Dynamics of the initiation of lipid peroxidation in membranes and the inhibition of it by α-tocopherol (α-Toc) were investigated under the biological condition. (1) First, we studied the initiation of lipid peroxidation in phosphatidylcholine (PC) liposomes. Membrane lipid peroxidation was not directly induced by superoxide (O_2^-), H_2O_2, and OH-radical (・OH) itself. The initiation of O_2^--dependent peroxidation required the presence of preformed PC-hydroperoxides (PC-OOH) and chelated metal catalyst such as Fe^<3+>-NTA (nitrilotriacetate). We proposed the mechanism that a radical group of PC-alkoxy radical (PC-O・), which was derived from degraded PC-OOH by the reaction with reduced Fe^<3+>-NTA by O_2^-, penetrates from membrane surface into the inner hydrophobic region and transfers "the initiation message of lipid peroxidation". (2) Second, we studied the location of α-Toc in liposome membranes, and the dynamics of its radical trapping and recycling by ascorbic acid (AsA). Little interaction of α-Toc with acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate into the phospholipid membranes, suggested little exposure of the OH-group of α-Toc at the membrane surface. The oxidation rate of α-Toc by positively charged Fe^<3+> was 150 times slower in negatively charged liposomes than in negatively charged SDS micelles, indicating that less than 0.65 mol% of the OH-groups of α-Toc was probably exposed at the membrane surface. The dynamic quenching abilities of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NS) labeled at different depths of membranes on the intrinsic fluorescence of α-Toc were in the order 5-NS>7-NS>12-NS>16-NS, indicating that the OH-group of α-Toc is located most at a position corresponding to 5-methylene carbon-inner from the surface and a little in hydrophobic region. AsA in bulk water phase completely suppressed the consumption of α-Toc during lipid peroxidation induced by 2,2'-azobis-(dimethylvaleronitrite) (AMVN), a hydrophobic radical precursor, in negatively charged liposomes, though negatively charged AsA could not penetrate into negatively and neutrally charged membranes (the ESR spectra of 5-NS and 16-NS labeled in these liposomes were no changed by the addition of AsA). These findings indicate that α-Toc is oxidized at inner region of the membranes by lipid radicals, and then floats up to the surface, where it is regenerated to α-Toc by AsA. (3) Finally, we measured the rate constant (k_s) of α-Toc for deactivating singlet oxygen (^10_2), which was generated by photoirradiation at the membrane surface, hydrophobic inner region and in bulk water. The following three factors ((1) the concentration and (2) mobility of α-Toc in membranes and of its active moiety at the membrane domains, and (3) the dielectric constant at membrane domains) are experimentally confirmed to be important for the consideration of k_s value of α-Toc in membranes: (1) the concentration of α-Toc, which was higher in membrane than in EtOH solution; the local concentration of the active moiety (OH-group) in membranes, which was 0%, 50-60%, and 40-50% at membrane surface polar zone (PZ), inner hydrogen belt (HB) and hydrophobic core (HC), (2) the mobility of α-Toc, which was higher in EtOH solution than in membranes, and higher at liquid crystalline state than at gel state of membrane phospholipids; the mobility of α-Toc, which was higher than that of β-carotene, because α-Toc locates at one half of the bilayer membrane but β-carotene locates across the bilayer; the local mobility of the OH-group of α-Toc, which was higher at HC region than at HB region, (3) the dielectric constant (micropolarity:ε), which reflects the reactivity of OH-group of α-Toc and ^10_2, at the membrane domains (εwas higher at HB region than at HC regions).

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Retinoic acid (RA), which is an active metabolite of vitamin A, plays an important role in the development and differentiation. RA is synthesized from retinal by retinal dehydrogenases (RALDH). Recent studies reported RALDHs mRNA expression in the small intestine. However, it is unclear whether retinal and/or retinol is converted to RA in the enterocytes. In this study, we investigated whether the expression of RA-target genes and their promoter activity are affected by RA or its precursors, i.e., retinal or retinol in the human colonic adenocarcinoma cell line C2_<BBe>1. The mRNA levels of retinoic acid receptor β(RARβ) and cellular retinol-binding protein type II (CRBPII) and their promoter activity were elevated by RA and by retinol and retinal as well. These results suggest that RA is effectively converted from retinol or retinal in C2_<BBe>1 cell, and that the locally produced RA may induce vitamin A-target gene expression.

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3日間の低C食を摂取させた健康な女子10名を被験者として,AsAまたはDAsAの結晶1mmol(AsA176mg,DAsA174mg)を経口負荷させた場合の尿中総C量および全血中総C濃度を測定した.被験者は両C型(AsAとDAsA)の負荷試験を実施するため,約1ヶ月の期間をあけて同じ代謝試験を2回行い,次のような結果を得た.1)通常期および欠乏期の24時間尿中総C量は,AsA群,DAsA群の間に有意な差は認められなかった.C負荷後24時間尿中総C量では,AsA群のC排泄量がDAsA群を有意に上回った(p<0.05).2)C負荷後の経時的尿中C排泄量は,負荷後3〜6時間尿,6〜9時間尿および9〜12時間尿において,AsA群の総C排泄量がDAsA群を有意に上回った(p<0.05).3)C負荷後の経時的尿中C排泄量には個人差が大きいことを認めた.4)C負荷直前,C負荷後1,2,3時間全血中総C濃度を解析した結果,両群間に有意な差は認められなかったが,増加量で比較すると,C負荷1時間後でDAsA群がAsA群を有意に上回った(p<0.05).5)C負荷後0〜3時間尿中総C量と負荷後1,2時間の全血中総C増加量の間に強い正の相関が認められた(p<0.01).また,C負荷後0〜24時間尿(=負荷後尿)と負荷3時間後の全血中増加量の間にも同様に強い相関が認められた(p<0.01).

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