高齢化社会の進捗，医療費の高騰に伴い，健康の維持増進や疾病の予防に関する国の普及啓発活動が推進され，セルフメディケーションへの関心が高まっている．特に疾病予防を目的として，生体に対して抗酸化作用を示す機能性食品が注目され，科学的根拠に基づいて，これらの抗酸化能を比較する統一的な評価方法の確立が望まれている．本研究では，リポソームを用いることにより，生体内に近い環境下での抗酸化能評価法を検討した．脂質二重膜内で生成する過酸化脂質と選択的に反応し，蛍光性酸化物を生成する蛍光プローブであるDPPP ( Diphenyl-1-pyrenylphosphine) を組み込んだL-α-Phosphatidylcholine dioleoyl (DOPC) リポソームを調製し，酸化剤 (Peroxynitrite) 存在下でDPPP oxideを生成させた．ここに抗酸化物質(α-Tocopherol; α-Toc, α-Tocotrienol; T3) を添加し，脂質膜過酸化抑制能を，DPPP oxideの生成量と，抗酸化物質濃度を，タンデム質量分析計を備えた液体クロマトグラフィー(LC-MS/MS)を用いて同時定量することにより評価した．リポソームに組み込んだDPPPと過酸化脂質との反応が化学量論的に進行することから，生成したDPPP oxideを定量することにより，過酸化脂質量を求めることができる．
これまでに蛍光検出器を備えた超高速LCを用い，抗酸化物質 (α-Toc, T3, epicatechin, resveratrol)，DPPP，DPPP oxideの同時定量を達成し，抗酸化物質の消費量に対応し,かつリポソーム膜への親和性を反映した実質的な脂質過酸化抑制能評価を可能にした．さらに，LC-MS/MSを用いることにより，蛍光を有しない抗酸化物質，DPPP，DPPP oxideを数ng/mLオーダーという高感度で同時分析することに成功した．その結果，α-TocまたはT3を単独で添加した試料では，コントロールと比較して酸化阻害作用が確認された．一方，α-Toc, T3を同時に添加した試料では，相加性はなく，単独使用との差がほとんど見られなかった．
LC-MS/MSを用いることで，誘導体化の必要がなく，UV吸収を持たない物質など，より幅広い抗酸化物質を対象とした測定が可能になる．本手法は，生体内に近い環境下での抗酸化物質の評価法として有用であると考える．
With the proliferation of aging society and concomitant increase of medical expenses, the government has promoted the enlightenment activity for the nation's health and disease prevention. Thus the interest toward self-medication increases among many Japanese people, and the functional foods which are claimed to have the antioxidant action attract attention for the purpose of disease prevention. Therefore, the establishment of the unified evaluation method to compare these antioxidant activity based on scientific grounds is needed for the appropriate use of these foods. In order to approach the issue, we have developed the measurement method of antioxidative activity in liposome, which afford the similar reaction conditions to those in biomembranes. The liposome of L-α-Phosphatidylcholine dioleoyl (DOPC) was prepared in the presence of DPPP (Diphenyl-1-pyrenylphosphine), which selectively reacts with lipid peroxide formed in the lipid bilayer. The lipid peroxide was produced in the presence of oxidizer (peroxynitrite), and DPPP oxide thus formed was used as a fluorescent probe.  The inhibition ability of lipid peroxidation was evaluated by assaying the production of DPPP oxide in the presence of an antioxidant (α-Tocopherol;α-Toc,α-Tocotrienol; T3) in liposomal solution.  The concentration of antioxidant was measured at the same time using liquid chromatography with the tandem mass spectrometer (LC-MS/MS).  Since DPPP incorporated in liposome and lipid peroxide were found to react stoichiometrically to give DPPP oxide, the amount of lipid peroxide thus formed was able to be measured by assaying formed DPPP oxide.
In a previous study, we carried out the simultaneous determination of the concentrations of antioxidants (α-Toc, T3, epicatechin, resveratrol), DPPP, and DPPP oxide using ultra high performance liquid chromatography with fluorescent detector (UHPLC-FLD), and achieved the evaluation of the inhibition activity of the antioxidants against the lipid peroxidation. It was found that the affinity of the antioxidant to lipid bilayers affected its activity.  Furthermore, we succeeded in analyzing antioxidant, DPPP, and DPPP oxide at the same time using LC-MS/MS, with high sensitivity of ng/mL order.  When α-Toc or T3 was added in the liposome, the antioxidative activity was confirmed. When these two antioxidants added in the reaction, however, no additive effect was observed. 
By using LC-MS/MS for analysis, the derivatization of the test materials with no UV absorption and the fluorescence was not necessary, and the application of the method for a wide range of antioxidants was enabled. We think this technique to be useful as the evaluation method of the antioxidant in the biomimetic environment.