L8 - Metabolic Pathways in RBCs PDF

8 Metabolic pathways in RBCs ILOs:By the end of this lecture, students will be able to 1. 2. 3. 4. Outline HMS pathway and its end products and regulation Outline glutathione peroxidase cycle Discuss the synthesis and functions of glucuronic acid Appraise the importance of HMS and uronic acid pathwa...

8 Metabolic pathways in RBCs ILOs:By the end of this lecture, students will be able to 1. 2. 3. 4. Outline HMS pathway and its end products and regulation Outline glutathione peroxidase cycle Discuss the synthesis and functions of glucuronic acid Appraise the importance of HMS and uronic acid pathways in relation to hemoglobin and RBCs 5. Correlate defects in HMS and uronic acid pathways to clinical conditions  As you recall from your glycolysis lecture in Block 2, ANAEROBIC glycolysis is the ONLY pathway for production of energy inside the RBCs, due to absence of mitochondria. You have also learned that defects in glycolytic enzymes, such as pyruvate kinase deficiency, can cause hemolytic anemia (refer to glycolysis lecture, block 2)  However, other metabolic pathways are of utmost importance either for keeping the integrity of the RBCs membrane, or for getting rid of the bilirubin released during hemoglobin breakdown. Such pathways include Hexose monophosphate shunt and uronic acid pathway, both of which occur in the CYTOPLASM Hexose monophosphate shunt (Pentose phosphate pathway) (Fig 1) Importance:  It is a parallel pathway to glycolysis that takes glucose 6-phosphate (G-6-P), produced by glycolysis, and converts it through a different series of reactions to fructose 6-phosphate and glyceraldehyde 3-phosphate while generating NADPH in the process.  The first step is the conversion of G-6-P to ribose 5-phosphate (R-5-P), which generates two NADPH. It includes an irreversible oxidative phase followed by a series of reversible sugar– phosphate interconversions (Figure 1) Figure 1: Hexose monophosphate shunt 1 Regulation of HMS  Glucose 6-phosphate dehydrogenase (G6PD) initiates this pathway, and comprises the rate-limiting and only regulated step in the whole pathway.  G6PD is feedback inhibited by NADPH, which competes directly with NADP+ for the enzyme active site. Therefore, the NADPH/NADP+ ratio ensures that the cell only produces the needed amount of NADPH. N.B. Insulin upregulates expression of the gene for G6PD, and flux through the pathway increases in the absorptive state (When insulin concentration increases) N.B: No ATP is directly consumed or produced in the pathway. Products of HMS and their importance 1- Ribose-5-P Produced by the HMS and has two potential fates: Substrate for the synthesis of nucleotides: it produces the ribose sugar backbone. Conversion through multiple steps to glycolytic intermediates (the shunt): 3 ribose 5-phosphate >>>>> 2 fructose 6-phosphate + glyceraldehyde 3-phosphate 2- NADPH The coenzyme NADPH differs from nicotinamide adenine dinucleotide (NADH) only by the presence of a phosphate group on one of the ribose units. This seemingly small change in structure allows NADPH to interact with NADPH-specific enzymes that have unique roles in the cell as follows: i) Reductive biosynthesis Such as the production of fatty acids in liver, adipose tissue, and the mammary gland; cholesterol in the liver; and steroid hormones in the placenta, ovaries, testes, and adrenal cortex ii) Hydrogen peroxide reduction (Role in erythrocytes)  Hydrogen peroxide (H2O2) is a member of the family of reactive oxygen species (ROS) that are formed from the partial reduction of molecular oxygen (O2).It can cause peroxidation of lipids present in cell membranes, such as RBCs membrane, causing their destruction, damage of cell membrane and hemolysis. (Figure 3)  The anitooxidant enzyme: glutathione peroxidase, defends the body against hydrogen peroxide. Glutathione peroxidase is a selenoprotein (contains selenium). It utilizes reduced glutathione to complete its protective reaction against H2O2.  Reduced glutathione (GSH),is a tripeptide-thiol (γ-glutamyl-cysteinyl-glycine) present in most cells, and can chemically detoxify H2O2.  Glutathione peroxidase reacts with the harmful hydrogen peroxide, to form oxidized glutathione (G-S-S-G), which no longer has protective properties.(figure 2) 2  The cell regenerates G-SH in a reaction catalyzed by glutathione reductase, using NADPH as a source of reducing equivalents. Thus, NADPH indirectly provides electrons for the reduction of H2O2. Figure 2: Glutathione peroxidase system iii) Role in white blood cells  Invading bacteria is recognized by the immune system and attacked by antibodies that bind it to a receptor on a phagocytic cell. After internalization of the microorganism has occurred, NADPH oxidase, located in the leukocyte cell membrane, is activated.  This enzyme uses the NADPH to reduce O2 from the surrounding tissue to superoxide anion (One of the ROS) (Figure 3). In this case, superoxide is used by the human body to kill the bacterial cell, then superoxide anion is removed by the endogenous antioxidant enzyme: superoxide dismutase  The rapid consumption of O2 that accompanies formation of is referred to as the respiratory burst. (Refer to block 3) Figure 3: Production of different ROS iv) Role in nitric oxide synthesis  Nitric oxide (NO) is the endothelium-derived relaxing factor that causes vasodilation by relaxing vascular smooth muscle. It also acts as a neurotransmitter, prevents platelet aggregation, and plays an essential role in macrophage function.  It has a very short half-life in tissues (3–10 seconds) because it reacts with O2 and and is converted into nitrates and nitrites  Note: NO is a free radical gas that is often confused with nitrous oxide (N2O), the “laughing gas” that is used as an anesthetic and is chemically stable. 3  Synthesis of nitric oxide requires the amino acid arginine, O2, and NADPH as substrates, and is catalyzed by nitric oxide synthase enzyme. v) Role in Cytochrome P450 mono-oxygenase system  This is the main system that helps the human body detoxify harmful agents such as drugs, food preservatives and others. Such harmful agents can destroy tissues, RBCs and other cells’ membranes  Monooxygenases (mixed-function oxidases) incorporate one atom from O2 into a substrate (creating a hydroxyl group), with the other atom being reduced to water (H2O). In the cytochrome P450 (CYP) monooxygenase system, NADPH provides the reducing equivalents (hydrogen) required by this series of reactions G6PD Deficiency (Favism)    G6PD deficiency is an X-linked hereditary condition, resulting from different point mutations in the gene coding for G6PD. It is the most common disease-producing enzyme abnormality in humans, affecting >400 million individuals worldwide (specially in the middle east) It is characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents. In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1–4 days after birth. The jaundice, which may be severe, typically results from increased production of unconjugated bilirubin Like Sickle cell trait and the thalassemias, G6PD deficiency also confers resistance to malaria. Pathogenesis     Diminished G6PD activity impairs the ability of the cell to form the NADPH that is essential for the maintenance of the G-SH pool. Since GSH is required for protection of cell membranes from the damaging effect of free radicals and peroxides formed within the cell, hence the lipids in RBCs membrane will be damaged resulting in hemolysis. ROS also oxidize the sulfhydryl groups in proteins, including hemoglobin. Oxidation of those sulfhydryl groups leads to the formation of denatured proteins that form insoluble masses (called Heinz bodies) that attach to RBC membranes Additional oxidation of membrane proteins causes RBC to be rigid (less deformable), and they are removed from the circulation by macrophages in the spleen and liver. Although G6PD deficiency occurs in all cells of the affected individual, it is most severe in RBC, where the pentose phosphate pathway provides the only means of generating NADPH. 4 Precipitating factors in G6PD deficiency Most individuals who have inherited one of the G6PD mutations do not show clinical manifestations (that is, they are asymptomatic), unless they are exposed to one of the following 1. Oxidant drugs: Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency are best remembered from the mnemonic AAA:    Antibiotics (for example, sulfamethoxazole and chloramphenicol), Antimalarials (for example, primaquine but not chloroquine or quinine), Antipyretics (for example, acetanilide but not acetaminophen). 2. Favism: Some forms of G6PD deficiency, for example, the Mediterranean variant, are particularly susceptible to the hemolytic effect of the fava bean. 3. Infection: Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The inflammatory response to infection results in the generation of free radicals in macrophages. The radicals can diffuse into the RBC and cause oxidative damage. Uronic acid pathway (Fig 5) Figure 5 :Uronic acid pathway    The main aim of this pathway is the production of Glucuronic acid. Glucuronic acid can be obtained in small amounts from the diet and from the lysosomal degradation of GAG. In the uronic acid pathway, glucose 1- phosphate reacts with uridine triphosphate (UTP) and is converted to UDP-glucose. (active form of glucose) Oxidation of UDP-glucose produces UDP-glucuronic acid, the form that supplies glucuronic acid. The end product of glucuronic acid metabolism in humans is D-xylulose 5-phosphate, which can enter the pentose phosphate pathway and produce the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate. 5 Importance of Uronic acid pathway 1. Synthesis of glucuronic acid which is used in conjugation of many agents(including drugs, hormones, food preservatives) as a step for detoxification of such agents. One of the agents that are conjugated by glucuronic acid is bilirubin. This helps excretion of bilirubin in urine and prevents development of jaundice. 2. Synthesis of GAGs and proteoglycans (refer to tissue organization block, C.T ground substance) 6