DOI: 10.1002/cphc.201402297 A Pulse Radiolysis Study of the Dynamics of Ascorbic Acid Free Radicals within a Liposomal Environment Kazuo Kobayashi,* [a] Yumiko Seike, [b] Akinori Saeki, [c] Takahiro Kozawa, [a] Fusako Takeuchi, [d] and Motonari Tsubaki [e] 1. Introduction Studies of cell-based chemical reactions have primarily focused on the aqueous solution regions of the cell. However, increas- ing attention has turned toward the chemistry that occurs within lipophilic membranes. [1] For example, the reactions of reactive oxygen species in membranes have been shown to occur readily within cellular membranes and are of significant biological importance. [2] It is important to understand that in vivo antioxidant capacity is determined by the reactivity of antioxidants toward radicals, in addition to several other fac- tors, which include the concentration, tissue distribution, cellu- lar localization, and fate of antioxidant-derived radicals, as well as the interactions with other antioxidants and active metabol- ic pathways. [3] Ascorbate (AsH  ) is involved in a wide range of biochemical processes. [4] It serves as a water-soluble antioxidant and is found throughout the human body. Brain and neuroendocrine tissues have the highest AsH  levels of any organ system. AsH  plays a unique role as an antioxidant in the brain extra- cellular microenvironment. [5] AsH  can potentially protect both the cytosolic and membrane components of cells from oxida- tive damage. Most oxidized free radicals generated by biologi- cal systems can promote the one-electron oxidation of AsH  to form the ascorbate radical (AsC  ). AsC  production and scav- enging have been observed to be both associated with, and independent of, enzymatic reactions, [6] and the detection of AsC  formation by using EPR spectra provides a measure of oxi- dative stress. [7] Pulse radiolysis is a powerful tool for investigat- ing the dynamics of AsC  in biological systems. [8] In the present work, this technique was extended to the cellular assembly system, and AsC  were generated in a model system containing membranes. 2. Results and Discussion Both oxidative and reductive species were expected to form during the radiolysis of an aqueous solution containing lipo- some vesicles, in both the exterior and interior aqueous phases of the liposome. Only one type of radical, AsC  , was generated within the liposomal bilayer under the experimental conditions examined here. Pulse radiolysis of a deaerated aqueous solution produces hydrated electrons, e  (aq) , and OHC [Eq. (1)]: H 2 O e  ðaqÞ þ OHC ð1Þ In the presence of N 2 O and 10 mm AsH  , AsC  radical anions are produced in the following reactions [Eqs. (2) and (3)]: e  ðaqÞ þ N 2 O þ H 2 O ! OHC þ OH  þ N 2 ð2Þ OHC þ AsH  ! AsC  þ H 2 O ð3Þ In the presence of Cl  , Cl 2  (the product of the reaction be- tween Cl  and OHC) reacts with AsH  to form AsC  . [8b] The initial transient increase in absorbance at 360 nm (A 360 nm ) indicated the formation of AsC  . The subsequent decay of AsC  was ob- [a] Dr. K. Kobayashi, Prof. T. Kozawa The Institute of Scientific and Industrial Research, Osaka University Mihogaoka 8-1, Ibaraki, Osaka (Japan) E-mail : kobayasi@sanken.osaka-u.ac.jp [b] Y. Seike Department of Life Science, Faculty of Science Himeji Institute of Technology, (presently) University of Hyogo Kamigori-cho, Akou-gun, Hyogo 678-1297 (Japan) [c] Dr. A. Saeki Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka (Japan) [d] Dr. F. Takeuchi Institute for Promotion of Higher Education 1-2-1 Tsurukabuto, Nada-ku, Kobe, Hyogo 657-8501 (Japan) [e] Prof. M. Tsubaki Kobe University, Graduate School of Science, Department of Chemistry 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501 (Japan) The dynamics of free-radical species in a model cellular system are examined by measuring the formation and decay of ascor- bate radicals within a liposome with pulse radiolysis tech- niques. Upon pulse radiolysis of an N 2 O-saturated aqueous so- lution containing ascorbate-loaded liposome vesicles, ascor- bate radicals are formed by the reaction of OHC radicals with ascorbate in unilamellar vesicles exclusively, irrespective of the presence of vesicle lipids. The radicals are found to decay rap- idly compared with the decay kinetics in an aqueous solution. The distinct radical reaction kinetics in the vesicles and in bulk solution are characterized, and the kinetic data are analyzed.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 0000, 00,1–5 &1& These are not the final page numbers! ÞÞ CHEMPHYSCHEM ARTICLES