Section 10: Nutrients and their functions

1. Section 10: Nutrients and their functions • Reactive oxygen species (ROS) and defenses 01/31/06

2. Reactive oxygen species (ROS) • ROS are highly reactive and indiscriminate. • The most destructive is superoxide anion radical, O2.-. • The damage that they do may contribute to diseases (cancer) and to aging. • There are three major sources of ROS production. • Neutrophils • Oxygen transport and use • Ionizing radiation 1

3. Neutrophils(inflammatory response) Neutrophils provide the body’s first line of defense against bacterial invasion. Circulating in the blood, they move into infected tissue, stimulating the complement system, leukotriene production and c-reactive protein (cRP) synthesis. • Bacteria are ingested by neutrophils and destroyed by intracellular granules containing proteases and antibiotics. • In addition, cell membrane-bound NADPH oxidase is activated to produce toxic O2.-, as shown. NADPH + H+ + 2 O2® NADP+ + 2 H+ + 2 O2.- • Chronic inflammation may induce constant production of O2.- with destructive consequences for the infected tissue and its surroundings. 2

4. Oxygen transport and use • Normal oxidative metabolism in all cells produces reactive oxygen species (ROS) as byproducts, which are toxic. • O2.- is produced from Hb. Red blood cells are estimated to produce between 1,000 and 1,000,000 O2.- per second. • O2.- is produced when e- “escapes” from the electron transport chain, especially in the two electron transfer from QH2 to cytochrome reductase (complex III). 3

5. Ionizing radiation • Destructive free radical ions, such as O2.-, can also be produced by high energy irradiation. • Radiation therapy uses the destructive power of such ionizing radiation to kill tumor cells. • X-rays are another example of useful ionizing radiation. • In all uses, it is important to minimize the exposure of vulnerable tissue to the radiation. 4

6. Protection again oxidative damage • Cells have strategies to combat the damage done by O2.- and by other reactive oxygen derivatives. • enzymatic • nonenzymatic • Nutritional components contribute to both categories. • micronutrients such as Se • vitamins such as C and E 5

7. Superoxide dismutase • Active site uses Cu(II), Zn(II) and his for two sequential reactions. 6

8. Catalase • Catalase converts peroxide to oxygen and water. • Together, catalase and superoxide dismutase minimize O2.- and subsequent toxic derivatives made from it. • Catalase is a heme enzyme. • Its rate is diffusion controlled. 7

9. Peroxidases • Organic peroxides are also destructive. • They are reduced by peroxidases, and also by alcohol dehydrogenases, to an alcohol and water. • Many structures, including ascorbate, serve as the reducing agent, AH2. 8

10. Glutathione peroxidase • Glutathione peroxidase uses the oxidation of the tripeptide glutathione (GSH = glu-cys-gly) to reduce organic peroxides. • Glutathione peroxidase requires Se in the form of the selenium analog of cysteine (Se replacing S). 9

11. Glutathione peroxidase cycle • Glutathione reductase converts GSSG back to 2 GSH, using NADPH generated by glucose 6-phosphate dehydrogenase in the pentose phosphate pathway (see section 6 lecture 4). 10

12. Ascorbate • Ascorbate (vitamin C) is oxidized in several reactions that reverse unwanted oxidative reactions. • For example, Fe(III) ® Fe(II), in the GI tract. • These are nonenzymatic. 11

13. Ascorbate requiring reactions • These enzymatic reactions require ascorbate, but its role is not fully understood. 12

14. Vitamin E derivatives IsomerR1R2R3 a CH3 CH3 CH3 b CH3 H CH3 g H CH3 CH3 d H H CH3 Fat-soluble uptake. Membrane bound. 13

15. a-D-tocopherol • a-tocopherol (vitamin E) reacts nonenzymatically to eliminate free radicals (R·) produced by unwanted oxidation reactions. • Other vitamin E derivatives are more effective for peroxides or other ROS. 14

16. Free radical chain reaction • Some oxidant, X, starts the reaction, creating R’. • If tocopherol, EH, is present, the reaction is reversed. • Without EH, the reaction proceeds via a peroxide free radical to form peroxide and a second free radical in the bilayer. • This free radical can react with oxygen and repeat the cycle to make many R’. • The tocopherol free radical, E’, is stable and is eliminated by a safe pathway to the quinone. 15

17. Vitamin E dietary requirements • There are four chiral centers; the naturally occurring compound is RRR-a-Tocopherol, or a-D-Tocopherol. • The synthetic product is all-rac-a-T, or a-DL-T. • b, d & g T’s are rated as RRR-a-T equivalents. • Dietary amount needed varies with intake of polyunsaturated fats, but 10mg for men and 8mg for women is the RDA for RRR-a-T equivalents. • Recent studies suggest that supplements may be more harmful than beneficial. 16

18. Bilirubin • Bilirubin, a heme degradation product, is produced in the liver. • Some is excreted after two glucuronic acid moieties are added (see S08L03). • Some circulates to peripheral cells where it serves as an anti-oxidant (coupled to A ® AH). • Inside cells, [bilirubin] is nM, which would be rapidly exhausted, but a bilirubin reductase restores it, using NADPH. • Bilirubin also circulates in the blood at much higher concentrations and may protect rbc membranes. 17

19. Urate • The purine degradation product, urate, has antioxidative properties similar to those of ascorbate and bilirubin. 18

20. Life-span and oxidative stress • For a variety of mammals, life-span varies inversely with metabolic rate (cal/g per day) and with superoxide anion levels (nmole/mg protein). from Sohal and Weindruch in Science 273:59-63 (1996) 19

21. Life-span and catalase & SOD levels • Overexpressing the genes for superoxide dismutase and catalase in fruit flies increases their life-span. 20

22. Life-span and caloric intake • Mice were maintained on diets of reduced total calories. • Life-span increases with reduced caloric intake. 21

23. Reduced caloric intake, primates A. 13.6 kg,19 year old rhesus monkey, control diet. B. 7.9 kg,19 year old, 30% calorie restricted (CR) diet. C. 6.8 kg, 38 year old, CR, died at 44 (longest life known). See (2004) Science305:1423. 22

24. Next topic: B vitamins

25. Superoxide anion • The most destructive reactive oxygen species is superoxide anion radical, O2.- , because of its exceptionally high and indiscriminant reactivity. • It produced enzymatically by neutrophils to kill bacteria as part of the inflammatory response. • It is produced by spontaneous unregulated mechanisms when O2 is being transported, transferred and participating in various reactions. • It is also produced by ionizing radiation (x-rays).

26. Life-span and Coenzyme Q • C. elegans (nematode) deprived of dietary coenzyme Q have 60% longer adult life-spans. • CoQ is a carrier in the electron transport chain, but the mechanism of action is not clear. • Reduced CoQ may inhibit aerobic or activate anaerobic pathways. • The production of antioxidant scavengers may be activated. • Uncoupling factors may be less activated when CoQ is lower. • from Larsen and Clarke (2002) Science 295:120-123. 20

27. 2002 Science 298:1745 Mechanism related to calorie restriction • Fruit fly. • Rpd3 is gene for Sir2, a NAD-dependent histone deacetylase. • DNA binds deacetylated histones more tightly and may be less available for aging-related expression or recombination. • Over expressing rpd3 increases lifespan. • Mutant does not respond to low calorie diet. • Mutation or low calorie diet increase Sir2 mRNA. 21

28. EXTRA CREDIT QUIZ TWO QUESTIONS SHOWN HERE FOR ONE MINUTE EACH Please move to the “quiz seating arrangement.” You will be given a scantron.

29. a. superoxide dismutase b. catalase c. glutathione peroxidase d. cytochrome reductase Two questions total on this quiz. 1. What enzyme has the tripeptide glu-cys-gly as a substrate?

30. a. ascorbate b. a-D-tocopherol c. bilirubin d. superoxide anion e. catalase Two questions total on this quiz. 2. Which antioxidant is most effective within membrane bilayers?