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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We provided a low-Vitamin C (C) diet to ten healthy females for three days before the experimental day and the orally administered 1mmol of Ascorbic Acid (AsA) crystal (176mg) or Dehydroascorbic Acid (DAsA) (174mg). Then we measured the total amount of C in urine and blood samples. This metabolism test was conducted by the crossover method. After ingesting AsA or DAsA, subjects underwent the same metabolic test taking the other C about one month later. 1) The 24-hour total amount of C in urine during the normal diet period and low-C diet period showed little or no difference between the AsA and DAsA group. After ingestion of C, the amount of C in urine of the AsA group was greater than that in the DAsA group (p<0.05). 2) The 3-6 hour, 6-9 hour, and 9-12 hour amount of C in urine after ingestion of C was significantly greater in the AsA group than in the DAsA group (p<0.05). 3) There was a large individual difference in the time-dependent urinary excretion of C after ingestion. 4) We serially analyzed the total amount of C in whole blood just before ingestion, 1 hour, 2 hours, and 3 hours after ingestion. These findings showed little or no difference between the AsA and DAsA group. Comparing the amount of increase relative to the concentration before ingestion of C, the DAsA group showed a significantly greater increase than the AsA group 1 hour after oral C loading (p<0.05). 5) After ingestion of C, there was a positive correlation between the level of blood C increase 1 hour after oral C loading and the amount of C in urine after 0-3 hour (p<0.01).

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