2Objectives of the Lecture 1- Understanding the main functional concepts for pentose phosphatepathway (also called hexose monophosphate pathway).2- Identify the main general uses of NADPH for normal cellularmetabolism with special focus on its rule for antioxidant mechanisms.3- Describing the genetic & biochemical basis of glucose 6 phosphatedehydrogenase deficiency (G6PD deficiency) with identifying its clinicalimplication in causing hemolytic anemia.4- Recognizing the precipitating factors for G6PD deficiency anemia5- Recalling classes of G6PD deficiency anemia ( as indicated by variants of G6Pdehydrogenase enzyme)6- Discussing the biochemical & clinical rationale for lines of diagnosis of G6PDdeficiency anemia.
3Pentose Phosphate Pathway (PPP) or Hexose Monophosphate Pathway (HMP) - is an alternative oxidative pathway for glucose rather glycolysis- results in no ATP production (not like glycolysis)- is the major pathway for NADPH production- produces ribose-5-phosphate for nucleotide synthesis (requiredfor DNA, RNA & others)
5Pentose Phosphate Pathway (PPP) or Hexose Monophosphate Pathway (HMP) as a source for NADPH are producedfor each one moleculeof glucoseGlucoseGlucose 6-phsphateDehydrogenaseFirst enzyme inthe pathwayPentose phosphate pathway (or hexose monophosphate pathway)occurs in the cytosol of many cells of the body including RBCs.
7Uses of NADPH in normal cellular metabolism 1- Role in fatty acid synthesis (reductive agent)2- Role in antioxidant mechanisms (part of glutathione system)3- Role in oxygen-dependent phagocytosis by WBCs4- Role in synthesis of nitric oxide (NO)
8Role of NADPH in antioxidant mechanisms IntroductionHydrogen peroxide (H2O2) is one member of the family of reactive oxygen species (ROS).Reactive oxygen species (ROS) are formed from partial reduction of molecular O2 i.e.adding electrons to oxygen leading to the formation of superoxide, hydrogen peroxideand hydroxyl radical.ROS are formed continuously from aerobic metabolism of drugs and environmental toxinsor diminished antioxidants. All these lead to oxidative stress.ROS cause damage to DNA, protein and unsaturated lipids of the cells including cellmembranes..They are implicated in cancer, chronic inflammatory disease and aging.
9Role of NADPH in antioxidant mechanisms (cont.) Introduction (cont.):The cell has different protective mechanisms that serve to minimize the toxic potential ofROS (antioxidant effect) as follows:A) Enzymes that catalyze antioxidant reactions:i- Catalase & superoxide dismutase enzymescatalyze the conversion of the toxic ROS to harmless products.ii- Glutathione reductasereduces oxidized glutathione to reduced glutathioneB) Antioxidant chemicals:Vitamins E, C and b-carotene (precursor of vitamin A) reduce ROS .
11Role of NADPH in antioxidant mechanisms (cont.) Introduction (cont.):Reduced Glutathione:Reduced Glutathione (G-SH) reduces hydrogen peroxide (H2O2) intooxidized glutathione (G-S-S-G) and water. The reaction is catalyzedby glutathione peroxidaseOxidized glutathione is reduced to reduced glutathione byglutathione reductase using NADPH as a source of reducingequivalents.
13Glucose 6-phosphate dehydrogenase deficiency (G6PD deficiency) DefinitionIt is an inherited disease characterized by hemolytic anemia caused byinability to detoxify oxidized agents.G6PD deficiency is the most common disease producing enzyme anomalies in humans, affecting more than 200 million individuals worldwide.The highest prevalence in the Middle East, tropical Africa & Asia.G6PD Deficiency is caused by 400 different mutations in gene coding for G6PD, only few of them causes the clinical symptoms of the disease.
14Mode of inheritance of G6PD Deficiency It is X- linked recessive genetic disorder (gene is carried on X-chromosome).The gene is present on the X chromosomeThe inheritance follows specific pattern:Males have one X chromosomeSo, they will be diseased if they have the affected gene (xY)Females have 2 X chromosomesmay be homozygous or heterozygousHomozygous: are diseased (xx)Heterozygous: are not diseased BUT: carriers (Xx)& can transfer the disease to their sons
15Heterozygous (carrier) NormalHeterozygous (carrier)Mother( Xx )XxXXXxYXYxYNormalFather(XY)50% ofDaughters are normalcarriersSons are affectedPunnet Square for X-linked Recessive Inheritance
16Decreased amounts of reduced glutathione due to decreased production of NADPH Reduction of amounts of NADPH in RBCs in G6PD deficiency causes decreasein reduction of oxidized glutathione to reduced glutathione.Role of reduced glutathione in RBCs:1- Reduced glutathione gets rid of ROS including hydrogen peroxide.2- Reduced Glutathione helps to keep sulfhydryl groups of hemoglobinprotein in the reduced state.
17Decreased amounts of reduced glutathione due to decreased production of NADPH Reduction of production of reduced glutathione results in:1- A decrease in detoxication of peroixides. This causes damage to RBCsmembrane and hemolysis (ending in hemolytic anemia).2- Hemoglobin protein is denatured forming insoluble masses (Heinzbodies). Heinz bodies attach to red cell membranes.Membrane proteins are also oxidized.Accordingly, red cells become rigid and removed from the circulationby macrophages in the spleen and liver ending in anemia
19Decreased amounts of reduced glutathione due to decreased production of NADPH Deficiency of G6PD occurs in all cells of affected individual. It is severe in RBCs because the only pathway to form NADPH in RBCs is pentose phosphate pathway (using G6PD).
20Precipitating factors in G6PD deficiency: Individuals who have inherited one of the many G6PD mutations do not showclinical manifestation.Some of patients with G6PD develop hemolytic anemia if they are exposed or ingest any of the followings oxidizing agents:1-Oxidant drugs:Antibiotics : e.g. sulfamethoxazoleAntimalarias : e.g. primaquineAntipyretics : e.g. acetanilid2- Favism:The hemolytic effect of ingesting of fava beans is not observed in allindividuals with G6PD deficiency but all patients with favism have G6PDdeficiency
21Most G6PD variants are caused by point mutations in the G6PD gene. Some of these point mutations do not disturb the structure of theenzyme's active site and hence, do not affect enzyme activity.Other point mutations may lead to production of mutant enzymes with one or more of the following:altered catalytic activity,decrease stabilityan alteration of binding affinity for NADP+ or Glucose 6-phosphate.The severity of diseases usually correlates with the amount ofresidual enzyme activity in the patient’s red cells.
22G6PD Variants can be classified into : Class III (G6PD Group A-) :A moderate form of the diseaseRBCs contain unstable G6PD enzyme, but normal activity in younger RBCs andreticulocytes.Accordingly, only older RBCs are hemolysed in a hemolytic episode.Class II mutations (G6PD Mediterranean):More severeG6PD enzyme shows normal stability but, very low activity in all RBCs.Class I mutations:It is often associated with chronic non spherocytic anaemia(occurs even in absence of oxidative stress).
23G6PD VariantsBoth G6PD Mediterranean and G6PD A- represent mutant enzymes that differ from the normal variants by a single amino acid. This change is due to DNA changes in the form of point mutations or missense mutations.Frame shift mutations or large deletions have not been identified indicating that the complete absence of G6PD is lethal.Mutation causing non spherocytic hemolytic anemia are clustered near the carboxyl end of the enzyme, whereas mutations causing milder forms of the disease tend to be located at the amino end of the enzyme.
24G6PD Deficiency Hemolytic Anemia Diagnosis ofG6PD Deficiency Hemolytic AnemiaDiagnosis of hemolytic anemiaCBC and reticulocytic countScreening:Qualitative assessment of G6PD enzymatic activity(UV-based test)Confirmatory test:Quantitative measurement of G6PD enzymatic activityMolecular test:Detection of G6PD gene mutation