抗氧化研究方法

1. 抗氧化研究方法

2. 氧化损伤的产生 • 线粒体呼吸链中，由于发生电子漏，导致氧分子经单电子转移反应，生成了超氧阴离子。 • 某些氧化酶催化氧化底物时，释放出活性氧，如黄嘌呤/黄嘌呤氧化酶体系放出超氧阴离子、葡萄糖/葡萄糖氧化酶体系放出过氧化氢。 • 巨噬细胞受刺激后呼吸爆发产生活性氧。

3. 体内自由基的产生 e e+H+ e+2H+ O2 O2－. H2O2 .OH H2O

4. 主要自由基简介 • 单线态氧 • 超氧阴离子 • 过氧化氢 • 脂质过氧自由基 • 碳中心自由基

5. 氧的状态 *2p p*2p p2p 2p *1s 2s *1s 1s Ground state O2 singlet O2 Superoxide O2- Peroxide ion O22-

6. 氧自由基的反应 超氧阴离子歧化 2O2-. + 2H+ H2O2 + O2 过氧化氢的反应： M (n+1)+ + .OH +OH- Mn+ + H2O2

7. 铁催化的羟自由基产生－Fenton Reaction and Harber-Weiss Reaction Fenton Reaction: Fe2+ + H2O2 Fe3+ + .OH + OH- (1) Fe3+ + O2-. Fe2+ + O2 Weiss Reaction: (2) Fe2+ + H2O2 Fe3+ + .OH + OH- O2-. +H2O2 O2+.OH +OH- (3) Net

8. 脂质过氧化 －CH2－ + .OH Initiation: － .CH2－ + H2O CH.+ O2 CHO2. Propagation: CHO2.+ CH2 CHO2H + CH. Termination: CH.+ CH. －CH－CH－

9. 抗氧化研究内容 • 清除自由基 • 抑制脂质过氧化 • 总抗氧化活力测定 • 抑制氧化导致的细胞损伤 • 缺血再灌注损伤及炎症

10. 1、清除自由基 a. 清除羟自由基 b. 清除超氧阴离子 c. 清除单线态氧 d. 清除DPPH自由基 e. 清除碳中心自由基

11. 电子顺磁共振法(Electronic Paramagnetic Resonance, EPR) • 以DMPO 捕获剂，测定羟自由基(Hydroxyl radical)和超氧阴离子自由基 • 以TEMP为捕获剂，测定单线态氧(Singlet oxygen) • 直接测定DPPH自由基 • 以4－POBN为捕获剂，测定碳中心自由基

12. 典型的羟自由基－DMPO加合物图谱

13. 典型的超氧阴离子自由基－DMPO加合物图谱

14. 典型的单线态氧－TEMP加合物图谱

15. 典型的碳中心自由基－4-POBN加合物图谱

16. 典型的DPPH自由基图谱

17. 化学法 (Hydroxyl radical) • Drug effects were tested on deoxyribose oxidation induced by 60 min incubation at 37C with nonchelated Fe2+, namely 20 M FeSO4, with or without 1.42 mM H2O2 , in PPB, pH7.4. The TBA-test, which detects aldehydic products, such as malondialdehyde (MDA), resulting from deoxyribose (or lipid) oxidation, was then carried out adding to 0.5 mL of sample, 1.0 mL of 2.8% trichloroacetic acid and 1.0 mL of 0.6% aqueous solution of TBA; tubes were heated at 95 C for30 min to develop the color, and successively cooled in ice. The red chromogen, expression of the TBA:MDA adduct formation, was extrtacted with n-butanol kept at 4C; after a brief centrifugation to favor organic phase separation, the upper n-butanol layer was removed and read spectrophotometrically at 532 nm against an appropriate blank. Results were caculated as nmol TBARS per mL, using a molar extinction coefficient of 154,000 at 532 nm.

18. Superoxide anion • To a mixture of tested compound (50 M), xanthine oxidase (25 munit/ml), and nitro blue tetrazolium (50 M) in Tris buffer (0.1 M, pH 7.4), and aliquot of 10l hypoxanthine (3.5 mM) was added, the total volume of the mixture was 1 ml. The absorbance of the reaction mixture was monitored spectrophotometrically at 560 nm for 10 min using a UV-VS spectrum meter.

19. Total antioxidant status (TAS) assay • ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS+) was produced by first adding MnO2 powder in the solution and keeping in the dark at room temperature for more than 12 h, then filtrated with syringe filter and kept in dark for another 6 hours. The obtained radical was stable in this form for more than two days when stored in the dark at room temperature. Stock solution of ABTS+ was diluted with PBS, pH 7.4, to an absorbance of 0.70 (0.02) at 734 nm and equilibrated at 30°C. After adding 1.0 ml of diluted ABTS+ to 10 l of serum or tissue homogenate supernate, the absorbance reading was taken at 30°C exactly 2 min after initial mixing. The total antioxidant capacity concentration was compared to equivalent antioxidant capacity of Trolox and is expressed in mols of Trolox/g of tissue.

20. Quenching DPPH radical • DPPH dissolved in ethanol to a concentration of 90M, tested compounds dissolved in ethanol or distilled water to 120 M solution, to 2.5 ml DPPH solution, the suitable amount of tested compound solution was added, the total volume was adjusted to 3 ml with ethanol and mixed thoroughly, the absorbency was recorded at 517 nm exactly at 10 min.

21. Xanthine oxidase inhibition assay • Hypoxanthine (35 M) was added to a mixture of xanthine oxidase (25 munit/ml) and tested compound (10-50 M) in Tris buffer (pH 7.4, 0.1 M). The change in absorbance was monitored at 293 nm at room temperature for 5 min.

22. TBARS Assay • Direct assay: • Four hundred microliter sample mixed with 400 l 10% trichloroacetic acid (TCA), 1 ml of 0.6% 2-thiobarbituric acid (TBA), and then incubated in a water bath at 95℃for 60 min. After cooling the sample with water, 2.0 ml of n-butanol were added and the mixture was shaken vigorously. After centrifugation at 5000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm.

23. Induced by other chemicals • For Fe2+-Ascorbate, usually incubated at 37℃for 30 min • For AAPH, usually incubated at 37℃for 2 hours

24. Cell culture • Mostly used oxidant is H2O2 • Direct addition • Enzymatic method Glucose/glucose oxidase system (H2O2) Xanthine/xanthine oxidase (O2-.)

25. Assay a. Morphological study b. MTT assay c. TBARS assay d. apoptosis

26. 缺血再灌注损伤 • 从89年到2002年，国内期刊上发表的有关缺血再灌注损伤的文章4308篇，其中2002年全年793篇

27. 机理 • 再灌流时，多种氧化酶系以氧气、NADPH、ATP等为底物，在Ca(II)、Fe(II)的催化介导下，产生大量的具有强烈生物氧化活性的自由基。氧自由基使不饱和脂肪酸部分激活，在金属复合物特别是铁的参与下，进而引起一系列的自由基反应，造成膜不饱和脂肪酸的脂质过氧化，导致脂膜局部破坏，直至溶解。 • 另外，自由基还可使磷脂酶改变活性，引起花生四烯酸(arachidonic acid, AA)代谢紊乱，导致微血栓形成

28. ATP xanthine dehydrogenase ADP 缺血 AMP xanthine oxidase Adenosine Carnine hypoxanthine O2-. O2 再灌注

29. 分类 • 脑缺血再灌注 • 心脏缺血再灌注 • 肾缺血再灌注 • 肝缺血再灌注

30. 生化检测指标 • TBARS • Lactic dehydrogenase (LDH) • SOD • GSH