PENTOSE PATHWAY & ANTIOXIDANTS

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

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 • 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. Glucose-6-P-DH NADP NADPH Glucose 6-P 6-Phosphogluconate Oxidative Branch NADP 6-Pgluconate DH NADPH CO2 Ribulose 5-P Nucleic acids Glyceraldehyde 3-P Xylulose 5-P Ribose 5-P TPP Transketolase Transketolase Non-oxidative Branch Glyceraldehyde 3-P Sedoheptulose-7-P Transaldolase Fructose 6-P Erythrose 4-P Fructose 6-P glycolytic intermediates Figure 1. The pentose phosphate pathway containing an oxidative and a non-oxidative branch

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

7. Oxidative Branch NADPH NADP 6-Phosphogluconate Glucose 6-P NADP NADPH CO2 Ribulose 5-P Ribose 5-P Nucleic acids 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).

8. NADPH NADP 6-Phosphogluconate Glucose 6-P (3) NADP Oxidative Branch NADPH CO2 Ribulose 5-P (3) Glyceraldehyde 3-P (1) Ribose 5-P (1) Xylulose 5-P (2) Non- oxidative Branch back to glucose-6-P or to glycolysis Glyceraldehyde 3-P (1) Sedoheptulose 7-P (1) Erythrose 4-P (1) Fructose 6-P (1) Fructose 6-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. NUTRITIONAL PREMISE: THIAMINE (VITAMIN B1) • 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

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

12.  Lipid (LH) L O2 OH H2O  Fe2+  H2O2 LOO 1O2  UV light heme Fe CoQ   H2O, H+ H+ O2- HOO   NADPH or CoQ H+ O2 Figure 5. Pathways for the formation of reactive oxygen species  lipid radical  Singlet oxygen  Peroxyl radical  Haber-Weiss reaction;  Fenton reaction  lipid peroxyl radical  Superoxide radical anion  Superoxide dismutase

13. H2O2 glutathione peroxidase 2 H2O GSSG 2 GSH glutathione reductase NADP+ NADPH + H+ pentose pathway Figure 6. Reactions of glutathione reduction and oxidation

14. SUMMARY OF ANTI-OXIDANT ENZYMES Glutathione peroxidase: 2 GSH + H2O2 GSSG + 2 H2O Uses selenium as a cofactor Catalase : 2 H2O2 H2O + O2 Lipid Peroxidase: removes LOOH Superoxide dismutase: 2 O2- + 2H+ H2O2 + O2 Mitochondrial - Mn2+ cofactor Cytoplasmic – Cu2+-Zn2+ cofactors; mutations associated with familial amyotrophic lateral sclerosis (FALS)

15. NUTRITIONAL CORRELATE: SELENIUM • 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

16. lipid peroxyl radical LOO LOOH VIT Eox Vit Ered hydroxyl radical (OH) superoxide radical (O2-) VIT Cox Vit Cred reduced products H2O2 +ROOH rxn 2 Glutathioneox (GSSG) Glutathionered (GSH) 2H2O NADP+ NADPH+ H+ Pentose phosphate pathway (rxn 8) Ribulose-5-P Glucose-6-P Figure 7. Antioxidant cascade Reduced forms/reduction Oxidized forms/oxidation rxn 5 rxn 6 rxn 9 rxn 4 rxn 7 rxn 1

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.