* Because of its dependence on the pentose monophosphate shunt, NADPH production is significantly impaired in individuals with deficiencies in G6PD.

Transcription

1 Glycolytic detours: A side reaction of glycolysis known as the pentose monophosphate shunt is key to supplying the NADPH. Required to remove cellular reactants such as H 2 2. NADPH is utilized by glutathione (GSH), GSH reductase and GSH peroxidase. NADPH also utilized by catalase (active catalase contains 4 tightly bound NADPH molecules). * Because of its dependence on the pentose monophosphate shunt, NADPH production is significantly impaired in individuals with deficiencies in G6PD. Inhibitors of glycolysis include fluoride, and arsenate. In the presence of arsenate, ATP normally formed in the conversion of 1,3-bisPG into 3-PG is lost (no net ATP prod.).

2 Pentose monophosphate shunt: GLYCLYSIS Glucose ATP Hexokinase ADP Glucose 6 P Fructose 6-phosphate + Glyceraldehyde 3 P PENTSE SHUNT * Ribose 5-phosphate 6 phosphogluconate phosphopentose Glucose 6-P (lactonase) dehydrogenase dehydrogenase isomerase 6-Phosphogluconate Ribulose 5-phosphate Fructose 6P NADP+ ATP ADP Fructose 1,6 bis-phosphate NADPH Glutathione reductase NADP + (FAD) GSSG GSH NADP + NADPH + C 2 H 2 Glutathione Peroxidase 2 + H 2 2 catalase (NADPH) Superoxide Dismutase (2H+) 2 + 2H 2 *Shunt provides needed NADPH (tumors) *Selenium is essential for glutathione (GSH) peroxidase activity. (2) 2 superoxide radical MetHb Hb + 2

3 Pentose monophosphate shunt: Xylulose (5C) + Ribose (5C) Ribose 5-phos. Ribulose 5-phosphate (5C) Transketolase Glyceraldehyde (3C) + Sedoheptulose (7C) (ribulose 5 phospate epimerase) Xylulose 5-phosphate (5C) Transaldolase Fructose (6C) + Erythrose (4C) Xylulose (5C) + Erythrose (4C) Stoichiometry 2 F6P + 1 G3P Transketolase Glyceraldehyde 3 P (3C) + Fructose 6-phosphate (6C) (Glycolysis)

4 DRUGS AND TXINS WHICH AFFECT ERYTHRCYTE FUNCTIN a) Complex hemoglobin Fe 2+ b) xidize hemoglobin Fe 2+ c) Hemolysis (Hb, RBC destruction) d) Genetic diseases (G6PD, HbS, Thal.)

5 Toxins which affect erythrocyte function: Carbon monoxide induces hypoxia by complexing Fe 2+ Hb (victims - bright red). - the avidity of heme group for C is 25,000 times greater than for oxygen. - the avidity of Mb / Hb for C is only 200x greater than 2 due to distal histidine. - C can thus be life threatening at relatively low conc. 25,000x 200x Voet Fig 7-13

6 xidation of Fe 2+. Excessive oxidation of Fe 2+ to Fe 3+ (oxyhemoglobin to methemoglobin) can induce hypoxia. deoxyhb: Fe 2+ (purple) oxyhb: Fe 2+ 2 (bright red) methb: Fe 3+ (brown) - 2 binds to Fe(III) irreversibly CyanHb: antidote for cyanide poisoning (amyl + Na nitrite) Why? Nitrite converts Fe 2+ to Fe 3+, creating binding sites for CN (better than Fe 3+ center on cytochrome oxidase). METHEMGLBINEMIA can be induced by a variety of drugs. These include: aniline drugs (dapsone, etc.) nitro aromatic drugs hydrazine drugs oxidants, chlorates, nitrites (cyanide antidote) quinones, naphthalene, benzene arsine

7 Role of GSH and catalase in detoxifying reactive oxygen species generated by genetic deficiency or xenobiotics: MAINTENANCE F ERYTHRCYTE GSH LEVELS 2H 2 2 catalase Drugs H 2 2 oxyhemoglobin methemoglobin 2 GSH Hemolysis Glutathione peroxidase - Se GSSG NAD+ methemoglobin reductase (cb5) NADH CELLULAR DAMAGE CAUSED BY MB NADPH dependent methemoglobin reductase NADPH methylene blue xyhemoglobin Fe 2+ 2 RED DRUGS (sulfonamides, dapsone primiquine, anesth.) and FAVA beans NADP+ Leucomethylene blue Methemoglobin _ Fe BRWN 2 + 2H 2 2H 2 H Fe 2+ Detoxified by GSH or Catalase Hemolysis (Black Urine) 2H Hydroxyl radical xidize Membrane Lipids

8 Hemolysis. Hypoxia can also be induced by premature destruction of erythrocytes or their progenitors (Anemia). Activated oxygen (oxygen free radicals) are released when oxyhemoglobin oxidized. These short-lived species can subsequently attack sites such as the erythrocyte membrane. This is frequently seen in cases of glucose 6-phosphate dehydrogenase deficiency (details later). HEMLYTIC ANEMIA (due to excessive free radical generation) can be induced by a variety of different drugs. These include: aromatic amines nitro compounds hydrazines antimalarial drugs Fava beans

9 Muscle fibers and oxidative metabolism: Fast twitch: - Used for rapid contractions of brief duration. - Energy primarily from anaerobic glycolysis, thus they can contract more rapidly than oxygen can be delivered to them. -They fatigue quickly and go into oxygen debt until the lactic acid produced via glycolysis can be re-oxidized after activity. - Glycogen content higher than slow fibers, mitochondrial content lower -Fast fibers contain little myoglobin and appear white (white meat). Slow twitch: - Used for sustained activity, do not fatigue easily (oxygen debt). - Derive energy from oxidative metabolism. - Richly supplied with blood vessels (oxygen delivery) - High mitochondrial content (oxidative metabolism) - Large amount of myoglobin - reddish color (dark meat)

10 Anesthetics and calcium: Malignant hyperthermia: Susceptible individuals: 1:12,000 in children to 1:40,000 1:70,00 in adults. Locus: chromosome 19 - Ryanodine receptor. Anesthetic produces excessive Ca 2+ release in skeletal muscle, resulting in the following: Excessive production of heat and lactic acid, ultimately resulting in acidosis and death. Condition is reversible if caught in the first several MINUTES (cooling, dantrolene) ther anesthetic risks: 20% of metabolism "toxic, can cause hepatic damage with repeated exposure. Risk of spontaneous abortion in pregnant R staff.

11 UNIT VERVIEW: XIDATIVE BILGY F THE ERTHRCYTE PRTECTIN AND MECHANISMS F INJURY Redox protection of erythrocytes (glutathione) Energy production for redox reaction (PMPS) Genetic disorders of erythrocyte function

12 xidative free radicals: Fenton Reaction: Fe 2+ + H 2 2 Fe 3+ + H - + H Haber-Weiss Reaction: [Fe 3+ Fe 2+ ] 2 + H H - + H Reaction Cycling: 2 + Fe Fe +2 Macrophage / WBC s: Cl - + H 2 2 H - + HCl Hypochloruous acid 2 + HCl HCl + Fe Cl - + H Fe +3 + Cl - + H 2 + N N - Nitric oxide - vasodilator, neurotransmitter (peroxynitrite - inflam./ atherosclerosis)

13 xidation and free radicals: - A secondary consequence of oxidative metabolism is the potential for the production of free radicals. - Free radical formation is exacerbated in presence of iron. - Thus sites such as the erythrocyte must possess mech. to deal with free radical formation. 4 e- + 4 H+ 2 2H 2 e SD e 2 Superoxide anion Cytochrome oxidases H 2 2 Hydrogen peroxide Cl- Catalase / GSH peroxidase e H Hydroxy radical e (Myeloperoxidase) HCl* H 2

14 Redox protection of erythrocytes (Glutathione - GSH) Cofactor for cellular defence against oxidative stress. oxidized H 2 2 reduced 2 GSH (GSH peroxidase) reduced H 2 GSSG oxidized

15 Properties of glutathione: - Principle antioxidant of the cell. - Tripeptide consisting of glutamate, cysteine, and glycine. - Glutathione is particularly important for erythrocytes and liver hepatocytes. In red blood cells, GSH exists at high concentrations (5-10 mm). - GSH is a cofactor for many cellular enzymes % of GSH exists in the cytosol, while 10-15% exists in the mitochondria. - Detoxifies reactive drug metabolites (acetaminophen).

16 Cellular roles of glutathione: - Scavenging activity on free radicals. - Maintains essential redox status of proteins by maintaining cysteine thiols in their reduced (SH) form. - Provides reservoir of cysteine for protein synthesis (not erythrocytes). - Modulates processes such as DNA synthesis (not erythrocytes), immune / microtubular processes. Curr. Top. In Cell. Regul., Vol 36:

17 GSH exits in two different forms in the cell: This carboxyl linkage can NLY be cleaved by the enzyme γ-glutamyl transpeptidase (GGTP) H H 2 C CH 2 NH 2 HN H g-glutamyl cysteinyl glycine NH 2 H N HS N H H HN CH 2 H H 2 C S S CH 2 NH H 2 C NH CH 2 H 2 C H NH 2 REDUCED GSH H XIDIZED GSH (GSSG) = 2 reduced GSH s covalently bonded by a disulfide bridge.

18 Glutathione synthesis and catabolism: Glutathione (GSH) Cysteine + ATP ADP + Pi Glycine + ATP ADP + Pi γ-glutamyl cysteinyl glycine Glutamate γ-glu γ-glutamyl-cysteine synthetase Cys Glutathione synthetase H NH 2 H N SH N H H buthionine sulfoximine (chemotherapy) free cysteine N-acetyl-cys - S - R dipeptidases (ectoenzyme) Cys Gly Cys - S - R γ-glutamyl-transpeptidase GGTP (ectoenzyme) dipeptidase R - S - G Drug (R) (electrophiles) glutathione-stransferase (GST) GGTP (AA) γ-glu- (AA) CoA Modified from Stryer acetyl-coa gly- (AA) (AA) Drug detoxification

19 Antioxidant role of GSH in erythrocytes: Methemoglobin Fe _ Hb + 2 H 2 2 catalase H CYTSL (Fe) H H GSH GSH peroxidase GSSG H 2 ATP ADP NADP + GSH reductase NADPH Pentose monophosphate pathway MRP Curr. Top. Cell. Regul. 36: (2000)

20 ther examples of GSH-mediated protection: (Liver) NHCCH 3 ACETAMINPHEN (paracetamol) NHCCH 3 Man Mouse Rabbit Rat NHCCH 3 S 3 PAPS -glucuronide SULFATE CNJUGATE (URINE) Sulfotransferase (60%) H GLUCURNIDE CNJUGATE (URINE) ER: N-acetyl cysteine overflow P450 (Cyp 2E1) NHCCH 3 Gly Cys H Glu GSH 1,4-Michael addition + NHCCH 3 Protein-SH LIVER NECRSIS Symptoms mild until 24 hrs post ingestion - nausea, vomiting, elevated AST. Resolution or death by 5 days. Doses of >140 mg/kg in children or >10g in adult are toxic. GSH CNJUGATE TXIC INTERMEDIATE

21 Genetic disorders of erythrocyte function a. Genetic and environmental causes of glucose 6-phosphate dehydrogenase deficiency b. Abnormal hemoglobins (HbS and the thalassemias)

22 Genetic G6PD deficiency: - Like a number of other disorders, G6PD deficiency is X-linked - FEMALES heterozygotes have 2 populations of red cells (wild-type and def.) - A - 16% of Africans and Black Americans - B - (numerous variations) Mediterranean: Greece, Turkey, Israel, Egypt, Italy. -It is estimated that worldwide ~400 million people are deficient in G6PD! - Type 1 <2% (Med.), Type II <10%, Type III 10-50% (type A), Type IV - normal X-chromosome - long arm: Colour Blindness Glucose-6-P Dehydrogenase Factor VIII (Haemophilia A) ptic atrophy Xm serum groups Kurdish Jews Percent G6PD deficient MALE population 62% Sardinia 30% Saudi 13% Sideroblastic anemia Muscular dystrophy U.S. blacks 11%

23 G6PD deficiency and oxidative damage: glyceraldehyde 3 phosphate PENTSE SHUNT Glyceraldehyde 3-P ATP ADP Glucose Hexokinase Glucose 6 P Glucose 6-P dehydrogenase 6-Phosphogluconate + Fructose 6-phosphate Ribose 5-phosphate NADP + NADPH NADP+ NADPH GSSG Carmustine (Chemotherapy -lymphoma, myelomas and brain tumors) GSH reductase NADP + H FAD GSH (Fe) H 2 GSH Peroxidase H catalase / NADPH Superoxide Dismutase SD superoxide radical MetHb 2 H Hb + 2

24 Structural mutations of the G6PD gene: - More than 100 amino acid mutations identified for G6PD identified. Two example G6PD mutations: N126D mutation V68M mutation only 8 angst. apart in crystal structure Asparagine 126 Aspartate Does not affect activity G6PD A - has 85% normal activity (G6PD B = normal = 100% activity) Valine 68 Methionine Increases rigidity in protein affects Lysine 205 (active site that binds glucose 6 phosphate protein folding) (G6PD A- 12% activity both mut.) Single letter amino acid codes: J. Biol. Chem. 275, (2000), Febs Lett. 366, 61-4 (1995)

25 Global G6PD deficiency and polymorphisms: Luzzatto & Notaro, Science. 293:442-3.

26 Epidemiology of Malaria:

27 Malaria - Mala (bad), Aria (air): 8000 BC Introduction of agriculture in Middle East & Africa, promoting conditions for spread of malaria 5700 BC Ancestral Plasmodium falciparum 1200 BC Heterozygote for G6PD exhibit malarial resistant (mis-sense mutations of small in-frame deletions) 476 AD Fall of the Western Roman Empire (malarial contribution) Luzzatto & Notaro, Science. 293:442-3.

28 Infection of red cells by malarial parasite: Plasmodium Falciparum (the most serious and prevalent form of malaria) is a protozoan parasite carried by mosquitoes. The parasite attacks red blood cell hemoglobin using a specialized food vacuole percent of the hemoglobin content in infected cells can be consumed by the parasite. Normal Red Cell Low glucose utilization - Low lactate formation Infected Red Cell High glucose utilization Malarial parasite needs ATP for RNA/DNA/Protein synthesis. ATP is taken from the erythrocyte by the synthesis of parasitic hexokinase.

29 G6PD deficiency combats malaria: ERYTHRCYTE G6P dehydrogenase NADP + NADPH H 2 + 1/2 2 H 2 2 GSH peroxidase (reductase) hemoglobin Malarial food vacuole hemoglobin (proteases) heme Fe 2+, TXIC Malarial parasite Amino hemazoin Acids Attack malarial parasite Fe 2+ 2 H 2 2 Fe 2+ H + H - Consequences of G6PD deficiency: Lower GSH:GSSG ratio in the cell, leading to higher levels of reactive oxygen species (RS) in the cell. Result: Less than ideal environment for survival of malarial parasite.

30 Malarial parasite biochemistry in red cells: GLUCSE ATP PARASITE HEXKINASE ADP PARASITE RNA & DNA SYNTHESIS PRTEIN SYNTHESIS GLUCSE-6-PHSPHATE 6-PHSPHGLUCNATE RIBSE PHSPHATE NADP+ GLYCERALDEHYDE-3-P NADPH LACTIC ACIDSIS PYRUVATE LACTATE NADH NAD+

31 Parasite biochemistry - cellular targets: 1. Infected red cells show increased glycolysis (30x). This can result in lactic acidosis due to pyruvate buildup (coma). 2. Red cell ATP hypoxanthine parasite purines RNA/DNA 3. Makes ATP by glycolysis (not citric acid cycle). Mitochondrial electron transport chain is for pyrimidine synthesis but acidosis inhibits erythrocyte glycolysis. 4. Degrades hemoglobin to release amino acids parasite protein synthesis (hemozoin) 5. Also makes NADPH through its own G6PDH (also glutamate dehydrogenase) Voet, Biochemistry; Pharmacol. Ther. 81, (1999).

32 NH 2 DRUG INDUCED HEMLYTIC ANEMIA and G6PD: Liver P450 H H N MDEL DRUG METABLITE (Phenylhydroxyamine) ERYTHRCYTE Aniline * Individuals with G6PD deficiency particularly susceptible to this mechanism of toxicity NADP+ NADPH REDUCTASE NADPH Membrane Damage Eventual Hemolysis REDX CYCLE N (Nitrosobenzene) H HYDRXY RADICAL Scavenged by : Vitamins C/E, GSH, etc. ( 2 ) 2H + Fe II/Cu I 2 GSH GSH peroxidase xyhemoglobin (H 2 ) Fe 2+ Fe 3+ Methemoglobin + 2 H 2 2 GSSG Disproport. (via SD) catalase Methemoglobin Reductase SUPERXIDE ANIN HYDRGEN PERXIDE H 2 + 1/2 2

33 Drugs which induce hemolytic anemia in G6PD deficient individuals In the modern world, individuals with G6PD deficiency typically exhibit few ill effects, until: Acetanilide Aminopyrine Aspirin Chloroquine Dapsone Dimercaprol Furazolidine Mepacrine Methylene Blue Naphthalene Nitrofurantoin Pamaquin, Pantaquin Phenacetin Phenylhydrazine Primaquine * Probenecid Salicylates Sulfa drugs Toluidine blue Reference: New Engl J Med. 324, (1990).