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

garry-buettner@uiowa.edu

SFRBM 2005 November

Workshop: Rigorous Detection and Identification of Free Radicals in Biology and Medicine

Ascorbic Acid Structure

(AscH2)

Ascorbic Acid

123

45

6

O

OH

O

HO

HHO

HHOH

H

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-

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

AscH- is a Donor Antioxidant

AscAscH

O

OH

OHO

O

OH

R+ RH+

O

O

HO

O

OH

O

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”.

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

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

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

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.

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

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.

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

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

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

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.

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

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

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

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.

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

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

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

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.

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-

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