1. DR. SAIDUNNISA, MD Professor of Biochemistry Hexose monophosphate shunt

2. Learning objectives At the end of the session student shall be able to: To understand the function of the pentose phosphate pathway in production of NADPH and ribose precursors for nucleic acid synthesis. To examine the importance of NADPH in protection of cells against highly reactive oxygen species. To relate defects in the pentose phosphate pathway to disease conditions. Interpret G6PD deficiency (hemolytic anemia and jaundice) and Wernicke-Korsakoff syndrome.

3. Case-1  A 25 year old African American was going on pleasure trip to Iraq. In preparation for his tour he is given a prophylactic dose of primaquine to prevent malaria. Several days after he began taking this drug he develops fatigue and hemolytic anemia.  Why Discuss?

4. Case-2  A native of East Africa presents with jaundice and spleenomegaly after eating fava beans.  Why?

5. Hexose Monophosphate shunt  Other names:  Pentose phosphate pathway  Phosphogluconate oxidative pathway  Shunt pathway: glucose instead of going through glycolysis it is shunted through this pathway.  10% of glucose molecules per day are entering this pathway

6. It’s a shunt

7. The pentose phosphate pathway has two main functions  Production of NADPH  Ribose 5P Site :  In the cytoplasm of specialized cells.

8. Generation of NADPH (Oxidative phase of HMP) mainly used for reductive syntheses of fatty acids. Not active in non-lactating mammary gland and has low activity in skeletal muscle.

9. Ribose 5P ( Non-Oxidative phase of HMP)  Are required for the synthesis of the nucleotides and nucleic acids (DNA, RNA).

10. Reactions of the pentose phosphate pathway occur in the cytosol in two phases  Phase-I :  Oxidative, non-reversible phase  Phase-II:  Non-oxidative, reversible phase  NADP +, not NAD +, is used as hydrogen acceptor

12. 5 carbon atoms Regulatory enzyme Oxidative pathway: The end products are; 1. One molecule of D-ribulose 5- phosphate 2. Two molecule of NADPH. 3. One molecule of CO 2

13. Don’t panic, you need not know all the reactions in detail; stay tuned. Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde at C1 of glucose-6-phosphate, to a carboxylic acid in ester linkage (lactone). NADP + serves as electron acceptor. 6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage (lactone) resulting in ring opening.

15. 1. Isomerization interconverts the stereoisomers. 2. Transketolase transfers a 2-C fragment from xylulose-5- phosphate to ribose-5- phosphate. It utilizes thiamine pyrophosphate (TPP), a derivative of vitamin B 1. 3. Transaldolase: catalyzes transfer of a 3-C dihydroxyacetone moiety, from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate. 4. Transketolase transfers a 2-C fragment from xylulose-5- phosphate to erythrose-4- phosphate.

16. Non oxidative phase  2 nd Transketolase reaction yields glyceraldehyde-3- phosphate and fructose-6- phosphate.  C 5 + C 5 C 3 + C 7 (Transketolase)  C 3 + C 7 C 6 +C 4 (Transaldolase)  C 5 + C 4 C 6 + C 3 (Transketolase) Overall:  3C 5 2 C 6 +1C 3

17. Mechanism of reaction need not know Transketolase transfers a 2-C Transaldolase transfer of a 3-C

18. Regulation of pentose phosphate pathway Glucose 6-phosphate dehydrogenase is the rate limiting enzyme. This enzyme is regulated by availability of the substrate NADP +. Is stimulated by insulin.

19. Glutathione and NADPH  What is glutathione?  Why is it important?  How is it related to NADPH?

20. Glutathione

21. Special functions of NADPH in RBC  Reduced glutathione (GSH) is essential to maintain the integrity of RBC membrane.  Is necessary to keep the iron in ferrous form i.e reduced (Fe +2 ) which can carry oxygen.

22. Pentose phosphate pathway protects cells against reactive oxygen species (ROS)  Reduction of molecular O 2 in a series of one-electron steps yields superoxide, hydrogen peroxide, hydroxyl radical, and water. The intermediate, activated forms of oxygen are known as reactive oxygen species (ROS )

23. Role of NADPH and glutathione in protecting cells against ROS  Reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hydroxyl free radicals by glutathione peroxidase.  Regeneration of GSH from oxidized form (GS-SG) requires the NADPH (produced in the glucose 6- phosphate dehydrogenase reaction) by glutathione reductase.

24. So, what happens if glucose 6-phosphate DH is defective? Insufficient production of NADPH. Which translates into insufficient glutathione. Is this a medical problem? YES

25. G6PD Deficiency Is the most common human enzyme deficiency in the world. Over 400 million people worldwide affected with highest prevalence in individuals of African, Mediterranean and Asian heritage. An X-linked disorder that therefore typically affects men.

26. G6PD Deficiency  Most G6PD-deficient individuals are asymptomatic  But when certain drugs are taken such as (sulfa antibiotics, antimalarials like primaquine) and toxic ingredient of fava beans, stimulate peroxide formation leading to sudden damage to RBC and clinical manifestations occur.

27. G6PD Deficiency  Distribution of G6PD deficiency coincides prevalence of malaria  G6PD deficiency imparts malaria resistance  Also sickle cell anemia

28. Glucose-6-phosphate dehydrogenase deficiency causes hemolytic anemia  Mutations in glucose 6-phosphate dehydrogenase, results in impairment of NADPH production.  Detoxification of H 2 O 2 is inhibited, and cellular damage results - leads to erythrocyte membrane breakdown and hemolytic anemia and jaundice.

29. Glucose-6-phosphate dehydrogenase deficiency causes hemolytic jaundice Normal value of G6PD in RBC is 6-12u/g of Hb.

30. Drug induced hemolytic anemia

31. Favism  Grown worldwide  Important in Middle East  High in protein  These two compounds deplete reduced glutathione leading to formation of free radicals and H 2 0 2 that cause haemolysis. Fava beans rich in two glycosidic compounds vicine, and convicine. Upon ingestion these are converted into divicine and isouramil

32. Glucose-6-phosphate dehydrogenase deficiency and resistance to malaria  G-6PD frequently in Africans-protects them from malaria.  Plasmodium falciparum is dependent on HMP shunt and reduced glutathione for their optimum growth in RBC.  One theory to explain this, is that cells infected with the Plasmodium parasite are cleared more rapidly by the spleen.

33. Wernicke-Korsakoff syndrome  Wernicke first described an illness consisted of ataxia, paralysis of eye movements (nystagmus) and mental confusion.  SS Korsakoff Russian psychiatrist described disturbance of memory in alcoholism.  Genetic disorder associated with transketolase activity results in Wernicke-Korsakoff syndrome.  Symptoms include mental disorder, loss of memory and partial paralysis.  These manifest in alcoholics whose diets are deficient in vitamin thiamine.  Transketolase is measured in RBC is an index of thiamine status of an individual