The Virtual Free Radical School
Ascorbate (Vitamin C),
its Antioxidant Chemistry
Garry R. Buettner and Freya Q. Schafer
Free Radical and Radiation Biology Program
Department of Radiation Oncology
The University of Iowa
Iowa City, IA 52242-1101
Tel: 319-335-6749
Email: garry-buettner@uiowa.edu or
freya-schafer@uiowa.edu
Ascorbate Chemistry
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Buettner & Schafer 1
Ascorbic Acid Structure
OH
O
HO
HO
O
OH
(AscH2)
Ascorbate Chemistry
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Buettner & Schafer 2
AscH2 is a Di-acid
OH
OH
O
HO
O
OH
HO
A scH 2
p K 1 = 4 .1
OH
O
HO
O
p K 2 = 1 1 .8
OH
O
A scH
-
O
HO
O
O
O
A sc
2-
At pH 7.4, 99.95% of vitamin C will be present
as AscH ; 0.05% as AscH2 and 0.004% as Asc2. Thus, the antioxidant chemistry of vitamin C is
the chemistry of AscH .
Ascorbate Chemistry
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Buettner & Schafer 3
OH
O
HO
HO
O
A scH 2
+H
+
-H
+
p K = 4 .1
OH
OH
O
HO
O
+H
O
A scH
+
-H
-e
O
HO
-O H
O
+
+H
p K = 1 1 .8
A sc
OH
+
-H
+
p K = -0 .8 6
OH
O
O
O
A scH
OH
HO
Forms of
Ascorbate
OH
O
2
-e
O
HO
O
OH
O
O
A sc
-e
O
HO
O
O
O DHA O
-H 2 O
OH
+ H 2O
HO
HO
O
HO
HO
O
OH
OH
D H A A (2 )
O
+ H 2O
-H 2 O
O
HO
O
OH
OH
D H A A (1 ) (> 9 9 % )
From Bors W, Buettner GR.
(1997) The vitamin C radical
and its reactions in Vitamin C in
Health and Disease, ed. by L.
Packer and J. Fuchs, Marcel
Dekker, Inc., New York,
Chapter 4, pp75-94. [PDF]
(p K ~ 8 -9 )
Ascorbate Chemistry
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Buettner & Schafer 4
OH
OH
O
HO
HO
-e -2 H +
O
A scH 2
+e +2 H
OH
O
HO
O
O
A sc
O
O
OH
OH
H
C
OH
C
O
H
C
OH
H
C
OH
HO
C
H
HO
C
H
L -xylo n ic
a cid
C H2OH
O
H
C
OH
HO
C
C H2OH
L -xylo se
Ascorbate Chemistry
O
O
+
C
OH
o xa lic a cid
OH
C
OH
C
O
+
HO
C
H
H
C
OH
HO
C
H
C H2OH
2 ,3 -d ike to -L g u lo n ic a cid
H
C
HO
C
OH
C
O
C H2OH
C
O
C
C
+H 2 O
O D HA O
O
O D HA O
C
O
O
+e
O
O
HO
-e
+
OH
HO
OH
OH
C H2OH
C H2OH
L -lyxo n ic a cid
Ascorbate
Falling
Apart
L -th re o n ic
a cid
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Buettner & Schafer 5
Kinetics of AscH- Reactions
k o b s/ M - 1 s-1
R adical
HO
1. 1 x 10
R O  (t er t - b u t y l a l k o x y l r a d i c a l )
1. 6 x 10
R O O  (a l k y l p er o x y l r a d i c a l , e.g . C H 3 O O  )
1- 2 x 10
10
9
6
These rate constants are
for the reaction:
Cl3CO O 
1. 8 x 10
G S  (gl u t a t h i y l r a d i c a l )
6 x 10
U H - (U r a t e r a d i c a l )
1 x 10
T O  (T o c o p h er o x y l r a d i c a l )
2 x 10 5 b
A s c - (d i s m u t a t i o n )
2 x 10 5
C PZ + (C l o r p r o m a zi n e r a d i c a l a c t i o n )
1. 4 x 10 9 (5 . 9 )
F e(I I I )E D T A / F e(I I )E D T A
 10
O 2 - / H O 2 
8
8


a
Estimated kobs for TO when in a biological membrane.
b
k is pH dependent, thus this is kobs at pH 7.4.
(5 . 6 )
6
2
2 . 7 x 10 5
F e(I I I )D es f er a l / F e(I I )D es f er a l
(p H 7 . 4 )
AscH + R  Asc  + RH
AscH reacts rapidly with
these and similar oxidants
making it an outstanding
donor antioxidant.
V er y s l o w
Adapted from: Buettner GR, Jurkiewicz BA. (1996) Catalytic metals, ascorbate, and free radicals:
combinations to avoid. Rad Research 145:532-541. [PDF]
Ascorbate Chemistry
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Buettner & Schafer 6
-
AscH is a Donor Antioxidant
OH
O
HO
OH
+
OH
O
A scH
O
HO
O
O
R
+
RH
O
O
A sc
AscH- donates a hydrogen atom (H or H+ + e-) to an
oxidizing radical to produce the resonance-stabilized
tricarbonyl ascorbate free radical. AscH has a pKa of
-0.86; thus, it is not protonated in biology and will be
present as Asc-.
Ascorbate Chemistry
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Buettner & Schafer 7
Dismutation of Ascorbate Radical
2 Asc- + H+
AscH- + DHA
kobs (7.4) = 1.4 x 105 M-1 s-1
This rate constant increases by a factor of 10 when phosphate is present.*
This dismutation reaction is the principal route to the elimination of
the Asc- in vitro. However, in vivo it is thought that reducing
enzymes are involved in the removal of this radical, resulting in
the recycling of ascorbate.**
*Reviewed in: Bors W, Buettner GR. (1997) The vitamin C radical and its reactions in Vitamin C in
Health and Disease, ed. by L. Packer and J. Fuchs, Marcel Dekker, Inc., New York, Chapter 4,
pp75-94. [PDF]
**Hossain MA, Asada K. (1985) Monodehydroascorbate reductase from cucumber is a flavin
adenine dinucleotide enzyme. J Biol Chem. 260:12920-12926.
Ascorbate Chemistry
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Buettner & Schafer 8
The Dismutation of Ascorbate Radical is
an Equilibrium Reaction
2 Asc- + H+
A scH2
D HA
K =
2
A sc
H
AscH- + DHA
+
to ta l
= 5 x 10
14
M
2
+
1 + H /K A s c H
2
Because of this equilibrium reaction, Asc- can be formed if both,
DHA and AscH- are present. Note that the equilibrium constant, K,
for this dismutation reaction is pH-dependent. Thus, the higher the pH
the more Asc- would be formed; however, at higher pH DHA is more
unstable. (KAscH2 is the first ionization constant of ascorbic acid.)
Reviewed in: Bors W, Buettner GR. (1997) The vitamin C radical and its reactions in Vitamin C in Health
and Disease, ed. by L. Packer and J. Fuchs, Marcel Dekker, Inc., New York, Chapter 4, pp75-94. [PDF]
Ascorbate Chemistry
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Buettner & Schafer 9
EPR Detection of
Asc
OH
6
HO
O
5
4
3
The ascorbate
radical is usually
observed as a
simple doublet
species by EPR.
O
1
2
O
O
A sc
aH
= 1.8 G
g = 2.0052
3476.0
3482.0
The intensity of the
EPR spectrum of
Asc- can be used
as an indicator of
oxidative stress in
vitro and in vivo.
Gauss
Ascorbate Chemistry
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Buettner & Schafer 10
Higher Resolution EPR
With appropriate
instrument settings
a more detailed
spectrum can be
observed by EPR.
[*1 0 ^ 3 ]
OH
6
1 2 .5
HO
O
5
4
3
1 0 .0
7 .5
O
1
2
O
O
A sc
5 .0
2 .5
0 .0
aH4 (1) = 1.76 G
-2 .5
aH5 (1) = 0.07 G
-7 .5
aH6 (2) = 0.19 G
-5 .0
-1 0 .0
-1 2 .5
3476
Ascorbate Chemistry
3477
3478
3479
3480
[G ]
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3481
3482
3483
Buettner & Schafer 11
Asc-, Real Time Marker of Oxidative Stress
Ascorbate Radical in Plasma
[Asc-]ss is
proportional
to the rate of
ascorbate
oxidation.
EPR Siganl Height of Asc
Radical/(A.U.)
35
25
15
5
0
2.5
5
7.5
10
[AAPH]/mM
[ Asc•-]ss in plasma is directly proportional to oxidative flux: EPR signal height of Asc•- (arbitrary units)
versus AAPH concentration. The solutions contained 58 µM ascorbate in plasma and various amounts
of the free radical-generator AAPH. From: Buettner GR, Jurkiewicz BA. (1993) The ascorbate free
radical as a marker of oxidative stress: An EPR study. Free Radic Biol Med 14: 49-55. [PDF][DOI]
Ascorbate Chemistry
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Buettner & Schafer 12
Asc-, as an indicator for adventitious
transition metals
The oxidation of ascorbate is very slow in
the absence of catalytic metals. This plot
shows [Asc-]ss with varying [Fe(III)] in two
different chelating environments. Because
the iron in EDTA is freely accessible to
reductants, Fe(III)EDTA is an excellent
catalyst for AscH oxidation. In contrast
Fe(III) in Desferal is not accessible to
reductants and thus Fe(III)Desferal does
not catalyze AscH oxidation.
150
100
.-
[Asc ]
Fe(III)EDTA
50
EPR as well as UV-Vis spectroscopy (265 =
14,500 M-1 cm-1 for AscH) have been used
to determine the metal content of buffer
solutions.
Fe(III)Desferal
0
0
5
[Fe(III)]/uM
10
Buettner GR. (1988) In the absence of catalytic metals, ascorbate
does not autoxidize at pH 7: Ascorbate as a test for catalytic metals.
J Biochem Biophys Meth 16: 20-40. [PDF]
Buettner GR. (1990) Ascorbate oxidation: UV absorbance of
ascorbate and ESR spectroscopy of the ascorbyl radical as assays for
iron. Free Rad Res Comm 10: 5-9 [PDF]
Ascorbate Chemistry
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Buettner & Schafer 13
Thermodynamics of Ascorbate
The unpaired electron of Asc- resides in the -system
that includes the tri-carbonyl moiety of ascorbate. This
results in a weakly oxidizing and weakly reducing
radical. Due to its -character Asc- does not react
with oxygen to form dangerously oxidizing peroxyl
radicals. Thermodynamically, it is relatively unreactive
with a one-electron reduction potential of only +282
mV. It is considered to be a terminal, small-molecule
antioxidant.
Buettner GR, Jurkiewicz BA. (1993) The ascorbate free radical as a marker of oxidative stress:
An EPR study. Free Radic Biol Med 14: 49-55. [PDF][DOI]
Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation,
-tocopherol, and ascorbate. Arch Biochem Biophy. 300:535-543. [PDF]
Ascorbate Chemistry
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Buettner & Schafer 14
The
Pecking
Order
Note that the donor
antioxidants are
found in the middle of
the “pecking order”.
R ed o x C o u p le
(o n e-electro n red u ctio n s)

+
H O , H /H 2 O

+
R O , H /R O H (alip ha tic alko xyl rad ic al)

+
R O O , H /R O O H (alkyl p e ro xyl rad ic al)


G S /G S (g lutathio ne )

+
P U F A , H /P U F A- H (b is -allylic -H)

+
T O , H /T O H (to co ph ero l)
+

H 2 O 2 , H /H 2 O , H O

+
A sc , H /A scH (As c or ba te )
+
C oQ , 2H /C oQ H 2
F e(III) E D T A/F e(II) E D T A
-
Buettner GR. (1993) The
pecking order of free radicals
and antioxidants: Lipid
peroxidation, -tocopherol,
and ascorbate. Arch Biochem
Biophys. 300:535-543. [PDF]
Ascorbate Chemistry
C oQ /C oQ

O 2 /O 2

P araq u at/P araq u at
F e(III)D F O /F e(II)D F O

R S S R /R S S R (G S H )

H 2 O /e aq
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E °'/m V
+ 2310
+ 1600
+ 1000
+ 920
+ 600
+ 480
+ 320
+ 282
+ 200
+ 120
- 36
- 160
- 448
- 450
- 1500
- 2870
Buettner & Schafer 15
C and E as Co-antioxidants
As seen in the thermodynamic pecking order
above, the tocopherol radical, TO, is more
oxidizing then Asc-. It is thought that ascorbate
contributes to the recycling of TO back to TOH.
TO + AscH-  TOH + AscThis mechanism is clearly important in protecting
LDL from unwanted oxidations, because LDL lacks
enzymes that could recycle TO. But its importance
in cells and tissues is still being debated.
Ascorbate Chemistry
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Buettner & Schafer 16
C and E as Co-Antioxidants (1)
H2O
OO
OH
R
R
R
O
Lipid
This cartoon represents
one leaflet of the lipid
bilayer of a membrane.
X H
X
O2
a
Ascorbate Chemistry
OO
b
Once oxygen reacts with
the lipid chain, the change
in dipole moment will cause
the peroxyl radical to “float”
to the interface.
Buettner GR. (1993)
Arch Biochem Biophys.
300:535-543. [PDF]
c
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Vitamin E
Buettner & Schafer 17
C and E as Co-Antioxidants (2)
-
x
A sc
F A -C o A
O
R
OH
R
R
R
PL A 2
OOH
R
Ph G Px
G
A sc H
P
H2O
R
O
O
Lipid
Vitamin E removes the
peroxyl radical; ascorbate
can recycle E; enzymes then
remove the damaged fatty
acid and insert a new one,
repairing the lipid.
Vitamin E
Buettner GR. (1993)
Arch Biochem Biophys.
300:535-543. [PDF]
d
Ascorbate Chemistry
e
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Buettner & Schafer 18
Recycling of Ascorbate
D on or
A scH
- 2e
- 2H
D on or H 2
+ 2e
+
+ 2H
+
DHA
The recycling of
ascorbate appears to
be an enzymedependent process.
The two electrons
required can come
from GSH.
May JM, Qu Z. Li X, (2001) Requirement for GSH in recycling of ascorbic acid in endothelial cells.
Biochemical Pharmacology. 62(7):873-81.
Vethanayagam JG, Green EH,. Rose RC, Bode AM. (1999) Glutathione-dependent ascorbate
recycling activity of rat serum albumin. Free Radical Biology & Medicine. 26:1591-8.
Mendiratta S, Qu ZC, May JM. (1998) Enzyme-dependent ascorbate recycling in human
erythrocytes: role of thioredoxin reductase. Free Radical Biology & Medicine. 25:221-8.
Ascorbate Chemistry
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Buettner & Schafer 19
Ascorbate, Summary
Ascorbate is a versatile, water soluble, donor,
antioxidant.
Thermodynamically, it can be considered to be the
terminal, small-molecule antioxidant.
OH
O
HO
OH
+
OH
O
A scH
Ascorbate Chemistry
O
HO
O
O
R
+
RH
O
O
A sc
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Buettner & Schafer 20