Ascorbate (Vitamin C), its Antioxidant Chemistry : 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 The Virtual Free Radical School
Ascorbic Acid Structure : Ascorbic Acid Structure (AscH2)
AscH2 is a Di-acid : AscH2 is a Di-acid 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- .
Forms of Ascorbate : Forms of Ascorbate From 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. [PDF]
Ascorbate Falling Apart : Ascorbate Falling Apart
Kinetics of AscH- Reactions : Kinetics of AscH- Reactions 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. [PDF] These rate constants are for the reaction:
AscH + R Asc + RH
AscH reacts rapidly with these and similar oxidants making it an outstanding donor antioxidant.
AscH- is a Donor Antioxidant : AscH- is a Donor Antioxidant 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-.
Dismutation of Ascorbate Radical : 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.* *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. [PDF]
**Hossain MA, Asada K. (1985) Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. J Biol Chem. 260:12920-12926. 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.**
The Dismutation of Ascorbate Radical is an Equilibrium Reaction : 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, pp75-94. [PDF] 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.)
EPR Detection of Asc- : 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.
Higher Resolution EPR : Higher Resolution EPR With appropriate instrument settings a more detailed spectrum can be observed by EPR. aH4 (1) = 1.76 G
aH5 (1) = 0.07 G
aH6 (2) = 0.19 G
Asc-, Real Time Marker of Oxidative Stress : 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. [PDF][DOI] [Asc-]ss is proportional to the rate of ascorbate oxidation.
Asc-, as an indicator for adventitious transition metals : Asc-, as an indicator for adventitious transition metals The oxidation of ascorbate is very slow in the absence of catalytic metals. This plot shows [Asc-]ss with varying [Fe(III)] in two different chelating environments. Because the iron in EDTA is freely accessible to reductants, Fe(III)EDTA is an excellent catalyst for AscH oxidation. In contrast Fe(III) in Desferal is not accessible to reductants and thus Fe(III)Desferal does not catalyze AscH oxidation.
EPR as well as UV-Vis spectroscopy (265 = 14,500 M-1 cm-1 for AscH) have been used to determine the metal content of buffer solutions.
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. [PDF]
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 [PDF] Fe(III)Desferal Fe(III)EDTA
Thermodynamics of Ascorbate : 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. [PDF][DOI]
Buettner GR. (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation, ‑tocopherol, and ascorbate. Arch Biochem Biophy. 300:535-543. [PDF]
The Pecking Order : 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. [PDF] Note that the donor antioxidants are found in the middle of the “pecking order”.
C and E as Co-antioxidants : 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.
C and E as Co-Antioxidants (1) : C and E as Co-Antioxidants (1) Buettner GR. (1993)
Arch Biochem Biophys.
300:535-543. [PDF] This cartoon represents one leaflet of the lipid bilayer of a membrane.
Once oxygen reacts with the lipid chain, the change in dipole moment will cause the peroxyl radical to “float” to the interface. Vitamin E Lipid
C and E as Co-Antioxidants (2) : C and E as Co-Antioxidants (2) Buettner GR. (1993)
Arch Biochem Biophys.
300:535-543. [PDF] Lipid Vitamin E Vitamin E removes the peroxyl radical; ascorbate can recycle E; enzymes then remove the damaged fatty acid and insert a new one, repairing the lipid.
Recycling of Ascorbate : Recycling of Ascorbate 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. The recycling of ascorbate appears to be an enzyme-dependent process. The two electrons required can come from GSH.
Ascorbate, Summary : Ascorbate, Summary Ascorbate is a versatile, water soluble, donor, antioxidant.
Thermodynamically, it can be considered to be the terminal, small-molecule antioxidant.