WBCs Metabolism By Dr. Samar Kassim

List the major metabolic pathways in neutrophils. Understand the defensive role of WBCs. Define the free radicals. Know that ROS may be good, may be bad. List the reactive oxygen species (ROS).

6. Understand the oxygen burst. 7 6. Understand the oxygen burst. 7. Understand the function of different enzymes involved in production and scavenging of ROS. 8. Understand the regulation of proteinase activity. 9. Know how anti-proteinase deficiency cause disease.

Neutrophils Have An Active Metabolism Major Biochemical Features of Neutrophils: Active aerobic glycolysis Active HMP Moderately active oxidative phosphorylation (because mitochondria are relatively few) High content of lysosomal enzymes Unique enzymes as myeloperoxidase and NADPH oxidase

Acute Inflammatory Response Neutrophils Contribute in Acute Inflammatory Response

Acute Inflammatory Response Increase of vascular permeability. Entry of activated neutrophils into the tissues. Activation of platelets. Spontaneous subsidence (resolution) if the invading microorganisms have been dealt with. One of the mechanisms of dealing with microorganisms is production of reactive oxygen species (ROS).

Reactive Oxygen Species ROS Trigger Inflammation

Reactive Oxygen Species ROS Powerful oxidants produced during the course of metabolism, Produced by blood cells and most other cells of the body. Superoxide (O2—∙) Hydrogen peroxide (H2O2) Peroxyl radicals (ROO•) Hydroxyl radicals (OH•) & OH-

Atoms or groups of atoms that have an Unpaired Electron Free Radicals (ROS) Atoms or groups of atoms that have an Unpaired Electron

ROS Play Important Role in Cellular Injury, e.g., resulting from administration of various toxic chemicals or ischemia, some of which can result in cell death. Several reactions are involved in their production and disposal.

Reaction 1 Superoxide (O2—∙) In RBCs: from Auto-oxidation of Hb -> Met Hb (about 3% daily). In other tissues: Formed by cytochrome p450 and xanthine oxidase

Reaction 2 Respiratory Burst Stimulated in neutrophils by contact with bacteria They produce superoxide in a reaction catalyzed by NADPH oxidase NADPH + 2O2 ↔ NADP+ + 2O2− + H+

Reaction 2 Respiratory Burst The NADPH is generated mainly by HMP whose activity increases markedly during phagocytosis NADPH + 2O2 ↔ NADP+ + 2O2− + H+

the rate of this same reaction is speeded up by Dismutation Spontaneous OR the rate of this same reaction is speeded up by

Many Fates for H2O2 Catalase (R4) Myeloperoxidase (R5) Glutathione peroxidase (R6) The Fenton reaction (R7) Haber-Weiss reaction (R8)

Reaction 4 Catalase

H2O2 and Halides produce Hypochlorus acid Reaction 5 Myeloperoxidase In Neutrophils H2O2 and Halides produce Hypochlorus acid

Glutathione peroxidase (Se) Reaction 6 Glutathione peroxidase (Se) Se

Reaction 7 The Fenton reaction

Reaction 8 Haber-Weiss reaction

Reaction 8 O2—∙ and Fe3+ are the substrates in the iron-catalyzed Haber-Weiss reaction, which also produces OH• and OH–

Reaction 8 O2—∙ can release iron ions from ferritin

Reaction 8 production of OH• may be one of the mechanisms involved in tissue injury due to iron overload in hemochromatosis

Any Questions

ROS Stress Superoxide (O2—∙) Hydrogen peroxide (H2O2) Peroxyl radicals (ROO•) Hydroxyl radicals (OH•) Chemical compounds and reactions capable of generating potential toxic oxygen species can be referred to as pro-oxidants.

Anti-oxidants Enzymes: SOD, Catalase, GSH peroxidase Non enzymes: NADPH, GSH, ascorbic acid, and vitamin E Compounds and reactions disposing of these Toxic Oxidant species, scavenging them, suppressing their formation, or opposing their actions.

In a normal cell, there is an appropriate pro-oxidant/antioxidant balance. However, this balance can be shifted toward the pro-oxidants in the following conditions: Increased production of oxygen species (e.g., following ingestion of certain chemicals or drugs). The state of diminished levels of antioxidants by inactivation of enzymes involved in disposal of oxygen species. This imbalance is called “oxidative stress” and can result in serious cell damage if the stress is massive or prolonged.

Oxidative stress Imbalance between production of toxic oxygen species & activity of different anti-oxidants

Neutrophil Reaction to OS RESPIRATOY BURST

RESPIRATOY BURST When neutrophils and other phagocytic cells engulf bacteria, they exhibit a rapid increase in oxygen consumption known as the respiratory burst. This is the rapid utilization of oxygen (following a lag of 15–60 seconds) and production of large amounts of reactive oxygen derivatives, such as O2—∙ , H2O2, OH•, and OCl− (hypochlorite ion). Some of these products are potent microbicidal agents.

RESPIRATOY BURST Neutrophils Contain Myeloperoxidase, Which Catalyzes the Production of Chlorinated Oxidants

RESPIRATOY BURST The enzyme myeloperoxidase is present in large amounts in neutrophil granules.

RESPIRATOY BURST It is responsible for the green color of pus, can act on H2O2 to produce hypohalous acids (hypochlorite)

Neutrophil Reaction to OS HOCl, the active ingredient of household liquid bleach, is a powerful oxidant and is highly microbicidal.

Neutrophil Reaction to OS When the Cl− is applied to normal tissues, its potential for causing damage is diminished. Cl− reacts with some amines to produce chloramines, which are also oxidants, though less powerful than HOCl.

disposed of by the action of glutathione peroxidase or catalase. Any superoxide that enters the cytosol of the phagocytic cell is converted to H2O2 by the action of superoxide dismutase, In turn, H2O2 is used by myeloperoxidase OR disposed of by the action of glutathione peroxidase or catalase.

Any Questions

Reaction 2 Respiratory Burst Stimulated in neutrophils by contact with bacteria They produce superoxide in a reaction catalyzed by NADPH oxidase NADPH + 2O2 ↔ NADP+ + 2O2− + H+

Reaction 2 Respiratory Burst The NADPH is generated mainly by HMP whose activity increases markedly during phagocytosis NADPH + 2O2 ↔ NADP+ + 2O2− + H+

Mutation in NADPH Oxidase

Mutation in NADPH Oxidase Mutations in the Genes for Components of the NADPH Oxidase System Cause Chronic Granulomatous Disease

Neutrophils Have An Active Metabolism Major Biochemical Features of Neutrophils: Active aerobic glycolysis Active HMP Moderately active oxidative phosphorylation (because mitochondria are relatively few) High content of lysosomal enzymes Unique enzymes as myeloperoxidase and NADPH oxidase

Proteinases of Neutrophils Proteinases of Neutrophils help getting rid of invading organisms by hydrolyzing elastin, various types of collagens, and other proteins.

Proteinases of Neutrophils If uncontrolled, they can Cause Serious Tissue Damage. Most of these proteinases are lysosomal enzymes and exist mainly as inactive precursors in normal neutrophils.

Proteinases of Neutrophils The activities of elastase and other proteinases are normally kept in check by a number of anti-proteinases present in plasma and the extracellular fluid.

Proteinases of Neutrophils Each of them can combine —usually forming a non-covalent complex— with one or more specific proteinases and thus cause inhibition.

Proteinases of Neutrophils Genetic deficiency of α1-antiproteinase inhibitor (α1-antitrypsin) permits elastase to act unopposed and digest pulmonary tissue, thereby participating in the causation of emphysema

List the major metabolic pathways in neutrophils. Understand the defensive role of WBCs. Define the free radicals. Know that ROS may be good, may be bad. List the reactive oxygen species (ROS).

6. Understand the oxygen burst. 7 6. Understand the oxygen burst. 7. Understand the function of different enzymes involved in production and scavenging of ROS. 8. Understand the regulation of proteinase activity. 9. Know how anti-proteinase deficiency cause disease.

Thx