Tocopherol Metabolism Using Thermochemolysis: Chemical and Biological Properties of γ-Tocopherol, γ-Carboxyethyl-hydroxychroman, and Their Quinones

Abstract
Identification and quantitative estimation of quinone metabolites of γ-tocopherol (γ-T) and its derivative γ-carboxyethyl hydroxychroman (γ-CEHC) are complicated by their functions as arylating electrophiles. We hypothesize that their biological properties are expressed through arylating quinone electrophile addition (Michael reaction) with thiol nucleophiles in cells and tissues. Glutathione (GSH) reacted with γ-tocopheryl quinone (γ-TQ) to form the hydroquinone adduct, which was identified by electrospray time-of-flight MS (ESI-TOF-MS). Tetramethylammonium hydroxide (TMAH) thermochemolysis reduced and methylated quinones and cleaved and methylated thioether adducts. These relatively nonpolar derivatives were readily separated by GC and identified by MS fragmentation patterns. γ-CEHC was synthesized and oxidized to a product identified as the quinone lactone (γ-CEHC-QL). TMAH methylated both γ-CEHC-QL and its GSH adduct without opening the lactone ring, and these products were separated by GC and identified by MS fragmentation patterns. γ-CEHC-QL reacted with both the cysteinyl enzyme papain and fetal bovine serum, and TMAH thermochemolysis showed that each product mixture contained unreacted precursor and thioether adduct. Cytotoxicities of phenolic precursors, γ-T and γ-CEHC, and their quinones, γ-TQ and γ-CEHC-QL, respectively, were compared in COS1, NT2, 3T3, and N2a cell lines. Phenolic precursor γ-T had a small effect only with NT2 and 3T3 cells while γ-CEHC had no effect in any cell line. Arylating quinones were highly cytotoxic in all cell lines with γ-TQ showing a significantly greater cytotoxicity than γ-CEHC-QL. These data are consistent with our arylating electrophile hypothesis as an explanation for some biological activities of Ts through their quinone metabolites.

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