Oxidation general concepts. Prof. Umberto Cornelli Loyola University Medical School-Chicago

Transcription

1 Oxidation general concepts Prof. Umberto Cornelli Loyola University Medical School-Chicago 1

2 Antioxidants a real mess! Is oxidative stress a real problem? Is every antioxidant good for health? Oxidation has to be faced in the same way in every district of the organism? Direct and indirect antioxidants 2

3 Oxygen is a biradical O2 (molecule) *2p 3

4 The Most Important ROMs = 2 * O2 singlet O2 superoxide H2O2 hydroperoxide OH hydroxyl radical 4

5 ROS Formation Pathways Energy (ATP) production Reactive Metabolic 5

6 Energetic Pathway 6

7 Energetic pathway in mitochondria O2 e- O2 e- H2O2 e- H2O e- OH 7

8 The quenching cycle superoxide O2 SOD superoxidodismutase H2O2 water Fe ² + Fenton reaction OH Catalase-peroxidase H2O 8

9 Reactive Pathway PMNF NADPH oxidase O2 O2 H2O2 Cu/Fe OH 9

10 Metabolic Pathway ipoxantine xantine dopamine O2 O2 arachidonic ac O2 norepinephrine O2 eicosanoids O2 O2 10

11 [OX] Oxidation paradigm death growth arrest mytogenesis incomplete growth 0 no growth 11

12 Different Cellular Localization of Enzymes Producing ROS Xantine oxidase NAD(P)H oxidase Heme oxigenase Lipoxigenase uncoupled enos mieloperoxidase peroxidase cyto P450 monoxigenase cycloxigenase 12

13 Sensitivity to oxidation Lipids Antiproteases Other proteins DNA Proteoglycans

14 Passive oxidation oxidation of functional components due to the environment This is the case of the propagation of oxidation due to lipids or protein oxidation. The antioxidant capacity is related to the antioxidant reserve available in that environment 14

15 Passive oxidation Ca++ Ca++ Ca++ Ca++ Na+Na+ Na+ Na+Na+ Na+ Na+Na+ Na+Na+ Na+K+ Na+ K+ O2 O2 Pump O2 O2 O2 O2 Pump kinases activation 15

16 Active oxidation due to the activation of receptors (such as MAP kinase receptors) wich determine a redox inflammatory condition. In this case the local antioxidants reserve is not sufficient to quench the process, and there is need to import antioxidant capacity. 16

17 example of redox inflammatory reaction macrophages (PMF) MAP receptors (eg tall-like) O2 MAP receptors PMF proteases O2 O2 O2 endothelial cell or lipoprotein 17

18 The two different types of oxidation Passive oxidation is typical of physiological conditions such as an increase of physical activity or even a meal (oxidized lipids intake) Active oxidation is not physiological and belongs to a disease or to common habits or treatments such as smoking, oral contraceptive, psychotropic drugs ) 18

19 Methods to determine oxidative stress (through oxidised markers 1) DNA Deoxyribonucleic acid 1 SPC Serum protein carbonyls 1 LHP Lipids hydroperoxides d-roms test 1 TBARS Thiobarbituric acid reacting substances 1 LNO 2 Nitrolinoleate 1 MDA Malondialdehyde 1 4-HNE 4-hydroxynonenal 1 IsoPs F 2 /D 2 /E 2 isoprostanes 1 NeuroPs F 3 /F 4 isoprostanes 1 H 2 O 2 Hydrogen peroxide 1 BH Breath hydrocarbons 1 19

20 Methods to determine OS (through spin traps 2, fluorescence 3, antioxidant capacity 4) ONOO Peroxynitrite 2 PTN Alpha-phenyl- N-tert- butylnitrone 2 AHS Aromatic hydroxylation of salicilate 2 TRAP Total peroxyl radical scavenging antioxidant capacity 3 TOSCA Total oxyradical scavenging capacity assay 4 SAT Saliva antioxidant test (hydrosoluble) 4 UAM Uric acid metabolite allantoin 4 TEAC Trolox equivalent antioxidant capacity 4 FRAP Ferric reducing ability 4 ORAC Oxygen radical absorbance capacity 4 DMPD N,N-dimethyl-p-phenylenediamine 4 DPPH 1,1-diphenyl-2-picrylhydrazyl 4 TRX Thioredoxine and glutaredoxine 4

21 Indirect Antioxidants Products (mainly drugs) that reduce the cellular reactivity or inhibit the reactivity chain: Steroids NSAIDS (non steroidal antiinflammatory drugs) Statins ACE (angiotensin converting enzyme) inhibitors

22 Steroids Mechanism of action: Reduction of NF-kB reactivity Reduction of circulating reactive cells (excluding neutrophiles) Increase of shock adsorbants: glucose, amminoacids, FFA (due to the facilitating lipolytic activiy of steroids on adrenaline and norepinephrine) 22

23 Steroids 4 groups of healthy volunteers (5M-5F) Aged between Fasting for 24 h Same food intake for 48 h Drugs administration: 8 a.m. with 120 ml of orange juice and 1 croissant d-roms test at baseline, 8h, 24h, 48h after drugs administration 23

24 Steroid Steroids and d-roms Test (U.CARR.) Dose mg Before After 8h After 24 h After 48 h cortisone ± ± 23 * 302 ± 29 * 281 ± 26 cortisol ± ± 29 * 306 ± 21 * 278 ± 17 prednisone ± ± 32 * 210 ± 29 * 285 ± 15 betamethasone ± ± 15 * 179 ± 16 * 283 ± 24 triamcinolone ± ± 7 * 159 ±13 * 267 ± 23 24

25 Conclusions/Steroids All steroids reduce the OS (at 8h) Betamethasone and triamcinolone are more effective than cortisone and cortisol, and show longer activity After 48 h the activity disappears Cortisol and cortisone seem to have a rebound effect not detected with betamethasone and triamcinolone 25

26 NSAIDs (Non Steroidal Antiinflammatory Drugs) Reduction of cellular reactivity (cycloxigenase and lipoxigenase inhibition) Reduction of expression of adhesion molecules Reduction of O2 generation; Reduction of NO synthesis, TNF- and IL-1 expression 26

27 NSAIDs 5 groups of healthy volunteers (5M-5F) Aged between Overnight fasting Drugs administration: 9 a.m. after breakfast with 120 ml of orange juice d-roms test at baseline (9 a.m) and at 3h after the administration of NSAIDs 27

28 NSAIDs and d-roms Test (U.CARR.) Drug dose Before After 3h Placebo 315 ± ± 38 ASA 500 mg 345 ± ± 31 * Naproxen 500 mg 330 ± ± 44 * Nimesulide 100 mg 326 ± ± 42 Celecoxib 100 mg 319 ± ± 35 * 28

29 Conclusions/NSAIDs NSAIDs excluding nimesulide seem to reduce the OS. ASA seems to be the most effective at least at short term 29

30 ACE Inhibitors (Angiotensin Converting Enzyme) Angiotensin II acts on AT1 and AT2 receptors These receptors activate phospholipases (PLA2), phosphokinases (PKC), nitrogen sinthase (NOs), NADPH oxidase. ACE inhibitors drastically reduce the activation of AT1 and AT2 receptors 30

31 ACE Inhibitors 33 hypertensive subjects started the treatment (never treated before) 11 cases/group (males only) Aged between d-roms test before and after 4 weeks Captopril (non selective) and losartan (AT1) 31

32 ACE Inhibitors and d-roms Test (U.CARR.) Product Before BP S BP D Placebo 93 ± ± 19 After 4 weeks BP S BP D 92 ± ± 20 Before D-ROMs After 4 weeks D-ROMs 378 ± ± 37 Captopril 94 ± ± ± 4 * 150 ± 12 * 375 ± ± 40 * Losartan 96 ± ± ± 3 * 145 ± 22* 380 ± ± 35 * 32

33 Conclusions /ACE Inhibitors Hypertension is characterized by OS ACE inhibitors reduce oxidative stress but in many subjects do not normalize the levels of the d-roms test 33

34 Statins Increase of HMGCoA reductase levels (up to times!) Most of the activity is due to the reduction of prenylation. Prenylation activates the molecules of the reactive chain (for examples NADPH oxidase). It is called pleiotrophic activity 34

35 Statins 36 dislipidemic subjects 12 cases/group (6 M-6 F) Aged between weeks treatment d-roms test before and after 4 weeks 35

36 Items Statins and d-roms Test (U.CARR.) (Simvastatin = Simva; Pravastatin = Prava) simva before simva after prava before prava after LDL 142 ± ± 41 * 158 ± ± 44 * Triglycerides 240 ± ± 42 * 236 ± ± 33 * HDL 37 ± ± ± ± 23 d-roms 352 ± ± 48 * 370 ± ± 44 * 36

37 Conclusions/Statins Statins reduce OS but do not normalize it 37

38 Direct Antioxidants 38

39 The Direct Antioxidant (AO) Hierarchy: Activity AO systems (catalase, peroxidase) Shock adsorbants AO (albumin, uric acid, transferrin ecc.) Essential AO (Vit E, Vit C,), mixed AO (CQ10, lipoic acid, squalene) Mx activity Other AO (carotenoids, flavonoids) 39

40 The Antioxidant (AO) Shield AO site Membrane Cytosol Circulating Enzymes (System) Type of AO Vit E, Vit A, squalene CQ10, lipoic acid Vit C, flavonoids Zn, Se, L-cysteine 40

41 Antioxidants Bioavailability and Potency Double-blind-cross-over study 14 healthy volunteers (M/F) 3 formulas with different AO F1, F2, F1+F2 Dry form (tablets) Vs fluid form (vials) 1 week treatment Activity determination through the d-roms test 41

42 Formula F1: Membrane and Enzymatic AO Zn Se L-cysteine Vit A Vit E Beta-carotene 5 mg 48 mcg 10 mg 0.4 mg 15 mg 50 mcg 42

43 Formula F2: Circulating and Cytosol AO Flavonoids from Citrus Vit C CQ10 Vit B6 30 mg 30 mg 10 mg 1 mg 43

44 d-roms Test: > 15 % Reduction in Serum Formula Fluid Dry p F1 77 % 31 % < 0.05 F2 31 % 7 % < 0.05 F1+F2 92 % 42 % < 0.05 Total 66 % 25 % <

45 The General Rule: Low Dosages Association of AOs OH O R R Vit E oxidized Vit E 45

46 Oxidation Cascade: To be Fully Operative Quenching Needs Support, Time, and Appopriate Location circulating AO membrane AO cytosol AO AO systems GSH efficiency 46

47 The Oxidation Cascade The most important terminal of the oxidation cascade is GSH, which must have the time to rigenerate and has to be available at the oxidation site quenching GSH + GSH GSSG GSH+GSH NADH+H+ 47

48 The Oxidation Cascade The antioxidant reserve increases when GSH has the time to rigenerate and when is spared. antioxidant reserve low GSH high GSH 48

49 The General Rule For Antioxidants.use them if you need them a simple and reliable method to determine oxidative stress is necessary They are Physiological Modulators 49

50 Some Antioxidant Combinations For vessels (ARD Stenovit - ARD Vessel) For the skin (ARD Esilen/ARD Esilen emulsion) For the gastrointestinal system (ARD Colostrum) For the brain (ARD Cogiton) 50