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![Page 1: Ascorbate Radical: A Realtime Indicator of Oxidative Flux Garry R. Buettner Free Radical & Radiation Biology and ESR Facility The University of Iowa Iowa.](https://reader035.fdocuments.in/reader035/viewer/2022062421/56649e265503460f94b1591f/html5/thumbnails/1.jpg)
Ascorbate Radical: A Realtime Indicator of
Oxidative Flux
Garry R. BuettnerFree Radical & Radiation Biology and
ESR FacilityThe University of Iowa
Iowa City, IA 52242-1101
SFRBM 2005 November
Workshop: Rigorous Detection and Identification of Free Radicals in Biology and Medicine
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Ascorbic Acid Structure
(AscH2)
Ascorbic Acid
123
45
6
O
OH
O
HO
HHO
HHOH
H
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AscH2 is a Di-acid
O
OH
OHO
HO
OHO
OH
OHO
O
OHO
O
OHO
O
OH
pK1 = 4.1 pK2 = 11.8
AscH2 AscH- Asc2-
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Forms of Ascorbate
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.
pK = 4.1
pK = 11.8
pK = 11.8
O O
OHO
OHOH
HO
+H+ -H+
+H+ -H+
+H+ -H+
-e
-e -e
+H2O-H2O
+H2O
-H2O
DHAA (2) DHAA (1)(>99%)(pK ~ 8-9)
OH
HO O O
HOHO
OHOH
Ascorbic Acid
AscH2
HO
OHO
OH
O
HO
AscH-
HO
OHO
OH
O
OAscH
HO
OHO
OH
O
O
Asc2
HO
OHO
O
O
OAsc
HO
OHO
O
O
ODHA
HO
OHO
O
O
O
123
45
6
O
OH
O
HO
HHO
HHOH
H
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AscH- is a Donor Antioxidant
AscAscH
O
OH
OHO
O
OH
R+ RH+
O
O
HO
O
OH
O
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The Pecking
Order
Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation, ‑tocopherol, and ascorbate. Arch Biochem Biophys. 300:535-543.
Redox Couple (one-electron reductions)
E°'/mV
HO, H+/H2O + 2310
RO, H+/ROH (aliphatic alkoxyl radical) + 1600
ROO, H+/ROOH (alkyl peroxyl radical) + 1000
GS/GS
(glutathione) + 920
PUFA, H+/PUFA-H (bis-allylic-H) + 600
TO, H+/TOH (tocopherol) + 480
H2O2, H+/H2O, HO
+ 320
Asc
, H+/AscH- (Ascorbate) + 282
CoQ-, 2H+/CoQH2 + 200
Fe(III) EDTA/Fe(II) EDTA + 120 CoQ/CoQ
- - 36 O2/O2
- 160 Paraquat/Paraquat
- 448
Fe(III)DFO/Fe(II)DFO - 450 RSSR/RSSR
(GSH) - 1500
H2O/e
aq - 2870
Note that the donor antioxidants are found in the middle of the “pecking order”.
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Kinetics of AscH- ReactionsAscH + R Asc + RH
Radical kobs/M-1 s-1 (pH 7.4)
a
HO
1.1 x 1010
RO (tert-butyl alkoxyl radical) 1.6 x 109
ROO (alkyl peroxyl radical,
e.g. CH3OO)
1-2 x 106
Cl3COO 1.8 x 108
GS (glutathiyl radical) 6 x 108 (5.6)
UH- (Urate radical) 1 x 10
6
TO (Tocopheroxyl radical) 2 x 105 b
Asc- (dismutation) 2 x 105 c
CPZ+ (Clorpromazine radical action) 1.4 x 109 (5.9)
Fe(III)EDTA / Fe(II)EDTA 102 d
O2- / HO2
2.7 x 105
Fe(III)Desferal / Fe(II)Desferal
Very slow
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EPR of Asc-
With appropriate instrument settings a detailed spectrum can be observed by EPR.
aH4 (1) = 1.76 G
aH5 (1) = 0.07 G
aH6 (2) = 0.19 G
[G] 3476 3477 3478 3479 3480 3481 3482 3483
-12.5
-10.0
-7.5
-5.0
-2.5
0.0
2.5
5.0
7.5
10.0
12.5
[*10^ 3]
Asc
O
O
OHO
O
OH
123
4
5
6
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EPR Detection of Asc-
The ascorbate radical is usually observed as a simple doublet species by EPR.
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
Asc
O
O
OHO
O
OH
H 123
4
5
6
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Dismutation of Ascorbate Radical
kobs (7.4) = 1.4 x 105 M-1 s-1
This rate constant increases by a factor of 10 when phosphate is present.*
2 Asc- + H+ AscH- + DHA
*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.
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The Dismutation of Ascorbate Radical is an Equilibrium Reaction
K =H+
= 5 x 1014 M2
DHA AscH2total
Asc2
1 + [H+]/KAscH2
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.
2 Asc + H+ AscH + DHA
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Asc-, Real Time Marker of Oxidative Stress
Ascorbate Radical in Plasma
5
15
25
35
0 2.5 5 7.5 10
[AAPH]/mM
EP
R S
igan
l Hei
gh
t of
Asc
Rad
ical
/(A.U
.)
[ 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-]ss is proportional to the rate
of ascorbate oxidation.
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Asc-, as an indicator for adventitious transition metals
0
50
100
150
0 5 10
[Fe(III)]/uM
[As
c.-
]/n
M
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\
Fe(III)Desferal
Fe(III)EDTA
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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
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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.
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Iron from Syringes
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.
Treatment, pH 7.4 PO4 [Fe]/M
Chelex 100 0.01 probably < 1 nM
Hamilton, 705-N 5.0 ± 2.9
Gas-Tight, Hamilton 1705-
TEF (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
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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-AscH-
Do not interrogate the solution continuously, photochemistry
Clean, clean, clean
ground glass is a disasterBuettner 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.
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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.
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Ascorbate radical, a tool
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.
aH = 1.8 G
g = 2.0052
3476.0 3482.0
Gauss
Asc
O
O
OHO
O
OH
123
4
56
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Ascorbate radical, a tool
Buettner, G.R. and Jurkiewicz, B.A. (1993) Free Rad. Biol. Med., 14: 49‑55.
pH
0
80
160
240
5 6 7 8 9 10
500 M AscH-
Sig
nal
Hei
gh
t/A
U
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0
25
50
75
0 5 10 15
(Power)1/2/mW
Sig
nal
Hei
gh
t/A
.U.
Ascorbate Power Saturation Curve
40 mW
Quantitation must also account for power saturation.
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.
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Realtime!!!!
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.
EPR of Asc Radical
h on
h on
h off h
off
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UV radiation increases the ascorbate radical signal in human skin graphs , Ascorbate radical
signal in graphs exposed to UV radiation (WG 305 nm cutoff and IR filters);
, Ascorbate radical signal in graphs exposed to visible light (400 nm cutoff and IR filters);
, Ascorbate radical signal in graphs exposed to room light only. Mean (n=4)
SEM. Jurkiewicz BA, Buettner GR. (1996) ESR detection of free radicals in UV-irradiated skin: Mouse versus man. Photochem Photobiol 64: 918-922.
0
10
20
30
40
50 S
ign
al H
eig
ht/
A.U
.
0 20 40 60
Time/min
h
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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.
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Whole Animal Studies
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.
Coronary sinus concentration of ascorbate free radical (Asc−) after ischemia-reperfusion sequences. The rise in Asc− concentration is significantly lower in the animals receiving the nitric oxide synthase inhibitor NG-
nitro—arginine.
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AscH- Recycles Tocopherol
10
20
30
40
50
60
0 50 100 150
time/min
As
co
rba
te R
ad
ica
l/nM
200
300
400
500
600
700T
OH
Ra
dic
al/u
M
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.
Plasma
Xanthine + Xanthine Oxidase
Asc-
TO-
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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.
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The End
3476.0 3482.0
Gauss
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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.
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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