1. PENTOSE PATHWAY & ANTIOXIDANTS BIOC 460 - DR. TISCHLER LECTURE 26

2. OBJECTIVES 1. For the pentose phosphate pathway: a. describe the oxidative and non-oxidative branches b. describe how the oxidative branch is regulated c. distinguish between the 3 modes in terms of the roles of the potential endproducts of each mode. 2.Describe the consequences of thiamine deficiency 3. In relation to antioxidant function in the body: a. list the major active (reactive) oxygen species, identify the antioxidant which reduces that species. b. describe the metabolism of glutathione c. identify the enzymes that remove peroxides and superoxide radicals from a cell and name their cofactor. d. describe the relationships between the components of the antioxidant cascade including the reactions involved. e. discuss why a defect of glucose-6-phosphate dehydrogenase in the red blood cell might lead to loss of membrane integrity.

3. PHYSIOLOGICAL PREMISE Do you have a partial enzyme deficiency about which you are unaware? There are circumstances where an individual may have such a partial deficiency but be unaware of the fact until a physiological event shifts the balance of metabolic processes. For example, individuals with malaria are given a drug called primaquine. When the body metabolizes primaquine it increases the demand for production of NADPH in most cells. A major source of NADPH is the glucose-6- phosphate dehydrogenase (G6PDH) reaction in the pentose phosphate pathway. In the red blood cell, this pathway is essential for removing peroxides, which can oxidize lipids in the plasma membrane causing the cell to become more fragile. Stressing the system with primaquine in an individual with a partial deficiency of G6PDH will lead to red cell destruction and hence the individual becomes anemic.

4. Functions of Pentose Phosphate Pathway 1)NADPH for biosynthetic pathways (e.g., synthesis of fatty acids and cholesterol); 2) NADPH for maintaining glutathione in its reduced state (see discussion of glutathione later); 3) Pentose sugar for synthesis of nucleic acids

5. glycolytic intermediates Glucose 6-P 6-Phosphogluconate NADPNADPH Glucose-6-P-DH Xylulose 5-P Ribose 5-P Oxidative Branch Non- oxidative Branch Nucleic acids Sedoheptulose-7-P Erythrose 4-P Transketolase Transaldolase Glyceraldehyde 3-P Fructose 6-P Glyceraldehyde 3-P TPP Transketolase Ribulose 5-P CO 2 NADPH NADP 6-Pgluconate DH Figure 1. The pentose phosphate pathway containing an oxidative and a non-oxidative branch

6. Ribulose 5-P Xylulose 5-P Ribose 5-P Sedoheptulose 7-P Erythrose 4-P Transketolase Transaldolase Glyceraldehyde 3-P Fructose 6-P Glyceraldehyde 3-P Transketolase Figure 2. Using the non-oxidative branch of the pentose pathway to produce ribose-5-phosphate for the nucleic acid pathways (Mode 1). Ribose-5-P is the sugar required for the synthesis of nucleic acids Nucleic acids Non- oxidative Branch

7. Glucose 6-P Ribulose 5-P 6-Phosphogluconate Ribose 5-P NADP NADPH CO 2 NADPH NADP Figure 3. Using the oxidative branch of the pentose pathway to produce NADPH for biosynthetic reactions and ribose-5- phosphate for producing nucleic acids (Mode 2). Nucleic acids Oxidative Branch

8. Oxidative Branch Non- oxidative Branch back to glucose-6-P or to glycolysis Glucose 6-P (3) Ribulose 5-P (3) 6-Phosphogluconate Xylulose 5-P (2) Ribose 5-P (1) Sedoheptulose 7-P (1) Erythrose 4-P (1) NADP NADPH CO 2 NADPH NADP Glyceraldehyde 3-P (1) Fructose 6-P (1) Glyceraldehyde 3-P (1) back to glucose-6-P or to glycolysis Figure 4. Using the oxidative branch to produce NADPH for biosynthesis and returning ribulose-5-P to glycolytic intermediates (mode 3)

9. used by transketolase, PDH,  KgDH deficiency affects nucleic acid synthesis/energy metabolism Wernicke-Korsakoff syndrome – observed in alcoholics due to poor diet thiamine deficiency in individuals on high CHO diet (e.g., rice) causes beriberi patients tire easily cardiac decompensation energy depletion on high CHO diet NUTRITIONAL PREMISE: THIAMINE (VITAMIN B 1 )

10. Brain atrophy due to Wernicke’s encephalopathy Slide to be shown in class

11. Table 1. Reactive Oxygen Species and Antioxidants that Reduce Them Reactive SpeciesAntioxidant Singlet oxygen 1 O 2 Vitamin A, vitamin E Superoxide radical (O 2 -  ) superoxide dismutase, vitamin C Hydrogen peroxide (H 2 O 2 ) Catalase; glutathione peroxidase Peroxyl radical (ROO  ) Vitamin C, vitamin E Lipid peroxyl radical (LOO  ) Vitamin E Hydroxyl radical (OH  ) Vitamin C

12. O2O2 UV light heme Fe CoQ 1O21O2  NADPH or CoQ  O2-O2-  H2O2H2O2 H+H+  H+H+ HOO   Lipid (LH) LL H2OH2O  O2O2 LOO    OH  Fe 2+ H 2 O, H + Figure 5. Pathways for the formation of reactive oxygen species  Superoxide dismutase  Haber-Weiss reaction;  Fenton reaction  Singlet oxygen  Superoxide radical anion  Peroxyl radical  lipid radical  lipid peroxyl radical

13. H2O2H2O2 glutathione peroxidase 2 H 2 O 2 GSH GSSG glutathione reductase NADPH + H + NADP + pentose pathway Figure 6. Reactions of glutathione reduction and oxidation

14. SUMMARY OF ANTI-OXIDANT ENZYMES Glutathione peroxidase: 2 GSH + H 2 O 2 GSSG + 2 H 2 O Uses selenium as a cofactor Catalase : 2 H 2 O 2 H 2 O + O 2 Superoxide dismutase: 2 O 2 -  + 2H +  H 2 O 2 + O 2 Mitochondrial - Mn 2+ cofactor Cytoplasmic – Cu 2+ -Zn 2+ cofactors; mutations associated with familial amyotrophic lateral sclerosis (FALS) Lipid Peroxidase: removes LOOH

15.  selenocysteine in glutathione peroxidase  intake may be related to lower cancer mortality cancer patients have lower plasma Se levels risk may be higher in those with low Se intake AZCC study – reduced incidence of prostate, colon, lung cancers  toxicity (> 1 mg/day) results in hair loss, GI upset, nerve damage NUTRITIONAL CORRELATE: SELENIUM

16. Vit E red VIT E ox Vit C red VIT C ox LOOH lipid peroxyl radical LOO  Glutathione red (GSH) NADP + NADPH + H + Glucose-6-P Ribulose-5-P Pentose phosphate pathway (rxn 8) +ROOH rxn 2 Glutathione ox (GSSG) H2O2H2O2 2H 2 O hydroxyl radical (OH  ) superoxide radical (O 2 -  ) reduced products Figure 7. Antioxidant cascade Reduced forms/reduction Oxidized forms/oxidation rxn 9 rxn 7 rxn 1 rxn 6 rxn 5 rxn 4

17. Medical Scenario: If the antioxidant protective system in the red blood cell becomes defective, hemolytic anemia occurs; that is red blood cells undergo hemolysis and their concentration in the blood decreases. Such is the case if glucose 6-phosphate dehydrogenase is defective in the pentose phosphate pathway. In individuals whose glucose 6-phosphate dehydrogenase is defective, there is insufficient NADPH produced in red blood cells to maintain the ratio of reduced glutathione to oxidized glutathione at its normal value of well over 100. Hence, peroxides destroy the red cell membrane because of the limited protective mechanism in these cells.