols are included as external/ internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with ␥ -tocopherol or ␦ -tocopherol. Although plasma levels of 13...

Inflammation can promote colon cancer. Mechanistic studies indicate that γ-tocopherol (γT), a major form of vitamin E in diets, has anti-inflammatory and anticancer properties. Here we investigated the effectiveness of γT and a mixture of tocopherols against colitis and colitis-promoted colon tumorigenesis in male BALB/c mice. γT or mixed tocopherols (at 0.1% diet) did not show any effect on colon tumorigenesis induced by azoxymethane (AOM, 10mg/kg) with three cycles of dextran sodium sulfate (DSS at 1.5–2.5%). γT failed to exhibit protection of severe colitis caused by three cycles of DSS at 2.5%. In contrast, when AOM-initiated carcinogenesis was promoted by relatively mild colitis induced by one-cycle DSS (1.5%), γT, but not mixed tocopherols, suppressed total multiplicity of macroscopic adenomas (P=0.06) and large adenomatous polyps (>2mm2, P<0.05) by 60% and 85%, respectively. γT also significantly decreased tumor multiplicity (>2mm2) induced by AOM with two cycles of 1.5% DSS even when dietary supplementation was started after AOM injection. Consistently, γT but not mixed tocopherols attenuated DSS (1.5%)-induced colon inflammation and damage as well as formation of atypical glandular hyperplasia. Mice supplemented with tocopherols had high fecal excretion of 13′-carboxychromanol, a long-chain vitamin E metabolite shown to have potent anti-inflammatory activities. Our study demonstrates that γT is able to alleviate moderate but not severe colitis and its promoted tumorigenesis, and indicates that inflammation severity should be considered in evaluating anticancer effectiveness of chemoprevention agents.