Carboxyethyl Hydroxychroman Research Articles

Metabolism of natural forms of vitamin E and biological actions of vitamin E metabolites

Natural forms of vitamin E comprise four tocopherols and four tocotrienols. During the last twenty years, there have been breakthroughs in our understanding of vitamin E metabolism and biological activities of vitamin E metabolites. Research has established that tocopherols and tocotrienols are metabolized via ω-hydroxylase (CYP4F2)-initiated side chain oxidation to form 13′-hydroxychromanol and 13′-carobyxychromanol (13′-COOH). 13′-COOHs are further metabolized via β-oxidation and sulfation to intermediate carboxychromanols, terminal metabolite carboxyethyl-hydroxychroman (CEHC), and sulfated analogs. Animal and human studies show that γ-, δ-tocopherol and tocotrienols are more extensively metabolized than α-tocopherol (αT), as indicated by higher formation of CEHCs and 13′-COOHs from non-αT forms than those from αT. 13′-COOHs are shown to be inhibitors of cyclooxygenase-1/-2 and 5-lipoxygenase and much stronger than CEHCs for these activities. 13′-COOHs inhibit cancer cell growth, modulate cellular lipids and activate peroxisome proliferator-activated receptor-γ and pregnane X receptor. Consistent with mechanistic findings, αT-13′-COOH or δTE-13′-COOH, respective metabolites of αT or δ-tocotrienol, show anti-inflammatory and cancer-preventive effects, modulates the gut microbiota and prevents β-amyloid formation in mice. Therefore, 13′-COOHs are a new class of bioactive compounds with anti-inflammatory and anti-cancer activities and potentially capable of modulating lipid and drug metabolism. Based on the existing evidence, this author proposes that metabolites may contribute to disease-preventing effects of γ-, δ-tocopherol and tocotrienols. The role of metabolites in αT's actions may be somewhat limited considering controlled metabolism of αT because of its association with tocopherol-transport protein and less catabolism by CYP4F2 than other vitamin E forms.

Tocopherols and Tocotrienols Are Bioavailable in Rats and Primarily Excreted in Feces as the Intact Forms and 13'-Carboxychromanol Metabolites

Vitamin E α-, γ-, or δ-tocopherol (αT, γT, δT) and γ- or δ-tocotrienol (γTE, δTE) are metabolized to hydroxychromanols and carboxychromanols including 13'-carboxychromanol (13'-COOH), 11'-COOH, and carboxyethyl hydroxychroman (CEHC), some of which have unique bioactivities compared with the vitamers. However, the bioavailability of these metabolites has not been well characterized. We investigated the pharmacokinetics (PK) of vitamin E forms and metabolites in rats. Six-week-old male Wistar rats received 1-time gavage of γT-rich tocopherols (50 mg/kg) containing γT/δT/αT (57.7%, 21.9%, and 10.9%, respectively) or δTE-rich tocotrienols (35 mg/kg) containing δTE/γTE (8:1). We quantified the time course of vitamin E forms and metabolites in the plasma and their 24-h excretion to the urine and feces. The general linear model repeated measure was used for analyses of the PK data. In the rats' plasma, Cmax of γT or δTE was 25.6±9.1 μM (Tmax=4 h) or 16.0±2.3 μM (Tmax=2 h), respectively, and sulfated CEHCs and sulfated 11'-COOHs were the predominant metabolites with Cmax of 0.4-0.5 μM (Tmax ∼5-7 h) or ∼0.3 μM (Tmax at 4.7 h), respectively. In 24-h urine, 2.7% of γT and 0.7% of δTE were excreted as conjugated CEHCs. In the feces, 17-45% of supplemented vitamers were excreted as unmetabolized forms and 4.9-9.2% as unconjugated carboxychromanols, among which 13'-COOHs constituted ∼50% of total metabolites and the amount of δTE-derived 13'-COOHs was double that of 13'-COOH derived from γT. PK data of vitamin E forms in rats reveal that γT, δT, γTE, and δTE are bioavailable in the plasma and are mainly excreted as unmetabolized forms and long-chain metabolites including 13'-COOHs in feces, with more metabolites from tocotrienols than from tocopherols.

γ−Carboxyethyl hydroxychroman, a metabolite of γ−tocopherol, preserves nitric oxide bioavailability in endothelial cells challenged with high glucose

Endothelial dysfunction occurs when there are imbalances between factors that regulate the synthesis and degradation of nitric oxide (NO•), and has been reported in patients with hyperglycemia and insulin resistance. We reported that supplementation with γ-tocopherol (γ-T) in humans limits impairments in endothelial function otherwise induced by postprandial hyperglycemia. Given the rapid metabolism of γ-T into γ-carboxyethyl hydroxychroman (γ-CEHC), we hypothesized that the vasoprotective activities of γ-T could be attributed to its metabolite γ-CEHC. To test this, human aortic endothelial cells (HAECs) treated with 0 (vehicle control) or 3 µM γ-CEHC for 24 h prior to incubation with normal (5 mM) or high (25 mM) glucose for 48 h. High-glucose increased levels of uncoupled endothelial nitric oxide synthase (eNOS) as evidenced by reduced ( p < 0.05) eNOS dimer:monomer. High glucose also prevented insulin-stimulated increases in p-AktSer473: total Akt, p-eNOSSer1177: total eNOS, and NO• production. These adverse changes were accompanied by increased ( p < 0.05) reactive oxygen species and mRNA expression of inflammatory mediators (VCAM-1, E-selectin, IL-8). However, each deleterious response evoked by high glucose was prevented when HAECs were incubated with γ-CEHC prior to the high glucose challenge. Taken together, our data support the hypothesis that vascular protection provided by γ-T invivo may be elicited through the bioactivity of its metabolite, γ-CEHC. Furthermore, it is possible that the antioxidant and anti-inflammatory activities of γ-CEHC may mediate this protective activity.

Abstract 2814: Inhibition of PhIP/DSS-induced colon carcinogenesis by different forms of tocopherols in CYP1A-humanized mice

Abstract Tocopherols (T), which exist as α-, β-, γ-, and δ-T are important dietary antioxidants. Our previous studies have demonstrated that a mixture of tocopherols (γ-TmT), δ-T, and γ-T effectively inhibited colon carcinogenesis in AOM/DSS-treated mice or AOM-treated rats. However, the effects of different forms of tocopherols on carcinogenesis in animal models and in humans remain controversial. In the present study, we investigated the inhibitory effects of α-T, γ-T, and δ-T on colon carcinogenesis induced by a dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and promoted by dextran sodium sulfate (DSS) in CYP1A-humanized (hCYP1A) mice. At 5-6 weeks age, male hCYP1A mice were fed the AIN93M diet or diet supplemented with 0.2% α-T, γ-T, or δ-T for 1 week before PhIP treatment (2x 100mg/kg b.w., 3 days apart), which was followed by DSS (1.5% in drinking water for 4 days) treatment. The mice were sacrificed 10 weeks after the first dose of PhIP. Significant inhibition of colon tumor incidences was caused by dietary δ-T and γ-T, but not by α-T. In addition, oxidative and nitrosative stress markers (i.e., 8-oxo-dG and nitrotyrosine) as well as pro-inflammatory stress markers (i.e., PGE2, LTB4, NFκB and p-STAT3) were significantly reduced by δ-T. Dietary treatment with δ-T and γ-T significantly increased the δ-T and γ-T levels, respectively, in the blood and tissues, but their levels were still lower than the levels of α-T. However, the levels of their side-chain degradation metabolites (δ-T and γ-T forms of carboxyethyl hydroxychroman and carboxymethylbutyl hydroxychroman) were much higher than their parent tocopherols in blood and tissues. Altogether, we demonstrated the inhibitory effects of δ-T and γ-T in a dietary carcinogen-induced colon carcinogenesis model. The inhibition is possibly due to the antioxidative, reactive nitrogen species-trapping, and anti-inflammatory activities δ-T and γ-T. (Supported by NIH grants F31CA168333, RO1CA120915 and RO1AT007036 as well as shared facilities funded by CA72720 and ES05022) Citation Format: Chung S. Yang, Jayson X. Chen, Anna B. Liu, Hong Wang, Marlon Lee, Siyuan Yu, Chunfang Zhao, Ying-Yi Kuo, Eric Chi, Nanjoo Suh. Inhibition of PhIP/DSS-induced colon carcinogenesis by different forms of tocopherols in CYP1A-humanized mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2814. doi:10.1158/1538-7445.AM2015-2814

Changes in the concentrations of vitamin E analogs and their metabolites in rat liver and kidney after oral administration.

Vitamin E analog, such as α- and γ-tocopherol, can undergo ω-oxidation without cleavage of the chroman ring, and this pathway is responsible for generation of the major urinary vitamin E metabolite, carboxyethyl hydroxychroman. However, it is still unclear how carboxyethyl hydroxychroman is changed in various tissues after vitamin E intake. We therefore investigated changes in the concentrations of α- and γ-tocopherol and their metabolites in rat liver and kidney. The concentration of α-tocopherol in rat liver increased until 6 h after oral administration, and then decreased. The change in the concentration of α-carboxyethyl hydroxychroman in rat liver in the α-Toc group slowly increased until 12 h after oral administration. Cytochrome P450 3A1 mRNA expression significantly increased from 12 h after the start of α-tocopherol administration. The change in the concentration of γ-carboxyethyl hydroxychroman in rat liver in the γ-Toc group markedly increased until 12 h after oral administration. On the other hand, γ-carboxyethyl hydroxychroman in rat kidney showed greater accumulation than α-carboxyethyl hydroxychroman from 3 h to 24 h after oral administration. From these results, we considered that γ-carboxyethyl hydroxychroman formed in the liver continues to be released into the bloodstream and is transported to the kidney rapidly.

The antioxidant and anti-inflammatory activities of tocopherols are independent of Nrf2 in mice

The present study investigated the antioxidant and anti-inflammatory actions of tocopherols in mice and determined whether the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is involved in these activities. A mixture of tocopherols (γ-TmT) that is rich in γ-tocopherol was used. Nrf2 knockout (Nrf2 -/-) and wild-type mice were maintained on 0.03, 0.1, or 0.3% γ-TmT-enriched diet starting 2weeks before the administration of dextran sulfate sodium (DSS) in drinking water (for 1week, to induce colonic inflammation), until the termination of the experiment at 3days after the DSS treatment. Dietary γ-TmT dose dependently lowered the levels of 8-oxo-deoxyguanosine, nitrotyrosine, inflammation index, and leukocyte infiltration in colon tissues, as well as 8-isoprostane and prostaglandin E2 in the serum, in both Nrf2 (-/-) and wild-type mice. No significant difference on the inhibitory actions of γ-TmT between the Nrf2 (-/-) and the wild-type mice was observed. The γ-TmT treatment significantly increased the serum levels of γ- and δ-tocopherols. Interestingly, the serum levels of tocopherol metabolites, specifically the γ- and δ-forms of carboxymethylbutyl hydroxychroman and carboxyethyl hydroxychroman, in Nrf2 (-/-) mice were significantly higher than those in wild-type mice. These findings suggest that the antioxidant and anti-inflammatory activities of γ-TmT in the colon are mostly due to the direct action of tocopherols in trapping reactive oxygen and nitrogen species, independent of the antioxidant enzymes and anti-inflammatory proteins that are regulated by Nrf2; however, Nrf2 knockout appears to affect the serum levels of tocopherol metabolites.

Effects of tocopherols and 2,2′-carboxyethyl hydroxychromans on phorbol-ester-stimulated neutrophils

Tocopherol vitamers [e.g., alpha-, gamma- and delta-tocopherol (α-TOC, γ-TOC and δ-TOC, respectively)] and their water-soluble 2,2′-carboxyethyl hydroxychroman metabolites (e.g., α-, γ- and δ-CEHC) all possess antioxidant properties. As a consequence, and similarly to other natural antioxidants, vitamin E compounds may be useful in preventing inflammatory and oxidative-stress-mediated diseases. In this study, we investigated the concentration-dependent effect of tocopherols and water-soluble metabolites on a key event in oxidative stress, for example, the oxidative burst in neutrophils. It was found that not only α-TOC but also γ-TOC and δ-TOC as well as α-, γ- and δ-CEHC at physiological concentrations inhibit superoxide anion (O 2 •−) production in phorbol-ester-stimulated neutrophils. This effect was mediated by the inhibition of the translocation and activation of protein kinase C (PKC) enzyme, which is the key event in the phorbol-ester signaling. Importantly, CEHCs were stronger inhibitors of PKC as compared with the vitamer precursors, and the gamma forms of both tocopherol and CEHC showed the highest inhibitory activities. Tocopherols, but not CEHCs, directly inhibit the fully activated nicotine–adenine–dinucleotide phosphate (NADPH) oxidase. However, none of the test compounds was able to directly scavenge O 2 •− when tested in a cell-free system. In conclusion, vitamin E compounds can control the neutrophil oxidative burst through the negative modulation of PKC-related signaling and NADPH oxidase activity. As an original finding, we observed that CEHC metabolites might contribute to regulate PKC activity in these cells. These results may have important implications in the anti-inflammatory and antioxidant role of vitamin E compounds.

Dietary flavonoids with a catechol structure increase α-tocopherol in rats and protect the vitamin from oxidation in vitro

To identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (-)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of alpha-tocopherol (alpha-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of alpha-T, the capacities of the flavonoids to i) protect alpha-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce alpha-tocopheroxyl radicals (alpha-T (.) ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected alpha-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of alpha-T (.) . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the alpha-T (.) by the flavonoids.

The Effect of Age on Vitamin E Status, Metabolism, and Function: Metabolism as Assessed by Labeled Tocopherols

The effects of age on vitamin E metabolism were studied in 97 healthy 20-75-year-old male nonsmoking Austrian volunteers of the VITAGE project. After a single oral intake of 30 mg d(6)-RRR-alpha- and d(2)-RRR-gamma-tocopheryl acetate, blood and 24-hour urine was collected. Deuterated tocopherols in plasma and deuterated urinary metabolites were analyzed by GC-MS. A first evaluation revealed a similar uptake of d(6)-alpha- and d(2)-gamma-tocopherol during the first 6 hours, and then d(2)-gamma-tocopherol started to decrease. Urinary d(2)-gamma- carboxyethyl hydroxychroman metabolites (CEHCs) exceeded those of d(6)-alpha-CEHCs by about 10 times. There was no effect of age. Thus, there might be no need for a higher vitamin E intake for healthy elderly nonsmoking men.

The effect of α- and γ-tocopherol and their carboxyethyl hydroxychroman metabolites on prostate cancer cell proliferation

It is known that γ-tocopherol inhibits human prostate cancer cell proliferation via down-regulation of cyclin-related signalling but tocopherol and tocotrienol metabolites with a shortened phytyl chain, carboxyethyl hydroxychromans, were not previously investigated as anti-proliferative agents. In this study, the effect of the two main tocopherols, namely, α-tocopherol and γ-tocopherol, and their corresponding metabolites (α- and γ-carboxyethyl hydroxychromans) was studied on proliferation and cyclin D1 expression of the prostate cancer cell line PC-3. The hydrosoluble vitamin E analogues Trolox and α-tocopherol succinate were also tested. The most effective inhibitors of PC-3 proliferation were γ-tocopherol and γ-carboxyethyl hydroxychroman. Their effect was discernable at 1 μM and reached a plateau at concentrations ⩾10 μM with maximal inhibition values ranging between 70 and 82%. α-Tocopherol, α-carboxyethyl hydroxychroman, and the analogue Trolox were much less effective; a weak effect was observed for concentrations ⩽10 μM and a maximal inhibition of less than 45% was found at 50 μM concentration. PC-3 cells showed higher inhibition, particularly by the γ derivatives, than HTB-82 and HECV cells. Tocopherols and carboxyethyl hydroxychromans exerted an inhibitory effect on cyclin D1 expression parallel to the retardation of cell growth. γ-Carboxyethyl hydroxychroman and γ-tocopherol showed effects also upstream of the cyclin modulation. Furthermore, the inhibition of cyclin D1 expression by γ-carboxyethyl hydroxychroman was competed for by α-carboxyethyl hydroxychroman. In conclusion, this study shows that carboxyethyl hydroxychroman metabolites are as effective as their vitamin precursors to inhibit PC-3 growth by specific down-regulation of cyclin expression, with the γ forms being the most effective ones. Although the inhibition of PC-3 cell growth and diminution of cyclin expression are clearly visible, more subtle mechanistic effects of tocopherols and their corresponding carboxyethyl hydroxychroman metabolites deserve further investigations.

Distribution and Metabolism of Tocopherols and Tocotrienols &lt;i&gt;In Vivo&lt;/i&gt;

We present here current data on the distribution and metabolism of vitamin E analogs in vivo. There are eight different naturally occurring forms of vitamin E: four tocopherols (α-, β-, γ-, and δ-Toc) and four tocotrienols (α-, β-, γ-, and δ-Toc-3). With regard to the bioavailability of vitamin E, it has been established that the affinity of various vitamin E analogs for α-tocopherol transfer protein (α-TTP), which may determine their plasma levels, is a major determinant of their biological activity. However, a novel function of Toc-3 has been noted as a result of its unique distribution in the skin and the adipose tissue. In addition, following the discovery that the final metabolites of Toc and Toc-3 are in the form of carboxyethyl hydroxychroman, it is now possible to examine the intermediary metabolites of vitamin E analogs. The metabolism of vitamin E is known to be involved in the actions of drug metabolic enzymes (CYP3A, CYP4F2). However, the relationship between α-TTP and the metabolic enzymes that are responsible for the regulation of vitamin E metabolism has yet to be clarified. Future research will focus on the elucidation of the vitamin E metabolic regulation system.

Vitamin E and drug metabolism

Tocopherols and tocotrienols are metabolized by side chain degradation initiated by cytochrome P450 (CYP)-catalyzed ω-hydroxylation followed by β-oxidation. Whereas α-tocopherol is only poorly metabolized, high amounts of the final products, carboxyethyl hydroxychroman (CEHC), are found from other tocols in HepG2 cells and in human urine. CYP3A4 and CYP4F2 were suggested to be involved in tocopherol degradation. CYP3A4 metabolizes most of the drugs and is induced by many of its substrates via the activation of the pregnane X receptor (PXR). Also tocopherols and in particular tocotrienols induce the expression of a PXR-driven reporter gene and the expression of endogenous CYP3A4 and CYP3A5 which is supported by sporadic publications spread over the last 30 years. The potential interference of vitamin E with drug metabolism is discussed in the light of related complications evoked by herbal remedies.

Identities and Differences in the Metabolism of Tocotrienols and Tocopherols in HepG2 Cells

The metabolism of alpha- and gamma-tocotrienol was investigated in HepG2 cells. Metabolites were identified by HPLC and gas chromatography/mass spectrometry. gamma-Tocotrienol was degraded to gamma-CEHC (carboxyethyl hydroxychroman), gamma-CMBHC (carboxymethylbutyl hydroxychroman), gamma-CMHenHC (carboxymethylhexenyl hydroxychroman), gamma-CDMOenHC (carboxydimethyloctenyl hydroxychroman) and gamma-CDMD(en)(2)HC (carboxydimethyldecadienyl hydroxychroman). alpha-Tocotrienol yielded alpha-CEHC, alpha-CMBHC, alpha-CMHenHC and alpha-CDMOenHC, whereas alpha-CDMD(en)(2)HC could not be detected. These findings demonstrate that the trienols are metabolized essentially like tocopherols, i.e., by omega-oxidation followed by beta-oxidation of the side chain. The failure to detect CMBHC with the original double bond in the side chain reveals that auxiliary enzymes are involved, as in the metabolism of unsaturated fatty acids. CMBHC were the most abundant metabolites obtained from the tocotrienols as well as from alpha-tocopherol. Quantitatively, the tocotrienols were degraded to a larger extent than their counterparts with saturated side chains. The pronounced quantitative differences in the metabolism between individual tocopherols as well as between tocotrienols and tocopherols in vitro suggest a corresponding lack of equivalence in vivo.