抗氧化研究方法氧化损伤的产生
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。
体内自由基的产生
O2 O2－,H2O2,OHe e+2H+ e+H
+
H2O
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
碳中心自由基氧的状态
1s
*1s
2s
*1s
2p
p2p
p*2p
*2p
Ground
state O2
singlet O2 Superoxide
O2-
Peroxide ion
O22-
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：
Mn+ + H2O2 M (n+1)+ +,OH +OH-
铁催化的羟自由基产生－ Fenton Reaction
and Harber-Weiss Reaction
Fenton Reaction,Fe2+ + H2O2 Fe3+ +,OH + OH-
Weiss Reaction,Fe3+ + O2-,Fe2+ + O2
Fe2+ + H2O2 Fe3+ +,OH + OH-
(1)
(2)
Net O2-,+H2O2 O2+.OH +OH- (3)
脂质过氧化
－ CH2－ +,OH －,CH2－ + H2OInitiation:
CH,+ O2 CHO2.
Propagation,CHO2,+ CHO2H +CH2 CH.
Termination,CH,+ CH,－ CH－ CH－
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
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，测定碳中心自由基典型的羟自由基－ DMPO加合物图谱典型的超氧阴离子自由基－ DMPO加合物图谱典型的单线态氧－ TEMP加合物图谱典型的碳中心自由基－ 4-POBN加合物图谱典型的 DPPH自由基图谱化学法 (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.
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.
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.
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.
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.
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,
Induced by other chemicals
For Fe2+-Ascorbate,usually incubated at
37℃ for 30 min
For AAPH,usually incubated at 37℃ for 2
hours
Cell culture
Mostly used oxidant is H2O2
a,Direct addition
b,Enzymatic method
Glucose/glucose oxidase system (H2O2)
Xanthine/xanthine oxidase (O2-.)
Assay
a,Morphological study
b,MTT assay
c,TBARS assay
d,apoptosis
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
ATP
ADP
AMP
Adenosine
Carnine
hypoxanthine
缺血
xanthine dehydrogenase
xanthine oxidase
O2
O2-.
再灌注分类
脑缺血再灌注
心脏缺血再灌注
肾缺血再灌注
肝缺血再灌注生化检测指标
TBARS
Lactic dehydrogenase (LDH)
SOD
GSH
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。
体内自由基的产生
O2 O2－,H2O2,OHe e+2H+ e+H
+
H2O
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
碳中心自由基氧的状态
1s
*1s
2s
*1s
2p
p2p
p*2p
*2p
Ground
state O2
singlet O2 Superoxide
O2-
Peroxide ion
O22-
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：
Mn+ + H2O2 M (n+1)+ +,OH +OH-
铁催化的羟自由基产生－ Fenton Reaction
and Harber-Weiss Reaction
Fenton Reaction,Fe2+ + H2O2 Fe3+ +,OH + OH-
Weiss Reaction,Fe3+ + O2-,Fe2+ + O2
Fe2+ + H2O2 Fe3+ +,OH + OH-
(1)
(2)
Net O2-,+H2O2 O2+.OH +OH- (3)
脂质过氧化
－ CH2－ +,OH －,CH2－ + H2OInitiation:
CH,+ O2 CHO2.
Propagation,CHO2,+ CHO2H +CH2 CH.
Termination,CH,+ CH,－ CH－ CH－
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
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，测定碳中心自由基典型的羟自由基－ DMPO加合物图谱典型的超氧阴离子自由基－ DMPO加合物图谱典型的单线态氧－ TEMP加合物图谱典型的碳中心自由基－ 4-POBN加合物图谱典型的 DPPH自由基图谱化学法 (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.
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.
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.
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.
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.
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,
Induced by other chemicals
For Fe2+-Ascorbate,usually incubated at
37℃ for 30 min
For AAPH,usually incubated at 37℃ for 2
hours
Cell culture
Mostly used oxidant is H2O2
a,Direct addition
b,Enzymatic method
Glucose/glucose oxidase system (H2O2)
Xanthine/xanthine oxidase (O2-.)
Assay
a,Morphological study
b,MTT assay
c,TBARS assay
d,apoptosis
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
ATP
ADP
AMP
Adenosine
Carnine
hypoxanthine
缺血
xanthine dehydrogenase
xanthine oxidase
O2
O2-.
再灌注分类
脑缺血再灌注
心脏缺血再灌注
肾缺血再灌注
肝缺血再灌注生化检测指标
TBARS
Lactic dehydrogenase (LDH)
SOD
GSH
