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 Oxygen Society Education Program Buettner & Schafer 1

 Ascorbic Acid Structure
OH

HO O
O

HO OH
(AscH2)

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 2

 AscH2 is a Di-acid
OH OH OH
HO O O pK = 4.1 HO O O
1
O pK2 = 11.8 HO O

HO OH O OH O O
AscH2 - 2-
AscH Asc

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 Oxygen Society Education Program Buettner & Schafer 3

 OH

HO O O

Forms of
HO OH
AscH2

+ +
+H -H pK = 4.1

HO
OH
O O HO
OH
O O
Ascorbate
-e

OH
AscH-
O O OH
AscH

+ +
+H+ -H+ pK = 11.8 +H -H pK = -0.86

OH OH OH
HO O O HO O O
O HO O
-e -e

O 2 O O O
Asc Asc O DHA O
From Bors W, Buettner GR.
-H2O +H2O (1997) The vitamin C radical
OH
HO and its reactions in Vitamin C
HO O
O
O
O in Health and Disease, ed. by
+H2O

-H2O
L. Packer and J. Fuchs, Marcel
HO OH O OH
HO OH HO OH Dekker, Inc., New York,
DHAA (2) DHAA (1) (>99%) Chapter 4, pp75-94.
(pK ~ 8-9)

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 4

 OH OH OH
HO O O -e -2H+ HO O O -e HO O O
+
+e +2H +e
HO OH O
AscH2 O
Asc O DHA O

O OH O OH O
OH C C OH
C
HO O O C O C OH HO C H
+H2O
C O C OH + H C OH
O DHA O H C OH HO C H HO C H
HO C H CH2OH CH 2OH
CH2OH L-xylonic L-lyxonic acid
acid
2,3-diketo-L-
gluconic acid

CH2OH
C O
O OH
O
C
OH
Ascorbate
H C OH C + H C OH
HO C
CH2OH O
C
OH
HO C
CH 2OH
Falling
L-xylose oxalic acid L-threonic
acid Apart

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 5

 Kinetics of AscH- Reactions
Radical kobs/M -1s-1 (pH 7.4)

HO• 10
1.1 x 10
RO • (tert -butyl alkoxyl radical) 1.6 x 109
ROO• (alkyl peroxyl radical, e.g. CH3OO•) 6
1-2 x 10
These rate constants are
Cl3COO • 1.8 x 108
for the reaction:
GS• (glutathiyl radical) 6 x 108 (5.6)
UH•- (Urate radical) 1 x 10
6 AscH− + R• → Asc• − + RH
TO• (Tocopheroxyl radical) 2 x 105 b
AscH− reacts rapidly with
Asc•- (dismutation) 2 x 105
these and similar oxidants
CPZ •+ (Clorpromazine radical action) 1.4 x 109 (5.9)
Fe(III)EDTA/ Fe(II)EDTA 2
making it an outstanding
≈10
donor antioxidant.
O2 •-/HO 2• 2.7 x 105
Fe(III)Desferal / Fe(II)Desferal Very slow

a Estimated kobs for TO• when in a biological membrane.

b k is pH dependent, thus this is kobs at pH 7.4.
Adapted from: Buettner GR, Jurkiewicz BA. (1996) Catalytic metals, ascorbate, and free radicals:
combinations to avoid. Rad Research 145:532-541.

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 6

 -
AscH is a Donor Antioxidant
OH OH
HO O HO O
O O
+ R + RH
O OH O O
AscH Asc

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 Oxygen Society Education Program 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.
**Hossain MA, Asada K. (1985) Monodehydroascorbate reductase from cucumber is a flavin
adenine dinucleotide enzyme. J Biol Chem. 260:12920-12926.

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 8

 The Dismutation of Ascorbate Radical is
an Equilibrium Reaction
2 Asc•- + H+ AscH- + DHA
DHA AscH2
total
K= 2
= 5 x 1014 M 2
Asc H+ 1 + H +/K AscH
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.

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 9

 EPR Detection of Asc•-
OH
6
5 O The ascorbate
HO O
4
3 2
1 radical is usually
O O
observed as a
Asc simple doublet
species by EPR.

The intensity of the

aH = 1.8 G EPR spectrum of
Asc•- can be used
g = 2.0052 as an indicator of
oxidative stress in
vitro and in vivo.
3476.0 3482.0
Gauss

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 10

 Higher Resolution EPR
With appropriate [*10^ 3]
OH
instrument settings 12.5
5
6
O
HO O
a more detailed 10.0 4
3 2
1

spectrum can be 7.5

O O
observed by EPR. 5.0 Asc

2.5

0.0

-2.5
aH4 (1) = 1.76 G -5.0

aH5 (1) = 0.07 G -7.5

-10.0

aH6 (2) = 0.19 G -12.5

3476 3477 3478 3479 3480 3481 3482 3483

[G]

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 11

 Asc•-, Real Time Marker of Oxidative Stress
Ascorbate Radical in Plasma

35 [Asc•-]ss is
proportional
EPR Siganl Height of Asc

to the rate of
Radical/(A.U.)

25 ascorbate
oxidation.

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.

Ascorbate Chemistry Oxygen Society Education Program 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
150 shows [Asc•-]ss with varying [Fe(III)] in two
different chelating environments. Because
the iron in EDTA is freely accessible to
Fe(III)EDTA reductants, Fe(III)EDTA is an excellent
100 catalyst for AscH− oxidation. In contrast
[Asc ]
.-

Fe(III) in Desferal is not accessible to

reductants and thus Fe(III)Desferal does
not catalyze AscH− oxidation.
50
EPR as well as UV-Vis spectroscopy (ε 265 =
Fe(III)Desferal 14,500 M-1 cm-1 for AscH− ) have been used
to determine the metal content of buffer
0 solutions.
0 5 10 Buettner GR. (1988) In the absence of catalytic metals, ascorbate
does not autoxidize at pH 7: Ascorbate as a test for catalytic metals.

[Fe(III)]/uM J Biochem Biophys Meth 16: 20-40.

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\

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 13

Thermodynamics of Ascorbate
The unpaired electron of Asc•- resides in the π-system
that includes the tri-carbonyl moiety of ascorbate. This
results in a weakly oxidizing and weakly reducing
radical. Due to its π-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.
Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation,
α-tocopherol, and ascorbate. Arch Biochem Biophy. 300:535-543.

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 14

 Redox Couple E°'/mV
The •
(one-electron reductions)
+
HO , H /H2O + 2310
Pecking • +
RO , H /ROH (aliphatic alkoxyl radical)
• +
+ 1600
ROO , H /ROOH (alkyl peroxyl radical) + 1000
Order • −
GS /GS (glutathione)
• +
+ 920
PUFA , H /PUFA-H (bis-allylic-H) + 600
Note that the donor TO•, H+/TOH (tocopherol) + 480
+ •
antioxidants are H2O2, H /H2O, HO + 320
found in the middle of Asc•− , H+/AscH- (Ascorbate) + 282
•- +
the “pecking order”. CoQ , 2H /CoQH2 + 200
Fe(III) EDTA/Fe(II) EDTA + 120
•-
CoQ/CoQ - 36
•−
O2/O2 - 160
Buettner GR. (1993) The •−
Paraquat/Paraquat - 448
pecking order of free radicals
and antioxidants: Lipid Fe(III)DFO/Fe(II)DFO - 450
peroxidation, α-tocopherol, •−
- 1500
and ascorbate. Arch Biochem RSSR/RSSR (GSH)
−
Biophys. 300:535-543. H2O/e aq - 2870

Ascorbate Chemistry Oxygen Society Education Program 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 Oxygen Society Education Program Buettner & Schafer 16

C and E as Co-Antioxidants (1)
H2 O

OO OH
R R

R
O

Lipid This cartoon represents

one leaflet of the lipid
X H bilayer of a membrane.
Once oxygen reacts with
X
the lipid chain, the change
in dipole moment will cause
O2 the peroxyl radical to “float”
OO
to the interface.

Buettner GR. (1993)

Arch Biochem Biophys.
Vitamin E
300:535-543.
a b c
Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 17
C and E as Co-Antioxidants
-
(2)
H2O AscH Asc FA-CoA

Px
G
OOH O OH

PhGPx
PLA2
R R R R

R R
O O

Lipid
Vitamin E removes the
peroxyl radical; ascorbate
can recycle E; enzymes then
remove the damaged fatty Vitamin E
acid and insert a new one,
repairing the lipid.

Buettner GR. (1993)

Arch Biochem Biophys.
300:535-543.
d e
Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 18
 Recycling of Ascorbate
Donor AscH
The recycling of
ascorbate appears to
be an enzyme-
- 2e + 2e dependent process.
- 2H+ + 2H+ The two electrons
required can come
DonorH2 DHA 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 Oxygen Society Education Program 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 OH
HO O HO O
O O
+ R + RH
O OH O O
AscH Asc

Ascorbate Chemistry Oxygen Society Education Program Buettner & Schafer 20