published_or_final_version / Anatomy / Master / Master of Philosophy

D-γ-tocotrienol (G-T3) chiefly present in palm oil has stolen the spotlight as a promising radiation protective agent for the treatment of acute radiation syndrome (ARS). Although G-T3 is a promising counteracting agent discovered, the separation and purification from its matrix are painstaking. These have limited its characterization, derivatization, and biomedical application. In this study, we developed chromatographic and distillation methods to enhance the purity and synthesis of G-T3 derivatives. Exactly 8% ethyl acetate (EA) in hexanes used in TLC resulted in right spots separation. Two gradient column chromatographic methods were examined using solvent mixtures of EA/hexanes and EA/petroleum ether (PE), respectively. We verified that a gradient elution with EA in PE led to the maximum purity (≥95%) based on the NMR and GC-MS outcomes. Also, G-T3 was stable in an emulsion to some extent, and some soluble G-T3 derivatives were synthesized, and their structure was confirmed

BACKGROUND: α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3. METHODS: The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control. RESULTS: The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT. CONCLUSIONS: c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

The clinical consequences of ionizing radiation exposure remain one of the leading causes of death in the United States. Much research has been carried out to discover a potential countermeasure for acute radiation syndrome (ARS) without success. The United States Food and Drug Administration (US FDA) has not accepted any effective and harmless ionizing radiation therapy agents (radioprotectors) for treating ARS. It has recently been discovered that g-tocotrienol (GT-3), one of the E vitamers chiefly present in palm oil, has radioprotective abilities in mice and nonhuman primate (NHP) models. Though GT-3 is one of the most promising countermeasures discovered, the separation and purification from other vitamers or its matrix is difficult. This has limited its characterization, derivatization, and biomedical application. We have therefore designed novel chromatographic methods to optimize separation and purification. Thin layer chromatography (TLC) was used to ascertain the best solvent system for column chromatography (CC). Exactly 8% ethyl acetate in hexane employed in TLC and CC resulted in good separation (Rf ≥ 0.3) and purification. Various fractions presumed to contain GT-3 were collected and analyzed to confirm the exact structure using 1H NMR, 13C NMR, DEPT, and GC-MS. Results obtained so far have revealed the exact structure of the compound. However, some traces of impurities have been indicated by the NMR outcomes; therefore, high-performance liquid chromatography (HPLC) will be used to maximize GT-3 purification. This present study will be instrumental in elucidating the biochemical structure of various complex plant bioactive components that are hard to isolate and analyze. It is envisioned that this work will help to erase the knowledge deficit in medicinal chemistry and assist in the development of new medications for ARS.

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Vitamin E exhibits anti-tumor activity by regulating pathways in cancer cells, potentially the lipoxygenase (LOX) pathway. We studied the effects of alpha tocopherol (AT), gamma tocopherol (GT), gamma tocotrienol (GT3), and an alpha-gamma tocopherol mixture (ATGT) on the production of the LOX metabolites 13-hydroxyoctadecaenoic acid (HODE), 15-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 5-HETE in colorectal cancer. These metabolites were examined in the HCT-116 cell line after 24 h treatment with select vitamin E isoforms and quantified by LC/MS/MS. Under physiological conditions, we find that treatment with varying vitamin E isoforms have different effects on the production of 13-HODE, 15-HETE, 12-HETE, and 5-HETE. GT increases 13-HODE and decreases 12-HETE. AT reverses the effects of GT regulation on the LOX pathway, while GT3 has no significant effect on the metabolites tested. GT shows superiority in regulating the LOX pathway as it increases 13-HODE and decreases 12-HETE for possible prevention of colorectal cancer.

[Truncated abstract] Vitamin E is comprised of a family of tocopherols (TOH) and tocotrienols. The most studied of these is [alpha]-tocopherol ([alpha]-TOH), as this form is retained within the body and any deficiency of vitamin E is corrected with this supplement. [alpha]-TOH is a lipid-soluble antioxidant required for the preservation of cell membranes and potentially acts as a defense against oxidative stress. Individuals who have a primary vitamin E deficiency such as low birth weight infants, secondary vitamin E deficiency due to fat malabsorption such as in abetalipoproteinaemia, or a genetic defect in TOH transport require supplementation. There is debate as to whether vitamin E supplementation in other patient groups is required. Vitamin E supplementation has been recommended for persons with FHBL, a rare disorder of lipoprotein metabolism that leads to low serum [alpha]-TOH and decreased LDL cholesterol and apolipoprotein B concentrations. We examined the effect of truncated apoB variants on vitamin E metabolism and oxidative stress in persons with heterozygous FHBL. We used HPLC with electrochemical detection to measure [alpha]- and [gamma]-TOH in serum, erythrocytes, and platelets, and GC-MS to measure urinary F2-isoprostanes and TOH metabolites as markers of oxidative stress and TOH intake, respectively. Erythrocyte [alpha]-TOH was decreased, but we observed no differences in lipid-adjusted serum TOHs, erythrocyte [gamma]-TOH, platelet [alpha]- or [gamma]-TOH, urinary F2-isoprostanes, or TOH metabolites. Taken together, our findings do not support the recommendation that persons with heterozygous FHBL should receive vitamin E supplementation. ... Sesame lignans are natural components of sesame seed oil and there is evidence that these lignans can inhibit CYP450 enzymes, in particular, those responsible for vitamin E metabolism. We hypothesised that sesame seed ingestion would increase serum [gamma]-TOH, lower plasma lipids and inhibit platelet function in human subjects with at least one cardiovascular risk factor. We used HPLC with electrochemical detection to measure [alpha]- and -TOH in serum and GC-MS to measure F2-isoprostanes and TOH metabolites as markers of oxidative stress and TOH intake, respectively. We used high-sensitive C-reactive protein as a measure of systemic inflammation. Platelet function was assessed using the PFA-100 platelet aggregation assay. Although serum [gamma]-TOH increased by 17%, we observed no effect on lipid metabolism, markers of inflammation, oxidative stress or platelet function following treatment with ~25 g/day sesame seeds for five weeks. Our findings challenge the hypothesis that sesame seed ingestion provides beneficial cardiovascular effects. In summary, we have studied the metabolism and transport of both [alpha]- and [gamma]-TOH in humans to evaluate the requirements for supplementation and the effects of vitamin E on platelet function and CYP3A4 activity. Specialised techniques using HPLC were developed to measure serum and cellular TOH concentrations both in supplemented and un-supplemented individuals. We also used GCMS to provide a sensitive, accurate assessment of TOH metabolites and midazolam pharmacokinetics in humans after vitamin E supplementation. We have examined the role vitamin E has on important biochemical endpoints, with emphasis on the implications for TOH supplementation in subjects at risk of CVD.

Thesis deals with the analysis of vitamin E in cereal samples, namely barley and wheat and malt produced from them. Selected samples were treated with low-temperature plasma, fungicide and biological treatment to elimination fungus and mycotoxins. The aim of the thesis was to find out the influence of the treatment, the combination of several types of treatment on vitamin E content in grain and malt. If the content of health-positive substances is not reduced and grain can be used for food, malt, or for feed purposes. The theoretical part focuses on the properties, occurrence, biochemistry and importance of vitamin E for living organisms. It also focuses on the properties of cereals that are a significant source of vitamin E. Malt production and sample treatment are also described. The experimental part describes the field experiment, individual types of treatment, extraction of vitamin E from the samples and determination of vitamin E content by high performance liquid chromatography with fluorescence detection. The results are compared with the current literature. It was found that the treatment did not have a significant negative effect on vitamin E content.

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No description available.