number 18 Done by Mahmoud Harbi Corrected by حسام أبو عوض Doctor Nayef Karadsheh Sources of Reactive Oxygen Species (ROS) 1 P a g e
1- Oxidases: there are some that produce hydrogen peroxide (H₂O₂) 2- Oxygenases: two types: monooxygenases and dioxygenases 3- Coenzyme Q step in the respiratory chain: some electrons escape from this step and goto O₂and form the superoxide ion 4- Phagocytosis 5- Ionizing radiations Cytochrome P450 (CYP)Monooxygenases It is a family of Oxygenases that contains relatedhemecontaining monooxygenaseswith almost the same structure but they differ in their specificity. The P450 in the name indicates that the heme absorbs light at 450nm. Their electron source is NADPH. Monooxygenases are found in 2 sites in the cell: 1- Mitochondria: they are used here to hydroxylate intermediates in the conversion of cholesterol into steroid hormones. They are also used in bile acid synthesis in the liver and they also activate (process) vitamin D. 2- Microsomal (ER) Fraction: they are used here to detoxify toxic compounds and activate/inactivate drugs by hydroxylation. Then these toxins or drugs (or other substrates) are solubilised and excreted with urine. Mechanism of action: R-H+ O₂+H++ NADPH ---> R-OH+ H₂O+ NADP+ 1- The substrate combines with the Cytochrome enzyme to form an enzyme-substrate complex. 2- The iron in the heme is in the ferric state (Fe+3). An electron donated from NADPH reduces the Ferric into ferrous (Fe+2). How are electrons transported from NADPH to the Cytochrome?Cytochrome is coupled to a reductaseenzyme that carries an electron from the NADPH into the Cytochrome P450. This reductase has electron carriers (FAD, FMN and sometimes an iron-sulfur complex). The electron moves from 2 P a g e
the NADPH to the FAD then to the FMN of the reductase and then to the heme iron. In the mitochondrial system, the electron moves from FAD in the reductase to an iron-sulfur complex then to the heme iron of the Cytochrome P450 3- O2binds to the enzyme-substrate complex and gains an electron from the iron to become O2-and the iron goes back to the ferric state. 4- Another electron is donated by NADPH, but this time it reduces the O₂not the iron, so it becomes O₂ ² 5- One oxygen atom is reduced to water and the other is incorporated into the substrate creating a hydroxyl group (ROH) and the cytochrome goes back to its normal state. -How does this produce ROS? Some electrons escape from the cycle and receive oxygen and this forms superoxide ions. Phagocytosis White blood cells kill microbes and foreign bodies by two ways: 1- Oxygen-dependent way In this mechanism the microbe is degraded in the macrophage. When the bacteria enter the body, it is attacked by an antibody. The antibody is now holding the bacteria. The antibody binds to the receptor on the 3 P a g e
macrophage surface and enters the cell with the bacteria it caught. A phagosome is formed and it combines with a lysosome to form a phagolysosome. - How does the phagolysosome kill the bacteria? It uses an enzyme called NADPH oxidase which converts O2 into superoxide ion by oxidising NADPH. This ion is converted spontaneously or by superoxide dismutase into hydrogen peroxide. H₂O₂ produces HOCl (hypochlorous acid) or produces an OH radical. To produce HOCl, H₂O₂ combines with Cl- catalyzed by an enzyme called myeloperoxidase. The HOCl produced kills the bacteria. To produce the OH radical,iron is used (Fe²+Feᵌ+). The OH radical produced kills the bacteria. Respiratory burst: the rapid production of ROS by consuming oxygen which happens in phagocytosis to digest the foreign organism. 2- Oxygen-independent: the phagolysosome kills the bacteria using ph. Nitric oxide (NO) - It is a ROS, free radical, neurotransmitter in the brain, vasodilator and it prevents platelet aggregation. So, it is functional and can be toxic if its concentration is high. - With high concentration, the NO reacts with the superoxide ion and forms Reactive nitrogen oxygen species (RNOS). - Formation of NO: It is formed by an enzyme called nitric oxide synthase which uses Arginine, oxygen and NADPH as substrates. - Nitric oxide synthase has 3 forms (isozymes), 2 of these 3 forms are constitutive (synthesized at a constant rate) and produce small amounts of NO just enough for vasodilation and neurotransmission. They are: 1. The neural nitric oxide synthase (nnos) 2. The endothelial nitric oxide synthase (enos). 3. The third form is called inducible nitric oxide synthase (inos) and it is not constitutive. The inos 4 P a g e
is found in the immune cells. Exposure of the immune cells to microorganisms induces the production of inos (that is why it is called inducible). The high number of inos enzymes produce a lot of NO. - NO combines with ROS and produces RNOS which also kills the bacteria in phagocytosis. - NO also works as a smooth muscle relaxant or vasodilator: it activates the enzyme guanylatecyclase which produces cgmp. cgmp activates protein kinase G which phosphorylates Ca²+channel proteins and decrease the calcium entry into the muscle cell. This causes relaxation. G6PD deficiency This enzyme is the rate limiting enzyme in the pentosephosphate pathway. Its deficiency is the most common intracellular enzyme deficiency amongst all enzymes (lactase enzyme is extracellular). It affects more than 400 million people worldwide (5-10% of world s population). It has the highest prevalence in the Middle East, tropical Africa and Asia and parts of the Mediterranean. The gene of this protein is X-linked. There are a lot of mutations for this gene, that s why there are a lot of variants of the deficiency. These mutations have been found using molecular biology (by using DNA sequencing) and more than 400 variants have been identified (Most common is the Mediterranean variant B-, around 65-70% of the patients). Most mutations are missense (point mutations) that do not inactivate the enzyme completely (The Mediterranean variant is a mutation that changes the base number 563 from cysteine to thymine, so proline is translated instead of phenylalanine). Large deletion or frameshift mutations have not been identified because the enzyme will be completely absent which is lethal. The deficiency has an advantage which is providing resistance against malaria like the sickle cell anaemia which also provides resistance against malaria. 5 P a g e
The most affected cell by this deficiency is the RBC because it has no other source of NADPH except the pentose-phosphate pathway. How does the low amount of NADPH cause haemolysis? When the patient is exposed to an infection (which produces ROS) or if the patient eats certain foods that have oxidants, the oxidants are reduced by glutathione peroxidase using glutathione. Glutathione now is oxidised and needs to be reduced, but glutathione reductase needs NADPH to reduce it. Because there is a low concentration of NADPH the glutathione will not be reduced so there will be a low amount of functional glutathione. This leads to low protection and more tendency for haemolysis upon exposure to oxidative stress (oxidative stress is when there are more oxidants than what the body s protective mechanisms can compensate for). Additional oxidation of membrane proteins causes RBC to be rigid (less deformable) and with a short half-life. Rigid cells are then removed from the circulation by macrophages. Some patients develop haemolytic anaemia if they are treated with an oxidant drug. Commonly used drugs that cause the haemolytic anaemia are: 1-Some antibiotics such as sulfamethoxazole and chloramphenicol 2-Some anti-malarias such as primaquine (was the main drug behind discovering the G6PD) 3-Some antipyretics such as acetanilide There are a lot of variants of the deficiency, but we can divide them to four classes: Class I (very severe): they cause chronic haemolytic anaemia. Their residual enzyme activity is less than 2%. The less the residual enzyme activity, the more severe the disease is. Class II (severe):a good example is Mediterranean (B-) G6PD. It causes acute haemolytic anaemia. Their residual enzyme activity is less than 10%. 6 P a g e
Class III (moderate): G6PD A- (African, two-point mutation) deficiency is a good example. Their residual enzyme activity ranges from 10%-60%. Class IV: there are mutations in the gene, but the enzyme is working. Their residual enzyme activity is above 60%. A good example is the variant A (African, one-point mutation 80%). Class II and III don t cause chronic haemolysis, they cause mild haemolysis that sometimes you don t notice. But the problem is when the patient is exposed to an oxidative stress, haemolysis happens. The normal enzyme with no mutations is given the symbol B As the red blood cell ages, the G6PD activity decreases. In the Mediterranean G6PD red cells, most young cells are not able to prevent oxidative damage because the enzyme activity is low in young cells. But in the A-,young cells are able to prevent oxidative damage because the enzyme activity is high in young cells. Diagnosis of the deficiency: Mediterranean: it is easily diagnosed because the activity is low from the start. The A-: it is hard to diagnose from the start because the enzyme activity is high at the beginning, like the normal enzyme, so you have to wait 2 to 3 weeks and ask for another diagnostic test. Alcohol metabolism 7 P a g e
- Ethanol is metabolised by alcohol dehydrogenase into acetaldehyde. NADH is also produced. Most of the acetaldehyde enters the mitochondria as acetate by the enzyme aldehyde dehydrogenase (NADH also produced). Acetate causes no problems, it goes to the blood and is distributed to different tissues. Acetate is taken up by different cells like muscle cells and cardiac cells and goes either to the mitochondria or stays in the cytosol of these cells. Acetate is then converted to acetyl CoA using acetyl CoA synthase. Acetyl CoA in the mitochondria goes to the Krebs cycle and the acetyl CoA in the cytosol is used in synthesising compounds like fat and cholesterol. The problem is when acetaldehyde (not acetate) enters the blood, there it has damaging effect. It causes liver cirrhosis and damages proteins. -There is an enzyme in the microsomal fraction called microsomal ethanol oxidizing enzyme which also metabolises alcohol producing acetaldehyde. It is one of the Cytochrome P450 enzymes and has the same mechanism of action as the Cytochrome. This enzyme has ahigh Km which means that it has low affinity. But for people that ingest alcohol, the enzyme metabolises more alcohol producing more acetaldehyde. Response of people to alcohol is different because of the polymorphism of this enzyme. -Biochemical consequences of ingesting lots of alcohol: It causes high NADH/NAD+ ratio. The high NADH/NAD+ ratio automatically causes high lactate, inhibits fatty acid oxidation and it inhibits gluconeogenesis. Gluconeogenesis is inhibited because pyruvate becomes lactate and the gluconeogenesis needs pyruvate to work. That is why drinking alcohol without eating enough amounts of sugars is bad. 8 P a g e