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: garry-buettner@uiowa.edu
SFRBM Sunrise Free Radical School
or freya-schafer@uiowa.edu
SFRBM Sunrise Free Radical School
1
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
SFRBM Sunrise Free Radical School
2
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?
SFRBM Sunrise Free Radical School
3
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.
SFRBM Sunrise Free Radical School
4
Elementary Lipid Peroxidation
L-H + X
 L + XH
L + O2
3 x 108 M-1 s-1
 LOO
LOO + L-H
 40 M-1 s-1
 L + LOOH
LOO + “R”
 LOOR
SFRBM Sunrise Free Radical School
Initiation
Propagation
Cycle
Termination
5
Preventive Antioxidants
Don’t let it get started.
SFRBM Sunrise Free Radical School
6
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, …
SFRBM Sunrise Free Radical School
7
Preventive Antioxidants:
Targeting Metals
Fe & Cu are the principal metals targeted–
loosely bound*
Proteins & metals –
Transferrin / Hemoglobin / Ceruloplasmin
Chelates –
Fe3+ – EDTA, DETAPAC (DTPA), Desferal
Fe2+ – Phenanthrolines, …
* “Loosely” bound iron on proteins, DNA as well as iron in hemes
can be dangerous.
8
SFRBM Sunrise Free Radical School
Preventive Antioxidants:
Why target metals?
Because they promote oxidant production.
Fe(II)chelate + H2O2  HO + Fe(III)chelate + OHor
Fe(II)chelate + LOOH  LO + Fe(III)chelate + LOH
and
Fe(II)chelate + O2  Oxidantsa
a
Qian SY, Buettner GR. (1999) Iron and dioxygen chemistry is an important route to initiation of
9
biological free radical oxidations: An electron paramagnetic resonance spin trapping
SFRBM Sunrise Free Radical School
study.Free Radic Biol Med, 26: 1447-1456.
Metal Deactivation
Fe(III)/Fe(II)
O
O
O C H 2C
N
CH2
CH2
EDTA
CH2
C O
E = + 120 mV
CH2
C O
Rxn with O2-
N
O C H 2C
O
O
EDTA Ethylenediaminetetraacetic acid anion
O
O
O C H2C
N (CH2)2
O C H2C
O
k = 106 M-1 s-1
N
CH2
(CH2)2
C O
CH2
C O
CH2
C O
N
O
O
DETAPAC
Diethylenetriaminepentaacetic
acid anion
SFRBM
Sunrise Free
Radical School
DETAPAC
E = + 30 mV
Rxn with O2k < 102 M-110s-1
Metal Deactivation: Why the difference?
H 2O
Fe(III)EDTA
Size -- too small,
leaving a site for H2O
SFRBM Sunrise Free Radical School
11
Metal Deactivation: Desferal
E (Fe(III)DFO/Fe(II)DFO) = - 450 mV
Kstability Fe(III)  1030.6
k (with O2-) < 103 M-1 s-1
Kstability Fe(II)  107.2
De-activates Fe(III) kinetically (no H2O of coordination)
and
thermodynamically.
H
H N
O
O
C NH
C NH
(CH2)5 (CH2)2 (CH2)5 (CH2) (CH2)5 CH
3
N C
N C
N C
HO O
HO O
SFRBM Sunrise Free Radical School
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, …
SFRBM Sunrise Free Radical School
13
Enzymes targeting peroxides: H2O2 , LOOH
Catalase:
2H2O2  2H2O + O2
GPx (GPx1):
H2O2 + 2GSH  2H2O + GSSG or
ROOH + 2GSH  H2O + ROH + GSSG
PhGPx (GPx4):
PLOOH + 2GSH  PLOH + GSSG + H2O
Prx (peroxidredoxins): H2O2 + Trx(SH)2  2H2O + Trx(SS)
1-cysPrx:
PLOOH + 2GSH  PLOH + GSSG + H2O
Non-enzymatic rxns H2O2 + 2GSH  2H2O + GSSG or
SFRBM Sunrise Free Radical School
ROOH + 2GSH  H2O + ROH + GSSG
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Glutathione (GSH)
NH3
O
H O
O
C CH CH2 CH2 C NH C C NH CH2 C
O
O
CH2
SH
O
glutamate
cysteine
Glutathione is a tri-peptide
SFRBM Sunrise Free Radical School
glycine
15
Preventive Antioxidants:
Removing Hydroperoxides
GSH will react directly with H2O2,
albeit very slowly.
2 GSH + H2O2  2 H2O + GSSG
kobs (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.
16
Hydroperoxide removal by GSH is mainly via
coupled enzyme reactions
2 GSH + ROOH  GSSG + H2O + ROH
GSH synthetic enzymes
inhibits
ROOH
2 GSH
NADPH
2e- GSSG
GPx
-
2e G-6-P
reductase
ROH
+ H 2O
GSSG
SFRBM Sunrise Free Radical School
primary
6-P-G
dehydrogenase
NADP+
G-6-P
17
stimulates
secondary
secondary
Pyruvate and H2O2
Pyruvate is a three-carbon ketoacid
produced during glycolysis.
Pyruvate can remove H2O2 by a
stoichiometric chemical reaction.
O
H3C C C
O
O
O
+ H2O2
Pyruvate
SFRBM Sunrise Free Radical School
H 3C
C
O
Acetate
+ CO2 + H2O
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, …
SFRBM Sunrise Free Radical School
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Singlet oxygen quenching,
avoiding peroxides
Singlet Oxygen 1O2, i.e. oxygen with extra energy
1
-1 above the ground state
O
23.4
kcal
mol
g 2
Singlet oxygen is electrophilic, thus it reacts with
the double bonds of lipids.
(No free radicals; hydroperoxides formed.)
k  2 x 105 M-1 s-1

SFRBM Sunrise Free Radical School
1O
2
+ PUFA
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PUFA-OOH
O
H
Linoleic acid
-H
1O2
B
A
OOH
B
A
9-OOH (30%)
+ O2, H
A
OOH
B
9-OOH (50%)
+
HOO
13-OOH (50%)
LOOHs:
1O vs
2
radicals
+
HOO
A
B
10-OOH (20%)
+
OOH
B
A
12-OOH (20%)
+
HOO
SFRBM Sunrise Free Radical School
13-OOH (30%)
21
Quenching of 1O2
Chemical quenching is a term used to signify
that an actual chemical reaction has occurred.
Hydroperoxide formation is chemical quenching.
1O
2
+ LH  LOOH
Physical quenching is the removal of the
excitation energy from 1O2 without any chemical
changes.
1O
2
+ -carotene  O2 + -carotene*
-carotene*  -carotene + heat
SFRBM Sunrise Free Radical School
22
Antioxidants, the road ahead
1. Overview and vocabulary
2. Preventive
3. Chain-breaking
4. Retarder vs true antioxidant
SFRBM Sunrise Free Radical School
23
Chain-Breaking Antioxidants
In general chain breaking antioxidants
act by reacting with peroxyl radicals,
ROO
SFRBM Sunrise Free Radical School
24
Chain breaking Antioxidants can be:
A) Donor antioxidant, e.g. tocopherol,
ascorbate, uric acid, …
B) Sacrificial antioxidant, e.g. nitric
oxide
SFRBM Sunrise Free Radical School
25
Peroxyl Radicals as Targets
RH + ROO

ROOH + R
R + O2

ROO
Peroxyl radicals, ROO, are often the
chain-carrying radical.


26
The
chain
SFRBM
Sunrisereaction
Free Radical can
Schoolalso be broken by intercepting R . In biology
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
SFRBM Sunrise Free Radical School
 LOONO
27
Characteristics of a Good Chainbreaking Antioxidant
a. Both Antioxidant & Antiox should be
relatively UN-reactive
b. Antiox - decays to harmless products
c. Does not add O2 to make a peroxyl
radical
d. Renewed (Recycled) – somehow
e. If the chain-breaking antioxidant is a
SFRBM Sunrise
Free Radical School atom donor, it should be in
hydrogen
the middle of the pecking order.
28
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
SFRBM Sunrise Free Radical School
Buettner GR. (1993) Arch Biochem Biophy. 300:535-543.
Termination
29
Redox Couple
(one-electron reductions)

+
HO , H /H2O

+
RO , H /ROH (aliphatic alkoxyl radical)

+
Depending on their
ROO , H /ROOH (alkyl peroxyl radical)


reduction potential,
GS /GS (glutathione)

+
antioxidants can
PUFA , H /PUFA-H (bis-allylic-H)

+
recycle each other.
TO , H /TOH (tocopherol)

+
For example,
H2O2, H /H2O, HO

+
ascorbate with a
Asc , H /AscH (Ascorbate)
+
reduction potential
CoQ , 2H /CoQH2
Fe(III) EDTA/Fe(II) EDTA
of +282 mV can
CoQ/CoQ
recycle TO (+480

O2/O2
mV) and urate
Paraquat/Paraquat
(+590 mV).
Fe(III)DFO/Fe(II)DFO

RSSR/RSSR
(GSSG)
Buettner GR. (1993)

Arch
Biochem
Biophy.
SFRBM
Sunrise
Free Radical SchoolH2O/e aq
The Pecking
Order
300:535-543.
E°'/mV
+2310
+1600
+1000
+920
+600
+480
+320
+282
+200
+120
-36
-160
-448
-450
-1500
2870
-30
Donor Antioxidant - Vitamin E
CH3
CH3
O
CH3
(CH2)3CH(CH2)3CH(CH2)3CH(CH3)2
CH3
CH3
O
Reaction with lipid peroxides:
CH3
LOO + TOH  LOOH + TO
TO
Recycling reaction with ascorbate
OH
O
HO
OH
O
HO
O
O
+ TO
OH
O
SFRBM Sunrise Free Radical School
AscH
+ TOH
O
O
Asc
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
H
N
HN
O
OH
N
H
N
UH3
UH2
pKa1 = 5.4
-
UH2pKa2 = 9.8
Uric acid
Normal urate concentrations in human plasma
range from 0.2 – 0.4 mM.
32
Ames
BN etFree
al. (1981)
acid provides an antioxidant defense in humans against oxidantSFRBM
Sunrise
Radical Uric
School
and radical-caused aging and cancer. A hypothesis. Proc. Natl Acad Sci. USA 78, 6858.
Uric Acid Reacts with Peroxyl Radicals
k = 3 x 106 M-1 s-1
ROO + UH2-  ROOH + UHRecycling by Ascorbate:
k = 1 x 106 M-1 s-1
UH- + AscH-  UH2- + Asc SFRBM Sunrise Free Radical School
33
Chain Breaking Antioxidant
Sacrificial – Nitric Oxide
radical,
reactive
but t1/2~s
small molecule,
fits everywhere
NO
uncharged,
diffusable
SFRBM Sunrise Free Radical School
lipid
soluble
34
Nitric Oxide as Antioxidant
Preventive: NO coordinates with heme-iron,
heme Fe2+ + NO
heme Fe2+ NO
We have used this for centuries in food preservation, the "sausage" effect.
Chain-breaking: NO can react with oxyradicals:
ROO + NO
RO + NO
NO
SFRBM
Sunrise Free
Radical School
upregulates
systems
that contribute
ROONO
RONO
to the antioxidant network:
heme oxygenase, ferritin, hsp70, and -glutamylcysteine synthetase
35
Fe2+
O2, H2O2
Nitric Oxide as ChainBreaking Antioxidant in
Lipid Peroxidation
[Fe2+-O2], OH
LH
O2
LOONO
L
OLOOH
LH
NO LOO
OLOONO
NO
LH
Sen
h 1 LH
O2
LOOH
O2
Asc
SFRBM Sunrise Free Radical School
AscH
OLOO
O2
Fe2+
LO
36
Fe3+
LO
Ld
Chain Breaking Antioxidant – Nitroxide
Example nitroxide
HO
NO
Tempol
A possible antioxidant cycle for a nitroxide
R2
NO
+
e-,H+
R1
R2
NOH
R1
nitroxide
hydroxylamine
R2
.R2
NOH
+ Rox
SFRBM Sunrise
R1 Free Radical School
RoxH
+
NO
R1
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.
SFRBM Sunrise Free Radical School
38
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 109 – 1010 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
= kb [Bulk] [HO]
Rate (HO + Antiox) = ka [Antiox] [HO]
39
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
RateBulk
= kb [Bulk] [HO]
RateAntiox = ka [Antiox] [HO]
we have
2
= kb [99%] [HO]
98 = ka
[1%] [HO]
SFRBM Sunrise Free Radical School
then, ka = 5 000 kb
40
Antioxidant vs Retarder
4. If ka = 5 000 kb and kb = 2 x 109 M-1 s-1,
then ka must be 1 x 1013 M-1 s-1
5. No way, not in water.
In H2O k must be < 1011 M-1 s-1
6. Because the a rate constant of 1013 M-1 s-1 in H2O 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.SFRBM
QED
Sunrise Free Radical School
41
Retarder
Oxidation products without
Oxidation
retarder
Oxidation
products
with
retarder
[Retarder]
SFRBM Sunrise Free Radical School
Oxidation
with retarder More
retarder
and lots
of it.
42
Time
Antioxidant - no recycling
Oxidation without
antioxidant
[Antioxidant]
Oxidation with
antioxidant
43
SFRBM Sunrise Free Radical School
Lag time due to
kinetic advantage
Time
What is a practical kinetic advantage?
Compare the pseudo first-order rate constants.
Rate (LOO + Antiox) = ka [Antiox] [LOO] = ka’ [LOO]
Rate (LOO + Bulk)
= kb [Bulk] [LOO]
= kb’ [LOO]
where ka’ = ka [Antiox] and kb’ = kb [Bulk]
If 1% “leakage” (damage) is acceptable,
then ka’ = 100 kb’
SFRBM Sunrise Free Radical School
If 0.01%, then ka’ = 10 000 kb’
44
LDL and TOH
Compare the pseudo first-order rate constants.
Rate (LOO + PUFA ) = 40 M-1 s-1 [PUFA] [LOO]
Rate (LOO + TOH ) = 105 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%
SFRBM Sunrise Free Radical School
Estimated from: Bowery VW, Stocker R. (1993) J Am Chem Soc. 115: 6029-6043
45
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.
SFRBM Sunrise Free Radical School
46
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. 47
SFRBM Sunrise Free Radical School
[C. Rice-Evans - next]