What is an antioxidant? How do antioxidants work?

Transcription

1 What is an antioxidant? How do antioxidants work? Garry R. Buettner and Freya Q. Schafer Free Radical and Radiation Biology Program and ESR Facility The University of Iowa Iowa City, IA Tel: or 1

2 Antioxidants, the road ahead This presentation focuses on the action/reaction of small molecule antioxidants. 1. verview and vocabulary 2. Preventive 3. Chain-breaking 4. Retarder vs true antioxidant 2

3 Antioxidants: A definition A substance when present in trace (small) amounts inhibits oxidation of the bulk. R A little bit goes a long way. So, what is a little bit? 3

4 Antioxidants: two broad classes Preventive and Chain-Breaking Preventive antioxidants intercept oxidizing species before damage can be done. Chain breaking antioxidants slow or stop oxidative processes after they begin, by intercepting the chain-carrying radicals. 4

5 Elementary Lipid Peroxidation L-H + X L + XH Initiation 3 x 10 8 M -1 s -1 L + 2 L Propagation L + L-H 40 M -1 s -1 L + LH Cycle L + R LR Termination 5

6 Preventive Antioxidants Don t let it get started. 6

7 Preventive antioxidants act by: A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, C. Quenching singlet oxygen, e.g. β-carotene, lycopene, bilirubin, 7

8 Preventive Antioxidants: Targeting Metals Fe & Cu are the principal metals targeted loosely bound* Proteins & metals Transferrin / Hemoglobin / Ceruloplasmin Chelates Fe 3+ EDTA, DETAPAC (DTPA), Desferal Fe 2+ Phenanthrolines, * Loosely bound iron on proteins, DNA as well as iron in hemes can be dangerous. 8

9 Preventive Antioxidants: Why target metals? Because they promote oxidant production. Fe(II)chelate + H 2 2 H + Fe(III)chelate + H - or Fe(II)chelate + LH L + Fe(III)chelate + LH and Fe(II)chelate + 2 xidants a a Qian SY, Buettner GR. (1999) Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: An electron paramagnetic resonance spin trapping 9 study.free Radic Biol Med, 26:

10 Metal Deactivation C H 2 C CH 2 C N CH 2 CH 2 N C H 2 C CH 2 C EDTA Ethylenediaminetetraacetic acid anion Fe(III)/Fe(II) EDTA E = mv Rxn with - 2 k = 10 6 M -1 s -1 C H 2 C CH 2 C N (CH 2 ) 2 N (CH 2 ) 2 N C H 2 C CH 2 C CH 2 C DETAPAC Diethylenetriaminepentaacetic acid anion DETAPAC E = + 30 mv Rxn with - 2 k < 10 2 M -1 s -1 10

11 Metal Deactivation: Why the difference? H 2 Fe(III)EDTA Size -- too small, leaving a site for H 2 11

12 Metal Deactivation: Desferal E (Fe(III)DF/Fe(II)DF) = mv K stability Fe(III) k (with 2 - )< 10 3 M -1 s -1 K stability Fe(II) De-activates Fe(III) kinetically (no H 2 of coordination) and thermodynamically. H H N (CH 2 ) 5 N C H C NH (CH 2 ) 2 (CH 2 ) 5 N C H Deferrioximine C NH (CH 2 ) (CH 2 ) 5 N C H CH 3 12

13 Preventive antioxidants act by: A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, C. Quenching singlet oxygen, e.g. β-carotene, lycopene, bilirubin, 13

14 Enzymes targeting peroxides: H 2 2, LH Catalase: 2H 2 2 2H GPx (GPx1): H GSH 2H 2 + GSSG or RH + 2GSH H 2 + RH + GSSG PhGPx (GPx4): PLH + 2GSH PLH + GSSG + H 2 Prx (peroxidredoxins): H Trx(SH) 2 2H 2 + Trx(SS) 1-cysPrx: PLH + 2GSH PLH + GSSG + H 2 Non-enzymatic rxns H GSH 2H 2 + GSSG or RH + 2GSH H 2 + RH + GSSG 14

15 Glutathione (GSH) C NH 3 CH CH 2 CH 2 C H NH C C CH 2 SH NH CH 2 C glutamate cysteine glycine Glutathione is a tri-peptide 15

16 Preventive Antioxidants: Removing Hydroperoxides GSH will react directly with H 2 2, albeit very slowly. 2 GSH + H H 2 + GSSG k obs (7.4) 1 M -1 s -1 * Appears to be too slow for biological significance. * Estimated from: Radi et al. (1991) J Biol Chem. 266:

17 Hydroperoxide removal by GSH is mainly via coupled enzyme reactions 2 GSH + RH GSSG + H 2 + RH GSH synthetic enzymes inhibits RH 2 GSH NADPH 6-P-G RH + H 2 GPx 2e - 2e - GSSG GSSG reductase NADP + G-6-P dehydrogenase G-6-P stimulates 17 primary secondary secondary

18 Pyruvate and H 2 2 Pyruvate is a three-carbon ketoacid produced during glycolysis. Pyruvate can remove H 2 2 by a stoichiometric chemical reaction. H 3 C C C + H 2 2 H 3 C C + C 2 + H 2 Pyruvate Acetate 18

19 Preventive antioxidants act by: A. Deactivating metals, e.g. transferrin, ferritin, Desferal, DETAPAC, EDTA, B. Removing hydroperoxides, e.g. catalase, glutathione peroxidases, pyruvate, C. Quenching singlet oxygen, e.g. β-carotene, lycopene, bilirubin, 19

20 Singlet oxygen quenching, avoiding peroxides Singlet xygen 1 2, i.e. oxygen with extra energy 1 g kcal mol -1 above the ground state Singlet oxygen is electrophilic, thus it reacts with the double bonds of lipids. (No free radicals; hydroperoxides formed.) k 2 x 10 5 M -1 s PUFA PUFA-H

21 A A - H + 2, H 9-H (50%) + H 13-H (50%) B H B 12 A Linoleic acid H H B 9-H (30%) + H A B 10-H (20%) + H A B 12-H (20%) H + 13-H (30%) LHs: 1 2 vs radicals 21

22 Quenching of 1 2 Chemical quenching is a term used to signify that an actual chemical reaction has occurred. Hydroperoxide formation is chemical quenching LH LH Physical quenching is the removal of the excitation energy from 1 2 without any chemical changes β-carotene 2 + β-carotene* β-carotene* β-carotene + heat 22

23 Antioxidants, the road ahead 1. verview and vocabulary 2. Preventive 3. Chain-breaking 4. Retarder vs true antioxidant 23

24 Chain-Breaking Antioxidants In general chain breaking antioxidants act by reacting with peroxyl radicals, R 24

25 Chain breaking Antioxidants can be: A) Donor antioxidant, e.g. tocopherol, ascorbate, uric acid, B) Sacrificial antioxidant, e.g. nitric oxide 25

26 Peroxyl Radicals as Targets # RH + R RH + R R + 2 R Peroxyl radicals, R, are often the chain-carrying radical. # The SFRBM chain 26 Sunrise reaction Free Radical can School also be broken by intercepting R. In biology this is rare, but in the polymer industry it can be very important.

27 Terminating the Chain, Peroxyl Radicals as Targets Tocopherol, a donor antioxidant - L + TH LH + T Nitric oxide, a sacrificial antioxidant - L + N LN 27

28 Characteristics of a Good Chainbreaking Antioxidant a. Both Antioxidant & Antiox should be relatively UN-reactive b. Antiox - decays to harmless products c. Does not add 2 to make a peroxyl radical d. Renewed (Recycled) somehow e. If the chain-breaking antioxidant is a hydrogen atom donor, it should be in the middle of the pecking order. 28

29 The Pecking rder Antioxidants have reduction potentials that places them in the middle of the Pecking order. This location in the pecking order provides antioxidants with enough reducing power to react with reactive oxidizing species. At the same time they are too weak to initiate reductive reactions. L + TH LH + T Termination Buettner GR. (1993) Arch Biochem Biophy. 300:

30 The Pecking rder Depending on their reduction potential, antioxidants can recycle each other. For example, ascorbate with a reduction potential of +282 mv can recycle T (+480 mv) and urate - (+590 mv). Redox Couple E '/mv (one-electron reductions) H, H + /H R, H + /RH (aliphatic alkoxyl radical) R, H + /RH (alkyl peroxyl radical) GS /GS (glutathione) +920 PUFA, H + /PUFA-H (bis-allylic-h) +600 T, H + /TH (tocopherol) +480 H22, H + /H2, H +320 Asc, H + /AscH - (Ascorbate) +282 CoQ -, 2H + /CoQH Fe(III) EDTA/Fe(II) EDTA +120 CoQ/CoQ /2-160 Paraquat/Paraquat -448 Fe(III)DF/Fe(II)DF -450 RSSR/RSSR (GSSG) Buettner GR. (1993) Arch 30 SFRBM Biochem Sunrise Biophy. Free Radical School H2/e aq :

31 CH 3 Donor Antioxidant - Vitamin E CH 3 CH 3 CH 3 CH 3 T (CH 2 ) 3 CH(CH 2 ) 3 CH(CH 2 ) 3 CH(CH 3 ) 2 CH 3 Reaction with lipid peroxides: L + TH LH + T Recycling reaction with ascorbate H H H H + T + AscH H Asc TH 31

32 Donor Antioxidant Uric Acid Uric acid is produced by the oxidation of xanthine by xanthine oxidase. At physiological ph it is ionized to urate. HN N H H N N H UH 3 UH 2 - pk a1 = 5.4 pk a2 = 9.8 UH 2- Uric acid Normal urate concentrations in human plasma range from mm. SFRBM Ames Sunrise BN et 32 Free al. (1981) Radical Uric School acid provides an antioxidant defense in humans against oxidantand radical-caused aging and cancer. A hypothesis. Proc. Natl Acad Sci. USA 78, 6858.

33 Uric Acid Reacts with Peroxyl Radicals k = 3 x 10 6 M -1 s -1 R + UH 2 - RH + UH - Recycling by Ascorbate: k = 1 x 10 6 M -1 s -1 UH - + AscH - UH 2- + Asc - 33

34 Chain Breaking Antioxidant Sacrificial Nitric xide radical, reactive but t 1/2 ~s small molecule, fits everywhere N uncharged, diffusable lipid soluble 34

35 Nitric xide as Antioxidant Preventive: N coordinates with heme-iron, heme Fe 2+ + N heme Fe 2+ N We have used this for centuries in food preservation, the "sausage" effect. Chain-breaking: N can react with oxyradicals: R R + N RN + N RN NSFRBM upregulates Sunrise Free systems Radical School that contribute to the antioxidant network: heme oxygenase, ferritin, hsp70, and γ-glutamylcysteine synthetase 35

36 Nitric xide as Chain- Breaking Antioxidant in Lipid Peroxidation Fe 2+ 2, H 2 2 [Fe ],H LN N hν 1 2 LH Sen 2 2 L LH L LH LH LH LH LN N L Asc Fe 2+ L 2 AscH Fe 3+ L 36 L d

37 Chain Breaking Antioxidant Nitroxide Example nitroxide H N Tempol A possible antioxidant cycle for a nitroxide R 2 N + e -, H + R 2 NH R 1 R 1 nitroxide hydroxylamine R 2 NH + R ox SFRBM Sunrise R 1 Free Radical School RoxH +.R 2 N R 1 37

38 Retarders vs Antioxidant Retarders suppress oxidations only slightly compared to a true antioxidant. A retarder is only able to make a significant change in the rate of oxidation of the bulk when present in relatively large amounts. Retarders are often confused with antioxidants. 38

39 Kinetic Comparison of Antioxidant and Retarder Theorem: There are no true antioxidants for H, only retarders. Proof: 1. The rate constants for nearly all reactions of H in biology are M -1 s -1. Thus, everything reacts rapidly with it and it will take a lot of a antioxidant to inhibit oxidation of the bulk. 2. Comparing rates: Rate (H + Bulk) = k b [Bulk] [H ] Rate (H + Antiox) = k a [Antiox] [H ] 39

40 Antioxidant vs Retarder 3. If we want 98% of the H to react with an antioxidant AND have only a little bit of antioxidant (1% of bulk), then using we have Rate Bulk = k b [Bulk] [H ] Rate Antiox = k a [Antiox] [H ] 2 = k b [99%] [H ] 98 = k a [1%] [H ] 40 then, k a = k b

41 Antioxidant vs Retarder 4. If k a = k b and k b = 2 x 10 9 M -1 s -1, then k a must be 1 x M -1 s No way, not in water. In H 2 k must be < M -1 s Because the a rate constant of M -1 s -1 in H 2 is not possible and is 100x larger than the upper limit for a rate constant in water, there are no true antioxidants for H, only retarders. 7. QED 41

42 Retarder xidation products without retarder xidation [Retarder] xidation with retarder xidation products with retarder More retarder and lots of it. Time 42

43 Antioxidant - no recycling xidation without antioxidant xidation [Antioxidant] xidation with antioxidant Lag time due to kinetic advantage Time 43

44 What is a practical kinetic advantage? Compare the pseudo first-order rate constants. Rate (L + Antiox) = k a [Antiox] [L ] = k a [L ] Rate (L + Bulk) = k b [Bulk] [L ] = k b [L ] where k a = k a [Antiox] and k b = k b [Bulk] If 1% leakage (damage) is acceptable, then k a = 100 k b If 0.01%, then k a = k b 44

45 LDL and TH Compare the pseudo first-order rate constants. Rate (L + PUFA ) = 40 M -1 s -1 [PUFA] [L ] Rate (L + TH ) = 10 5 M -1 s -1 [TH] [L ] If [PUFA] in LDL 1.5 M & [TH] in LDL 0.02 M,* then k TH = 30 k PUFA Leakage about 3% 45 Estimated from: Bowery VW, Stocker R. (1993) J Am Chem Soc. 115:

46 Parting Thoughts 1 To test a compound for possible efficacy as a donor, chain-breaking antioxidant, studying its reactions with R would be much more appropriate than with H. 46

47 Parting Thoughts 2 Antioxidants come in all colors and flavors. Picture stolen from C. Rice-Evans. Keep in mind that besides possible antioxidant activity, the primary bio-activity of the antioxidant may be very different. 47 [C. Rice-Evans - next]