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 SFRBM 2005 November Workshop: Rigorous Detection and Identification of Free Radicals in Biology and Medicine

Ascorbic Acid Structure ( AscH 2 )

AscH 2 is a Di-acid

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

AscH - is a Donor Antioxidant

The Pecking Order Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation,  ‑ tocopherol, and ascorbate. Arch Biochem Biophys. 300: Note that the donor antioxidants are found in the middle of the “pecking order”.

Kinetics of AscH - Reactions AscH  + R   Asc   + RH

EPR of Asc  - With appropriate instrument settings a detailed spectrum can be observed by EPR. a H 4 (1) = 1.76 G a H 5 (1) = 0.07 G a H 6 (2) = 0.19 G

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. a H = 1.8 G g = Gauss

Dismutation of Ascorbate Radical k obs (7.4) = 1.4 x 10 5 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, pp **Hossain MA, Asada K. (1985) Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. J Biol Chem. 260:

The Dismutation of Ascorbate Radical is an Equilibrium Reaction 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, pp Asc   + H + AscH  + DHA

Asc  -, Real Time Marker of Oxidative Stress [ 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 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: 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: ( 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: ( and the Pinawa Meeting, 1977) "Citation Classics", selection by the Institute for Scientific Information, the publishers of Current Contents

Iron, how much is there? ReagentTreatment [Fe]/  M[Cu]/  M 50 mM PO 4, pH 7.0none  mM PO 4, pH 7.0 Chelating resin < 0.1  mM KClnone M NaClnone--  mM PO 4, pH 7.4/4.0 mM KCl none mM EDTAnone XO at 20 mU/mLnone 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:

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.

The Ascorbate Test Ascorbic acid solution (3.5  L of 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 -1 cm -1 ); A loss of more than 0.5% in this time indicates significant metal contamination; goal <0.05%. Tips : use AscH 2, not Na-AscH - Do 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:

Ascorbate, suggestions for making a stock solution Tips: 1. use AscH 2, not Na-AscH -; Na-AscH -; is already partially oxidized. 2.We prepare our ascorbate stock solutions as 100 mM in DI water. [O 2 ] 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:

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. a H = 1.8 G g = Gauss

Ascorbate radical, a tool Buettner, G.R. and Jurkiewicz, B.A. (1993) Free Rad. Biol. Med., 14: 49 ‑ 55. pH  M AscH - Signal Height/AU

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: EPR of Asc Radical h on 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:

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:

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: 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 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 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:

Instrument Settings: A starting point with the peddle-to-the-metal 1.Scan range, centered at g  2.005: a. Bruker:  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;  H pp  0.65 G 4. Nominal power with TM Cavity = 40 mW