Transcript What is an antioxidant? How do antioxidants work? Garry R. Buettner and Freya Q.
What is an antioxidant? How do antioxidants work? Garry R. Buettner and Freya Q. Schafer
Free Radical and Radiation Biology Program and ESR Facility The University of Iowa Iowa City, IA 52242-1101 Tel: 319-335-6749 Email: [email protected] SFRBM Sunrise Free Radical School or [email protected]
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Antioxidants, the road ahead
This presentation focuses on the action/reaction of small molecule antioxidants.
1. Overview and vocabulary 2. Preventive 3. Chain-breaking 4. Retarder vs true antioxidant
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Antioxidants: A definition
A substance when present in trace (small) amounts inhibits oxidation of the bulk. OR A little bit goes a long way.
So, what is a little bit?
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Antioxidants: two broad classes
Preventive and Chain-Breaking Preventive
antioxidants intercept oxidizing species before damage can be done.
Chain breaking
antioxidants slow or stop oxidative processes after they begin, by intercepting the chain-carrying radicals .
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Elementary Lipid Peroxidation L-H + X
L
+ XH
Initiation
L
+ O 2 LOO
+ L-H 3 x 10 8
M -1 s -1 LOO
Propagation
40 M -1 s -1
L
+ LOOH
Cycle
LOO
+ “R
”
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LOOR Termination 5
Preventive Antioxidants
Don’t let it get started.
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Preventive antioxidants act by:
A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, … B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, … C. Quenching singlet oxygen, e.g.
-carotene, lycopene, bilirubin, … 7
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Preventive Antioxidants: Targeting Metals
Fe & Cu are the principal metals targeted – loosely bound* Proteins & metals – Transferrin / Hemoglobin / Ceruloplasmin Chelates – Fe 3+ – EDTA, DETAPAC (DTPA), Desferal Fe 2+ – Phenanthrolines, … * “Loosely” bound iron on proteins, DNA as well as iron in hemes can be dangerous.
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Preventive Antioxidants: Why target metals?
Because they promote oxidant production.
Fe(II)chelate + H or 2 O 2
HO
+ Fe(III)chelate + OH Fe(II)chelate + LOOH
LO
+ Fe(III)chelate + LOH and Fe(II)chelate + O 2
Oxidants a a
Qian SY, Buettner GR. (1999) Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: An electron paramagnetic resonance spin trapping SFRBM Sunrise Free Radical School
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study.
Free Radic Biol Med
,
26
: 1447-1456.
Metal Deactivation O O O C H 2 C CH 2 C O O N CH 2 CH 2 N C H 2 C CH 2 C O O O EDTA Ethylenediaminetetraacetic acid anion Fe(III)/Fe(II) EDTA E = + 120 mV Rxn with O 2
k = 10 6 M -1 s -1 O O O C C O H 2 C N H 2 C (CH 2 ) 2 N CH 2 (CH 2 ) 2 C O O N CH 2 CH 2 O C O C O O DETAPAC E = + 30 mV Rxn with O 2
k < 10 2 M -1 10 s -1
Metal Deactivation: Why the difference? H 2 O Fe(III)EDTA Size -- too small, leaving a site for H 2 O 11
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Metal Deactivation: Desferal
E
(Fe(III)DFO/Fe(II)DFO) = - 450 mV
K
stability
Fe(III) 10
30.6
K
stability
Fe(II) 10
7.2
k
(with O 2 ) < 10
3
M -1 s -1 De-activates Fe(III) kinetically (no H 2 O of coordination) and thermodynamically.
H H O O N (CH 2 ) 5 N C C (CH 2 ) 2 NH (CH 2 ) 5 N C C (CH 2 ) NH (CH 2 ) 5 N C CH 3 HO O
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HO O Deferrioximine HO O 12
Preventive antioxidants act by:
A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, … B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, … C. Quenching singlet oxygen, e.g.
-carotene, lycopene, bilirubin, … 13
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Enzymes targeting peroxides: H 2 O 2 , LOOH Catalase: 2H 2 O 2
2H 2 O + O 2 GPx (GPx1): H 2 O 2 + 2GSH
2H 2 O + GSSG or ROOH + 2GSH
H 2 O + ROH + GSSG PhGPx (GPx4): PLOOH + 2GSH
PLOH + GSSG + H 2 O Prx (peroxidredoxins): H 2 O 2 + Trx(SH) 2
2H 2 O + Trx(SS) 1-cysPrx: PLOOH + 2GSH
PLOH + GSSG + H 2 O Non-enzymatic rxns H 2 O 2 + 2GSH
2H 2 O + GSSG or
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ROOH + 2GSH
H 2 O + ROH + GSSG
Glutathione (GSH) O C O NH 3 CH CH 2 O CH 2 C H O NH C C CH 2 SH NH CH 2 O C O glutamate cysteine glycine Glutathione is a tri-peptide
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Preventive Antioxidants: Removing Hydroperoxides GSH will react directly with H 2 O 2 , albeit very slowly .
2 GSH + H 2 O 2
2 H 2 O + GSSG
k obs
(7.4)
1 M -1 s -1 * Appears to be too slow for biological significance.
SFRBM Sunrise Free Radical School * Estimated from: Radi
et al.
(1991) J Biol Chem. 266: 4244-4250.
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Hydroperoxide removal by GSH is mainly via coupled enzyme reactions 2 GSH + ROOH
GSSG + H 2 O + ROH GSH synthetic enzymes inhibits ROOH
GPx
ROH + H 2 O
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primary 2 GSH 2e -
GSSG reductase
GSSG NADPH 6-P-G 2e -
G-6-P dehydrogenase
NADP + G-6-P stimulates secondary secondary 17
Pyruvate and H
2
O
2 Pyruvate is a three-carbon ketoacid produced during glycolysis.
Pyruvate can remove H 2 O 2 by a stoichiometric chemical reaction.
H 3 C O C C O O + H 2 O 2 Pyruvate
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H 3 C C O O + CO 2 + H 2 O Acetate 18
Preventive antioxidants act by:
A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, … B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, … C. Quenching singlet oxygen, e.g.
-carotene, lycopene, bilirubin, … 19
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Singlet oxygen quenching, avoiding peroxides Singlet Oxygen 1 O 2 , i.e. oxygen with extra energy 1
g O 2 23.4 kcal mol -1 above the ground state Singlet oxygen is electrophilic, thus it reacts with the double bonds of lipids.
(No free radicals; hydroperoxides formed.) k
2 x 10 5 M -1 s -1 1
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O 2 + PUFA
PUFA-OOH 20
A A + O 2 H , H OOH 9-OOH (50%) + HOO B B 13-OOH (50%)
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O H Linoleic acid 1O2 A OOH B 9-OOH (30%) + HOO A A 10-OOH (20%) + OOH B B 12-OOH (20%) HOO + 13-OOH (30%) LOOHs: 1 O 2
vs
radicals 21
Quenching of
1
O
2 Chemical quenching
is a term used to signify that an actual chemical reaction has occurred. Hydroperoxide formation is chemical quenching.
1 O 2 + LH
LOOH Physical quenching
is the removal of the excitation energy from 1 O 2 changes.
without any chemical
1 O 2
+
-carotene -carotene*
O 2 +
-carotene* 22
-carotene + heat
Antioxidants, the road ahead
1. Overview and vocabulary 2. Preventive 3. Chain-breaking 4. Retarder vs true antioxidant
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Chain-Breaking Antioxidants
In general chain breaking antioxidants act by reacting with peroxyl radicals, ROO
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Chain breaking Antioxidants can be:
A) Donor antioxidant , e.g. tocopherol, ascorbate, uric acid, … B) Sacrificial antioxidant , e.g. nitric oxide 25
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Peroxyl Radicals as Targets
RH + ROO
R
+ O 2
ROOH + R
ROO
Peroxyl radicals, ROO
, are often the chain-carrying radical.
The chain reaction can also be broken by intercepting R
.
this is rare, but in the polymer industry it can be very important.
Terminating the Chain, Peroxyl Radicals as Targets Tocopherol, a donor antioxidant LOO
+ TOH
LOOH + TO
Nitric oxide, a sacrificial antioxidant LOO
+
NO
LOONO
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Characteristics of a Good Chain breaking Antioxidant a. Both Antioxidant & Antiox
relatively UN-reactive should be b. Antiox
- decays to harmless products c. Does not add O 2 radical to make a peroxyl d. Renewed (Recycled) – somehow e. If the chain-breaking antioxidant is a 28 the middle of the pecking order.
The Pecking Order Antioxidants have reduction potentials that places them in the middle of the Pecking order.
This location in the pecking order provides antioxidants with enough reducing power to react with reactive oxidizing species. At the same time they are too weak to initiate reductive reactions.
LOO
+ TOH
LOOH + TO
Termination
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The Pecking Order Depending on their reduction potential, antioxidants can recycle each other. For example, ascorbate with a reduction potential of +282 mV can recycle TO
(+480 mV) and urate
(+590 mV).
Buettner GR. (1993)
Arch Biochem Biophy.
300:535-543.
Redox Couple (one-electron reductions)
HO , H + /H 2 O RO , H + /ROH (aliphatic alkoxyl radical) ROO , H + /ROOH (alkyl peroxyl radical) GS /GS (glutathione) PUFA , H + /PUFA-H (
bis
-allylic-H)
TO
, H + /TOH (tocopherol)
H 2 O 2 , H + /H 2 O, HO
Asc
, H + /AscH - (Ascorbate)
CoQ , 2H + /CoQH 2 Fe(III) EDTA/Fe(II) EDTA CoQ/CoQ O 2 /O 2 Paraquat/Paraquat Fe(III)DFO/Fe(II)DFO RSSR/RSSR (GSSG) H 2 O/e aq
E°'/mV
+2310 +1600 +1000 +920 +600 +
480
+320 +
282
+200 +120
-
36
-
160
-
448
-
450
-
1500
-
2870
CH 3 O Donor Antioxidant - Vitamin E CH 3 CH 3 TO
O CH 3 CH 3 (CH 2 ) 3 CH(CH 2 ) 3 CH(CH 2 ) 3 CH(CH 3 ) 2 CH 3 Reaction with lipid peroxides
:
LOO
+ TOH
LOOH + TO
Recycling reaction with ascorbate HO OH O O + TO O OH
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AscH HO O OH O Asc O O + TOH 31
Donor Antioxidant – Uric Acid Uric acid is produced by the oxidation of xanthine by xanthine oxidase. At physiological pH it is ionized to urate.
O HN H N O N H N OH UH 3 pK a1 = 5.4
UH 2 pK a2 = 9.8
UH 2 Uric acid Normal urate concentrations in human plasma range from 0.2 – 0.4 mM.
Ames BN
et al.
and radical-caused aging and cancer. A hypothesis. Proc. Natl Acad Sci. USA 78, 6858.
Uric Acid Reacts with Peroxyl Radicals k = 3 x 10 6 M -1 s -1 ROO
+ UH 2 -
ROOH + UH
Recycling by Ascorbate: k = 1 x 10 6 M -1 s -1 UH
+ AscH -
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UH 2 + Asc
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Chain Breaking Antioxidant Sacrificial – Nitric Oxide radical, reactive but t 1/2 ~s small molecule, fits everywhere NO uncharged, diffusable
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lipid soluble 34
Nitric Oxide as Antioxidant
Preventive:
NO coordinates with heme-iron,
heme Fe 2+ + NO heme Fe 2+ NO We have used this for centuries in food preservation, the "sausage" effect.
Chain-breaking:
NO can react with oxyradicals:
ROO + NO ROONO RO + NO RONO
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heme oxygenase, ferritin, hsp70, and -glutamylcysteine synthetase
Nitric Oxide as Chain Breaking Antioxidant in Lipid Peroxidation O 2 NO LOO Sen h
O 2 1 O 2 LH LOOH Asc Fe 2+
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AscH Fe 3+ Fe 2+ O 2 , H 2 O 2 [Fe 2+ -O 2 ], OH LH L OLOOH LH NO OLOONO LH LO OLOO LO O 2 36 L d
Chain Breaking Antioxidant – Nitroxide Example nitroxide HO NO Tempol A possible antioxidant cycle for a nitroxide R 2 NO R 1 + e - , H + R 2 R 1 NOH nitroxide hydroxylamine R 2 NOH R 1 + R ox RoxH + .R
2 R 1 NO 37
Retarders vs Antioxidant
Retarders suppress oxidations only slightly compared to a true antioxidant. A retarder is only able to make a significant change in the rate of oxidation of the bulk when present in relatively large amounts.
Retarders are often confused with antioxidants.
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Kinetic Comparison of Antioxidant and Retarder
Theorem: There are no true antioxidants for HO
, only retarders.
Proof:
1. The
rate constants
for nearly all reactions of
HO
in biology are
10 9 – 10 10 M -1 s -1
. Thus, everything reacts rapidly with it and it will take a lot of a “antioxidant” to inhibit oxidation of the bulk. 2.
Comparing rates
: Rate (HO + Bulk) = SFRBM Sunrise Free Radical School
k b
Rate (HO + Antiox) =
k a
[Bulk] [HO ] [Antiox] [HO ]
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Antioxidant vs Retarder 3. If we want 98% of the HO
to react with an “antioxidant” AND have only a little bit of antioxidant (1% of bulk), then using Rate Bulk Rate Antiox = k
b
[Bulk] [HO
] = k
a
[Antiox] [HO
] we have 2 = k
b
[99%] [HO
] 98 = k
a
[1%] [HO
] 40
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then,
k
a = 5 000 k
b
Antioxidant vs Retarder
4. If
k
a = 5 000 k
b
and k
b
= 2 x 10 9 M -1 s -1 , then k
a
must be 1 x 10 13 M -1 s -1 5. No way, not in water.
In H 2 O k must be <
10 11 M -1 s -1 6. Because the a rate constant of 10 13 M -1 s -1 in H 2 O is not possible and is 100x larger than the upper limit for a rate constant in water, there are no true antioxidants for HO
, only retarders.
7. QED
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[Retarder]
Retarder
Oxidation products without retarder Oxidation products with retarder Oxidation with retarder More retarder and lots of it.
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Time
Antioxidant - no recycling
Oxidation without antioxidant [Antioxidant] Oxidation with antioxidant 43
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Lag time due to kinetic advantage Time
What is a practical kinetic advantage?
Compare the pseudo first-order rate constants.
Rate (LOO
+ Antiox) = k
a
Rate (LOO
+ Bulk) = k
b
where
k a ’
= k
a
[Antiox] [LOO [Bulk] [LOO [Antiox] and
k b
’ = k
b
] = ] =
k
[Bulk]
k a ’ b
’ [LOO
] [LOO
] If 1% “leakage” (damage) is acceptable, then
k a
’ = 100 k
b
’
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If 0.01%, then
k a
’ = 10 000 k
b
’ 44
LDL and TOH
Compare the pseudo first-order rate constants.
Rate (LOO
+ PUFA ) = 40 M -1 s -1 [PUFA] [LOO
] Rate (LOO
+ TOH ) = 10 5 M -1 s -1 [TOH] [LOO
] If [PUFA] in LDL
1.5 M & [TOH] in LDL
0.02 M,* then
k’ TOH
= 30 k ’
PUFA
Leakage about 3% 45
SFRBM Sunrise Free Radical School Estimated from: Bowery VW, Stocker R. (1993) J Am Chem Soc. 115: 6029-6043
Parting Thoughts 1 To test a compound for possible efficacy as a donor, chain-breaking antioxidant, studying its reactions with ROO
would be much more appropriate than with HO
. 46
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Parting Thoughts 2 Antioxidants come in all colors and flavors.
Picture stolen from C. Rice-Evans.
Keep in mind that besides possible antioxidant activity, the primary bio-activity of the “antioxidant” may be very different.
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47 [C. Rice-Evans - next]