Ascorbate Radical:
A Realtime Indicator of
Oxidative Flux
Garry R. Buettner
Free Radical & Radiation Biology and
ESR Facility
The University of Iowa
Iowa City, IA 52242-1101
garry-buettner@uiowa.edu
SFRBM 2005 November
Workshop: Rigorous Detection and Identification of Free
Radicals in Biology and Medicine
Ascorbic Acid Structure
OH
H
H
6
H
HO
5
H
HO
O
4 1
3 2
O
OH
Ascorbic Acid
(AscH2)
AscH2 is a Di-acid
HO
OH
O
O
OH
HO
AscH2
OH
O
HO
pK1 = 4.1
O
O
OH
AscH-
pK2 = 11.8
HO
OH
O
O
O
O
Asc2-
HO
OH
O
OH
H
H
6
H
HO
5 4
O
OH
HO
AscH2
H
+
+H+ -H pK = 4.1
HO
OH
O
O
-e
HO
OH
O
AscH-
HO
O
O
O
Asc2
O
OH
HO
Ascorbic Acid
Forms of
Ascorbate
O
OH
O
AscH
+H+ -H+ pK = 11.8
+
+H+ -H pK = 11.8
OH
O
OH
O
O
1
3 2
-e
HO
OH
O
O
O
O
Asc
-e
OH
O
HO
O
O
O
DHA
-H2O +H2O
OH
HO
O O
O O +H2O
HO
-H2O
O
HO
OH
OH
HO OH
HO OH
DHAA (2)
DHAA (1)(>99%)
(pK ~ 8-9)
See: Buettner GR, Schafer
FQ. (2004) Ascorbate (Vitamin
C) as an Antioxidant. in Vitamin
C:its Functions and
Biochemistry in Animals and
Plants. Ed May JM, Asard H,
Smirnoff N. BIOS Scientific
Publishers. pp 173-188.
-
AscH is a Donor Antioxidant
HO
OH
O
HO
O
+ R
OH
O
AscH
OH
O
O
+ RH
O
O
Asc
Redox Couple
(one-electron reductions)
HO, H+/H2O
RO, H+/ROH (aliphatic alkoxyl radical)
ROO, H+/ROOH (alkyl peroxyl radical)
GS/GS (glutathione)

+
PUFA
,
H
/PUFA-H (bis-allylic-H)
Note that the

+
TO
,
H
/TOH (tocopherol)
donor antioxidants
H2O2, H+/H2O, HO
are found in the
Asc , H+/AscH- (Ascorbate)
middle of the
“pecking order”.
CoQ-, 2H+/CoQH2
Fe(III) EDTA/Fe(II) EDTA
CoQ/CoQ
Buettner GR. (1993) The
pecking order of free
O2/O2
radicals and
Paraquat/Paraquat
antioxidants: Lipid
peroxidation,
Fe(III)DFO/Fe(II)DFO
-tocopherol, and
ascorbate. Arch
RSSR/RSSR  (GSH)
Biochem Biophys.
H2O/e aq
300:535-543.
The
Pecking
Order
E°'/mV
+ 2310
+ 1600
+ 1000
+ 920
+ 600
+ 480
+ 320
+ 282
+ 200
+ 120
- 36
- 160
- 448
- 450
- 1500
- 2870
Kinetics of AscH- Reactions
AscH + R  Asc  + RH
Radical
HO
RO (tert-butyl alkoxyl radical)
ROO (alkyl peroxyl radical,
e.g. CH3OO)
Cl3COO
GS (glutathiyl radical)
UH- (Urate radical)
-1
kobs/M s
-1
(pH 7.4)
1.1 x 1010
1.6 x 109
1-2 x 106
1.8 x 108
6 x 108 (5.6)
6
1 x 10
2 x 105 b
TO (Tocopheroxyl radical)
2 x 105 c
Asc- (dismutation)
9
CPZ+ (Clorpromazine radical action) 1.4 x 10 (5.9)
Fe(III)EDTA / Fe(II)EDTA
102 d
2.7 x 105
O2- / HO2
Very slow
Fe(III)Desferal / Fe(II)Desferal
a
EPR of Asc6
[*10^ 3]
With appropriate
12.5
instrument settings 10.0
a detailed
7.5
spectrum can be
observed by EPR. 5.0
HO
OH
5
O
4
3
O
1
2
O
O
Asc
2.5
aH4 (1) = 1.76 G
aH5 (1) = 0.07 G
0.0
-2.5
-5.0
-7.5
aH6 (2) = 0.19 G -10.0
-12.5
3476 3477 3478 3479 3480 3481 3482 3483
[G]
EPR Detection of Asc6
OH
5
HO
O
4
3
H
The ascorbate
radical is usually
observed as a
simple doublet
species by EPR.
O
1
2
O
O
Asc
The intensity of the
EPR spectrum of
Asc- can be used
as an indicator of
oxidative stress in
vitro and in vivo.
aH = 1.8 G
g = 2.0052
3476.0
3482.0
Gauss
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.*
*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.
The Dismutation of Ascorbate Radical is an
Equilibrium Reaction
2 Asc + H+
AscH2
DHA
K=
2
Asc
AscH + DHA
+
H
total
= 5 x 1014 M2
1 + [H+]/KAscH
2
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.
Asc-, Real Time Marker of Oxidative Stress
Ascorbate Radical in Plasma
EPR Siganl Height of
Asc Radical/(A.U.)
35
[Asc-]ss is
proportional
to the rate
of
ascorbate
oxidation.
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.
Asc-, as an indicator for adventitious
transition metals
Fe(III)EDTA
100
.-
[Asc ]/nM
150
50
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.
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\
Iron, a bit of history
1. Iron contaminates buffers, 0.1 – 1 or more M;
2. Choice of chelating agent can change observations;
3. DETAPAC (DTPA) introduced to free radical
community;
4. Iron a big player in spin trapping;
5. Everything goes better with DETAPAC.
Buettner, G.R. and Oberley, L.W. (1978) "Considerations in the spin trapping of superoxide and hydroxyl radicals
in aqueous systems using 5,5-dimethyl-1-pyrroline-1-oxide." Biochem. Biophys. Res. Commun. 83: 69-74. (
and the Pinawa Meeting, 1977)
Buettner, G.R., Oberley, L.W., and Leuthauser, S.W.H.C. (1978) "The effect of iron on the distribution of superoxide
and hydroxyl radicals as seen by spin trapping and on the superoxide dismutase assay." Photochem.
Photobiol. 28: 693-695. ( and the Pinawa Meeting, 1977)
"Citation Classics", selection by the Institute for Scientific Information, the publishers of Current Contents
Iron, how much is there?
Reagent
Treatment
[Fe]/M
[Cu]/M
50 mM PO4, pH 7.0
none
0.3 - 0.7
 0.13
50 mM PO4,
pH 7.0
Chelating
resin
< 0.1
 0.001
100 mM KCl
none
2.5
--
1 M NaCl
none
--
 0.001
67.5 mM PO4, pH
7.4/4.0 mM KCl
none
3.5 - 8.9
--
50 mM EDTA
none
9.7 - 19.4
--
XO at 20 mU/mL
none
0.004 - 0.7
--
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.
Iron from Syringes
Treatment, pH 7.4 PO4
Chelex 100
[Fe]/M
 0.01 probably < 1 nM
Hamilton, 705-N
5.0 ± 2.9
Gas-Tight, Hamilton 1705TEF (22S Steel needle)
0.18 ± 0.12
1705-TEF (Teflon needle)
0.14 ± 0.03
1725-TEF LL (Steel needle)
0.061 ± 0.008
1725-TEF LL (Teflon needle)
0.015 ± 0.007
Buettner, G.R. (1990) Ascorbate oxidation: UV absorbance of ascorbate and
ESR spectroscopy of the ascorbyl radical as assays for iron. Free Rad
Res Commns, 10: 5-9.
The Ascorbate Test
Ascorbic acid solution (3.5 L of 0.100 M) is added to
3.00 mL of near-neutral buffer solution;
Absorbance is followed for 15 min at 265 nm (AscH265 = 14,500 M-1cm-1);
A loss of more than 0.5% in this time indicates
significant metal contamination; goal <0.05%.
Tips : use AscH2, not Na-AscHDo not interrogate the solution continuously, photochemistry
Clean, clean, clean
ground glass is a disaster
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.
Ascorbate, suggestions for making
a stock solution
Tips:
1. use AscH2, not Na-AscH-; Na-AscH-; is already partially
oxidized.
2. We prepare our ascorbate stock solutions as 100 mM in DI
water. [O2]i = 0.25 mM, but soon is 0 mM with loss of <1% of
AscH-; the pH 2, helping with stability.
3. Clean, clean, clean
4. Ground glass can be a disaster.
5. In the spectrometer, do not interrogate the solution
continuously --- photochemistry
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.
Ascorbate radical, a tool
HO
OH
6
5
O
4
3
O
1
2
O
O
Asc
aH = 1.8 G
g = 2.0052
3476.0
3482.0
Gauss
Buettner GR, Jurkiewicz BA. (1993) The ascorbate free radical as a marker of oxidative stress:
An EPR study. Free Rad Biol Med 14: 49-55.
Ascorbate radical, a tool
240
500 M AscH-
160
80
0
5
6
7
pH
8
9
10
Buettner, G.R. and Jurkiewicz, B.A. (1993) Free Rad. Biol. Med., 14: 49-55.
Ascorbate Power Saturation Curve
Signal Height/A.U.
75
Quantitation
must also
account for
power
saturation.
50
25
40 mW
0
0
5
10
(Power)1/2/mW
15
Buettner GR, Kiminyo KP
(1992) Optimal EPR
detection of weak nitroxide
spin adduct and ascorbate
free radical signals. J
Biochem Biophys Meth 24:
147-151.
Realtime!!!!
EPR of Asc
Radical
h
off
h
off
Buettner GR,
Doherty TD,
Bannister TB.
(1984) Hydrogen
peroxide and
hydroxyl radical
formation by
methylene blue in
the presence of
ascorbate. Rad
Environ Biophys
23: 235-242.
h
on
h
on
UV radiation increases the ascorbate radical signal
in human skin graphs , Ascorbate radical
50
signal in graphs
exposed to UV radiation
(WG 305 nm cutoff and
IR filters);
Signal Height/A.U.
40
, Ascorbate radical
signal in graphs
exposed to visible light
(400 nm cutoff and IR
filters);
30
h
20
, Ascorbate radical
signal in graphs
exposed to room light
10
only. Mean (n=4)  SEM.
0
0
20
Time/min
40
60
Jurkiewicz BA, Buettner
GR. (1996) ESR
detection of free radicals
in UV-irradiated skin:
Mouse versus man.
Photochem Photobiol 64:
918-922.
Whole Animal Studies
Sharma MK, Buettner GR, Spencer K, Kerber RE. (1994) Ascorbyl free radical as a real-time marker of
free radical generation during myocardial reperfusion: An electron paramagnetic resonance study.
Circulation Research 74: 650-658.
Whole Animal Studies
Coronary sinus concentration
of ascorbate free radical
(Asc−) after ischemiareperfusion sequences. The
rise in Asc− concentration is
significantly lower in the
animals receiving the nitric
oxide synthase inhibitor NGnitro—arginine.
Zhang Y, Bissing JW, Xu LJ, Ryan AJ,
Martin SM, Miller FJ, Kregel KC,
Buettner GR, Kerber RE. (2001) Nitric
oxide synthase inhibitors decrease
coronary sinus-free radical concentration
and ameliorate myocardial stunning in an
ischemia-reperfusion model.
J Am Coll Card. 38:546-554
.
60
Asc-
Plasma
700
50
600
40
500
30
400
TO-
20
10
0
Xanthine +
Xanthine Oxidase
50
100
time/min
300
TOH Radical/uM
Ascorbate Radical/nM
AscH- Recycles Tocopherol
Sharma MK, Buettner
GR. (1993) Interaction
of Vitamin C and
Vitamin E during free
radical stress in
plasma: An ESR
study. Free Rad Biol
Med 14: 649-653.
200
150
Interpretation - Summary
1. [Asc•–]ss = f([AscH–], oxidative flux, metals,
comproportionation; true autoxidation);
2. Best done at pH < 7.5 to avoid true autoxidation;
3. Comproportionation usually small contributor;
4. Controls can be a challenge; easiest when sample
is its own control;
5. Realtime indicator of oxidative flux, NOT what
happened in past;
6. Technical: To determine [Asc] account for saturation effects in
EPR spectra of Asc and standard; we use 3-carboxy proxyl.
The End
3476.0
3482.0
Gauss
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.
Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation,
-tocopherol, and ascorbate. Arch Biochem Biophy. 300:535-543.
Instrument Settings:
A starting point with the peddle-to-the-metal
1. Scan range, centered at g  2.005:
a. Bruker:  8 - 10 G
b. Varian, on paper: 40 G
2. Scan rate: Varian 1 G/(12 or 24 s) gives 22 or 45
s between lines - use   1.5 s; Bruker varies,
but maximize  to 1/5 to 1/4 of time to go
through line (peak-to-peak)
3. Mod Amp = 0.65 – 0.8 G; Hpp  0.65 G
4. Nominal power with TM Cavity = 40 mW