G6PD case and discussion on biochemical pathways.pptx

G6PD deficiency case preparation and discussion in biochemical pathways including HMP shunt, glutathione reduction and general overview

1. Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency Case presented by: Dr. Ritika Mukherjee JRA (1 st year) Biochemistry, RIMS, Ranchi

2. CASE HISTORY A 14 years old boy was admitted with complaints of headache, anorexia, nausea and intermittent fever followed by chills and rigors. Malaria was suspected and confirmed by identification of the Plasmodium vivax parasite in the blood smear. The patient was treated with ACT ( Artemisinin based combination therapy) and P rimaquin . Three days later the patient complained of weakness, abdominal pain, passing red coloured urine. On general examination : patient was found to be icteric and pale. On systemic examination : no organomegaly was found. On routine investigation: Haemoglobin: 8g/dl RBC : 2.26/cc TB: 5.6 mg/dl DB: 0.8 mg/dl AST: 55 U/L ALT : 60 IU/L Ur: 25 mg/dl Cr: 1mg/ dlfg *ACT = Artemisinin combination therapy ( Artesunate + lumefantrine )

3. Provisional diagnosis : Glucose 6 phosphate dehydrogenase deficiency. Differential diagnosis : ADR of ACT Complicated malaria Other types of hemolytic anemia (Spherocytosis, Sickle cell disease, Thalassemia, Paroxysmal nocturnal hemoglobinuria , Pyruvate kinase deficiency, Immune mediated hemolysis , Hypersplenism etc.)

4. Peripheral blood smear under light microscope: RBC with Heinz bodies . ( Hemoglobin denaturated and adhere to membrane, stained with basic dye.)

5. Confirmatory test : (Gold standard) Quantitative determination of Glucose-6-phosphate dehydrogenase in erythrocytes. UV method in semi-auto-analyser: Principle: The enzyme activity is determined spectrophotometrically by measurement of the rate of absorbance change due to reduction of NADP + . It is measured at 340 nm filter. Sample: Erythrocytes. Reference range: 8-14 IU/g of hemoglobin . Other methods: FST ( Fluoroscent spot test), RDT(rapid diagnostic test kit – semiquantitative ).

6. Biochemistry behind this case: Pentose phosphate pathway (PPP) or Hexose monophosphate pathway (HMP shunt) – Oxidative phase Re-generation of reduced glutathione Presence of oxidative stress as precipitating factor Absence of other NADPH generating pathways in RBC

7. HMP Shunt : Main source of NADPH in non-photosynthetic organisms. Two phases: 1. oxidative generation of NADPH (Irreversible) 2. Non-oxidative inter-conversion of sugars (reversible) The 1 st phase reactions: Net reaction: G6P + 2NADP + + H 2 O  Ribulose-5P + 2NADPH + 2H + + CO 2 G6PD is the rate limiting enzyme in first phase.

8. Non-oxidative phase of HMP shunt: 1 . and 3. 3. 4. Net reaction: 3C 5 ⇌ 2C 6 +C 3

9. Relevance of HMP shunt (Can occur in cytoplasm of all cell)

10. Re-generation of reduced Glutathione: 1. 2. *FAD is cofactor of glutathione reductase Glutathione in RBC has two roles: Deal with oxidative stress: help to neutralize reactive oxygen species that potentially damage membrane Maintain structure of Hemoglobin – GSH serves as sulfhydryl buffer that keeps residues of Hb in reduced sulfhydryl form. In absence of GSH, residues of Hb cross linked with each other, results in denaturation and insoluble depositions (Heinz bodies)

11. Presence of oxidative stress: examples of precipiating factors are - certain drugs: Primaquine , Dapsone , Acetylphenylhydrazine , Bactrim , Nitrofurantoin , Rasburicase , Sulfonamides , Aspirin etc. Food: Fava beans or broad beans, Soy, peanuts Infections: Hepatitis, Cholera *Prx2= Peroxiredoxin 2 These above mentioned examples are oxidative agents that generate reactive oxygen species or free radicles and peroxides. G6PD deficiency become evident in the RBC in the presence of such stressors.

12. Absence of other NADPH-generating pathways in RBC: The other lesser known pathways of NADPH generatioin involve mitochondria. The three major routes catalysed 1. NADP-linked malic enzyme 2. NADP-linked isocitrate dehydrogenase 3. nicotinamide nucleotide transhydrogenase . RBC lacks mitochondria. So, its only source for NADPH is HMP pathway. Thus free radicles and precipitated hemoglobin damage RBC membrane and integrity leads to abrupt onset hemolysis triggered by oxidative factors. That’s the pathogenesis behind manifestation of G6PD eficiency .

13. G6PD enzyme: Predominantly homo-dimer enzyme, containing 514 amino-acid residues. The gene is located on X chromosome (Xq2.8). Inheritance pattern is X linked recessive. G6PD deficiency is the most common enzymopathy . More than 400 biochemical variants derived from 230 mutations have been described. Mostly are asymptomatic. Depending on level of enzyme activity and clinical phenotype, the variants are grouped into 5 categories. Class I : severe deficiency assiciated with chronic hemolytic anemia Class II : severe deficiency (<10%) without hemolytic anemia Class III : moderate to mild deficiency (10% - 60% activity) Class IV : very mild or no deficiency Class V : increased activity (G6PD Hektoen )

14. Geographical distribution and advantage : G6PD deficiency is mostly present in Africa, middle east and southeast A sia and wherever people from these regions have migrated. In several of these areas, specially in malaria prone regions frequency of G6PD deficiency gene is as high as 20% or more. G6PD gene can be considered as an example of genetic polymorphism in human species. Advantage: G6PD deficiency provides resistance to Plasmodium falciparum malaria. P. falciparum has two redox system to deal with oxidative stress, those are – thioredoxin and glutathione. So it also required NAPDH and/or GSH for its survival and growth. Otherwise increasing oxidative stress within the cell causes death of the parasite and prevent spread of the infection.

15. Management : Symptomatic management and avoidance of precipitating factors. Screening : Fluoroscent spot test (FST)