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1 .0 number 37 Done by حسام ا بو عوض Corrected by Doctor Diala Heme Degradation 1 P age

2 In the last lecture, we introduced this topic and now we ll go into its details. The red blood cells have a life span of approximately 120 days, this does not mean that every 120 days ALL of our red blood cells will die and new ones are to be made, every day there are some red blood cells that die and others being made (as not all of the red blood cells were produced at the same time) (some heme degradation occurs due to the rupturing of immature RBCs (15%)). When an RBC dies it releases its contents to the plasma, including haemoglobin. Haemoglobin cannot be recycled (recycling means that the molecule would simply be picked up by another cell and used directly) and so, it must get degraded (haemoglobin has a protein part and the heme part, protein degradation was discussed previously so we will only dig into the details of heme degradation here). The heme degradation process mainly occurs in the spleen (which functions as a blood filter, so if you ever dissect a spleen you ll find that it is very red from inside due to the red-blood-cells) and some of the degradation occurs in the reticuloendothelial system of the liver (we ll only discuss the spleen s). The heme is acted upon by the enzyme heme-oxygenase changing it into biliverdin (simply, the porphyrin ring of the heme gets broken resulting in several ring structures bound linearly to each other (see the diagram)). In the process, NADPH + H+ is changed to NADP+, Fe³+ is released and one atom of the O₂ used participates in changing the reactant to the product, while the other atom exits as a CO. Biliverdin is green in colour. After that, Biliverdin Reductase enzyme uses NADPH + H+ to change biliverdin to bilirubin (redorange in colour) (NADP+ is also produced). The only difference between biliverdin and bilirubin is the addition of a hydrogen to the carbon in the middle of the structure (see the diagram) and the change in the position of the double bonds in the ring that was bound to that carbon by a double bond (results in addition of a hydrogen to the nitrogen atom in that ring, see the diagram to understand). As you can tell, there are only two oxygens in the structure of bilirubin, i.e. the molecule is highly hydrophobic. As mentioned in the last sheet, bilirubin is used in colouring and most of the things it colours are in the liquid state, this means that bilirubin has to be changed to a more hydrophilic form (bilirubin and its 2 P age

3 derivatives are known as bile pigments. Another use for bilirubin is as an antioxidant (oxidised to biliverdin)). The changing of bilirubin to a soluble form is done by the liver, so some transportation must be done. Bilirubin leaves the spleen and binds to albumin in the blood (not a covalent binding, if it was so then bilirubin wouldn t be released) where it travels to the liver and enters via facilitated diffusion (no energy)[remember to never give drugs that bind to albumin at the same site of bilirubin to children, those anionic drugs (e.g. salicylates and sulphonamides) would displace bilirubin increasing its concentration in the blood leading to bilirubin s entry to the CNS causing mental retardation (neural damage) to the child]. In the liver, bilirubin binds to some intracellular proteins (e.g. ligandin). Bilirubin is acted upon (in the liver) by the enzyme bilirubin glucuronyl-transferase which adds 2 glucuronic acid molecules (from 2 UDPglucuronic acid) (remember; glucuronic acid is the carboxylic acid form of glucose) to our bilirubin changing it to the more soluble Bilirubin Diglucuronide [a deficiency in the enzyme bilirubin glucuronyl-transferase is associated with two syndromes: Crigler-Najjar (I and II) syndrome and Gilbert syndrome, the first (Crigler-Najjar) is a severe syndrome that really makes life difficult, the second, though, is rarely diagnosed, even if present, as patients live a completely normal life with it (might just feel extremely tired when fasting)]. Bilirubin diglucuronide is also known as conjugated bilirubin. This conjugated bilirubin now wants to leave the liver and enter the bile canaliculi and then into the bile. However, leaving the liver (by conjugated bilirubin) can only be done المواصلات بدها ليرات والتكسي (الكبد) بياخد مصاري ( energy) via active transport (needs so this transport process is the rate limiting step in heme,(وانت طالع مش وانت داخل! degradation. A deficiency in the transport protein of conjugated bilirubin results in a syndrome called the Dubin-Johnson syndrome. Unconjugated bilirubin is not normally secreted. (Remember; bile acts as an emulsifier that eases the mixing and degradation processes of food). Some conjugated bilirubin goes through side-chains of the biliary duct to other areas to do its coloration job there. 3 P age

4 In the bile, the bilirubin enters the intestines giving a yellowish colour. Bacteria (normal flora) in the intestines hydrolyze and reduce the conjugated bilirubin changing it to urobilinogen which is colourless. Urobilinogen now has two possible pathways: 1- The bacteria in the intestines change it to stercobilin (gives the faeces its brown colour). 2- Urobilinogen gets reabsorbed in the gut and enters the portal blood, which takes it back to the liver, there some urobilinogen enters (again) into the bile via the biliary duct (enterohepatic urobilinogen cycle) and the remainder gets transported with the blood to the kidneys where it is converted to urobilin (yellow) and gets excreted with urine giving it its characteristic yellow colour. Jaundice Jaundice (or icterus) ا و الصفار) (اليرقان is not a disease, it is a symptom of some diseases that result from problems in the bilirubin pathway which lead to the accumulation of bilirubin. There are several types of jaundice (according to what accumulates, bilirubin or conjugated bilirubin). I- Hemolytic Jaundice When there are diseases that cause haemolysis (e.g. sickle cell anemia, pyruvate kinase deficiency or glucose-6-phosphate dehydrogenase deficiency) then too many red blood cells die resulting in amounts larger than normal of heme being released. The liver capacity for conjugating bilirubin is ~3000mg/day, if the unconjugated bilirubin (produced from heme) made in the spleen (and reticuloendothelial cells in the liver) exceeds this amount unconjugated bilirubin will accumulate and lead to jaundice. II- Hepatocellular Jaundice This type of jaundice occurs due to some damage in the liver cells making the liver unable to conjugate the same amounts of bilirubin it does normally leading to the accumulation of the unconjugated bilirubin in the blood. It is noted that urobilinogen increases in the urine (becomes darker) in this type of jaundice (the enterohepatic circulation decreases) and that stools may have a pale, clay colour. III- Obstructive Jaundice 4 P age

5 This type of jaundice occurs when the biliary duct gets obstructed (extrahepatic cholestasis) due to a tumour in the duct or to gall-bladder stones preventing the entry of conjugated bilirubin into the intestines. Here, there is no overproduction of bilirubin or a decreased conjugation of it. Signs and Symptoms: GI pain, nausea, pale clay coloured stool and urine that darkens upon standing. Hyperbilirubinemia (high levels of bilirubin in the blood) and excretion of bilirubin with urine are seen (conjugated bilirubin does not get changed to urobilinogen as the biliary duct is blocked). The prolonged obstruction of the biliary duct may damage the liver leading to increased levels of unconjugated bilirubin. IV- 5 P age Jaundice in New-borns We cannot call this a symptom of a disease as newborns (especially if born immature) often accumulate bilirubin as not enough enzymes (bilirubin glucuronyltransferase) have been made yet leading to jaundice. Within 4 weeks the adult levels of the enzyme are reached. More bilirubin than what the albumin can bind may result in bilirubin s diffusion into the basal ganglia causing toxic encephalopathy (kernicterus). To treat this condition, we expose the child to a bluefluorescent light that converts bilirubin to some more polar (water-soluble) isomers. These resulting photoisomers can be excreted into the bile without the need of conjugation. Bilirubin is usually measured using the van den Bergh reaction (Diazotized sulfanilic acid + bilirubin Red azodipyrroles). The colour change is measured using colorimetry. Two measurements are done: 1- A direct reacting measurement of conjugated bilirubin (aqueous solution) (conjugated bilirubin directly reacts with the diazotized sulfanilic acid). 2- An indirect reacting measurement of TOTAL bilirubin (the solvent used is methanol, both, conjugated and unconjugated bilirubin dissolve and so can react with the diazotized sulfanilic acid). Using the results of the two measurements we can get the amount of unconjugated bilirubin (Total bilirubin = Conjugated bilirubin + unconjugated bilirubin). In normal plasma, only 4% of the bilirubin is conjugated (direct-reacting) as most of the conjugated type is secreted into bile.

6 Synthesis of Other Nitrogen Containing Compounds From Amino Acids We ll be talking about catecholamines, histamine, serotonin, creatine, melanin and some other small, but important, molecules. Catecholamines: As you can see in the diagram, catechol is the (ortho)hydroxyphenol-group and for any molecule to be called a catecholamine it must contain the catechol group and an amine group (as the name implies). The catecholamines synthesised normally in the body are dopamine, norepinephrine and epinephrine. The brain synthesises dopamine and norepinephrine which function as neurotransmitters. The adrenal-medulla synthesises epinephrine and norepinephrine (outside the nervous system, norepinephrine and epinephrine are hormone regulators of carbohydrate and lipid metabolism). In response to fear, low blood glucose, exercise, cold, etc., the adrenal medulla secretes epinephrine and norepinephrine to increase the degradation of glycogen and triacylglycerols, increase the blood pressure and increase the cardiac output (prepares the body for the fight or flight reaction). Synthesis of catecholamines Tyrosine, the amino-acid that contains a phenol group, serves as an excellent precursor for the catecholamines due to its similarity to their structures. Tyrosine hydroxylase adds the second OH to tyrosine s phenol ring changing it into DOPA (3,4-dihydroxyphenylalanine), tetrahydrobiopterin (BH₄) + O₂ are changed to Dihydrobiopterin (BH₂) + H₂O in the process. Note that DOPA is not a catecholamine as it still contains a COO group, the next step is to remove this group using DOPA decarboxylase and the coenzyme PLP (vitamin B6) to produce Dopamine, our first catecholamine. If norepinephrine is needed, dopamine is hydroxylated (remember the catechol group cannot be changed, so the OH to be added is to be added on the carbon skeleton) using the enzyme Dopamine β-hydroxylase (if the first carbon next to the amino-group is called the α-carbon, then the one next to it is the β-carbon; the OH is added to this β-carbon) and the coenzymes ascorbate 6 P age

7 (vitamin C, the first reaction that we encounter that uses this coenzyme) and Cu²+ are needed, in the process, O₂ is changed to H₂O and ascorbate to dehydroascorbate. If epinephrine is needed is methylated using the enzyme phenylethanolamine- N-methyltransferase and the methyl carrier S-adenosyl-methionine (SAM) (changed to S-adenosyl-homocysteine). Parkinson disease, a neurodegenerative disease, occurs as a result of the low levels of dopamine in the brain (due to an idiopathic loss of the dopamineproducing cells in the brain). Giving L-DOPA is the most common treatment. Degradation of Catecholamines There are two possible routes for the degradation of any of our three catecholamines; both routes lead to the same products (but different intermediates). There are two enzymes working in this process, MAO (monoamine oxidase, catalyses the oxidative deamination reactions) and COMT (catechol-omethyl transferase, catalyses the O-methylation reactions using SAM as the methyl donor). Either MAO begins the degradation and COMT finishes it or COMT begins and MAO finishes (so each route has only two steps) (when MAO begins, it produces aldehyde intermediates, which get oxidised to the corresponding acids). VMA and HVA (the products) are excreted in the urine (VMA is increased with pheochromocytoma, an adrenal tumour that increases catecholamine production). (The doctor said that the intermediates are not to be memorised, but she might ask about the reactant and its corresponding product). 7 P age

8 MAO Inhibitors These are drugs that inhibit the action of MAO preventing the degradation of the catecholamines. This means that the catecholamines will still be available in the synaptic cleft to transmit the signal, this leads to overstimulation. MAO also degrades serotonin, so giving MAO inhibitors increases the effects of serotonin and curing depression, this is a good effect, but many problems are associated with such drugs. Histamine: It is a chemical messenger that mediates many cellular responses and it is well-known for its effect (vasodilation, leads to leakage of liquids which leads to edema which, in turn, narrows the airways creating breathing problems) in allergic reactions. Other than allergic reactions, histamine mediates gastric acid secretions and neurotransmission in parts of the brain. Histamine is secreted by mast cells as an allergic reaction or in response to a trauma. Histamine is synthesised from (guess from the names ) histidine. As the name implies, histamine is an amine, so we simply remove the COOH group of histidine to produce histamine. The enzyme is a decarboxylase and the coenzyme is PLP. Serotonin: Tryptophan is the precursor of serotonin, which is synthesised and stored in several sites of the body (mostly intestinal mucosal cells and, in smaller amounts, in the CNS (as a neurotransmitter) and the platelets). Serotonin s physiologic roles include pain perception, regulation of sleep, appetite, temperature, blood pressure, cognitive functions and mood (causes a feeling of wellbeing). In fact, the sleep order is controlled by melatonin not serotonin (like if you travel to USA you d need some time to get accustomed to the timing their and fix your sleeping time accordingly). Serotonin is converted to melatonin (at night) in the pineal gland (close to the pituitary gland) by a methylation reaction (add CH₃) and an acetylation reaction (add CO-CH₃). (The details in the diagram ARE TO BE MEMORISED). Creatine 8 P age

9 Creatine Synthesis Arginine and Glycine react using the enzyme amidinotransferase producing ornithine and guanidinoacetate. A methyl group is transferred to guanidinoacetate using a methyltransferase and SAM, this produces creatine. Creatine kinase uses an ATP molecule to produce creatine phosphate from creatine (ADP and H+ are released), the reaction is reversible and does produce ATP in the reverse direction. Presence of creatine kinase in the plasma indicates heart damage and this test is used in diagnosing MI. Creatine phosphate (or phosphocreatine) is a high energy molecule present in the muscles and provides a small, but rapidly mobilizable, reserve of high energy phosphates. The amount of creatine phosphate in the body is proportional to the muscle mass. Creatine Degradation When the phosphate group of creatine phosphate is released (to produce energy for the muscle) the creatine is changed into creatinine. (Creatine can also be changed to creatinine by a hydration reaction). Creatinine is used in the kidney function test and a high creatinine concentration is a late marker of renal failure (too late to do anything). The production of creatinine from creatine phosphate or creatine is called (cyclization, a cyclic structure is formed, see the diagram). Creatinine is excreted in the urine and its excreted amount is proportional to the total creatine phosphate content of the body and thus this amount can be used in estimating the muscle mass of the patient. When the muscle mass decreases (paralysis or muscular dystrophy) the creatinine content of the urine decreases. Melanin: It is a pigment in many tissues (eye, hair and skin). It is synthesised from tyrosine in the epidermis by melanocytes. Melanin protects the underlying cells from the harmful effects of the sunlight (UV-light). A defect in melanin 9 P age

10 leads to albinism (the most common form is due to defects in copper-containing tyrosinase). The diagram shows the synthesis pathway of melanin in the melanosome (the structure that synthesises melanin in the melanocyte). There are two types of melanin: pheomelanin (higher concentrations give a red-colour) and eumelanin (real melanin; higher concentrations give a darker colour and lower concentrations give a lighter colour). As you see in the diagram, the synthesis of both, eumelanin and pheomelanin, begins via the same pathway (L-tyrosine L-Dopa Dopaquinone) and split at the point of dopaquinone. Dopaquinone may change to cysteinyldopa (the precursor of pheomelanin) or it might change to dopachrome which changes to DHICA which changes to 5,6-indoelquinone carboxylic acid, this second pathway gives us eumelanin. The produced melanin has to leave the melanosome and reach the superficial layers (if we are talking about skin colour for example) to give its effect (the colour). This melanin transport process is light sensitive, if there is more light more melanin is secreted and one becomes darker and in winter, when the light intensity is lower, the amount of melanin secreted decreases and one becomes lighter. The Other Molecules section was not explained by the doctor in her lectures and I don t know whether is required for exam purposes or not. I solely depended on the slides there. Other Molecules Thyroxine is synthesised from tyrosine. Glutathione (the reductant we studied about a lot) is synthesised from glutamate, cysteine and glycine [it has other functions like conjugation of some drugs (i.e. making them more polar), transport of amino acids across the cell membrane, acting as a cofactor and facilitation of the rearrangement of the protein sulphide bridges). Serine is changed to ethanolamine which reacts with choline and acetyl-coa to produce acetylcholine. Glycine and succinyl CoA synthesise the heme group (as we saw in the previous sheet). 10 P a g e

11 Tryptophan, with the help of the GI bacteria, produces the indoles. Hepatic tryptophan produces nicotinamide. Glutamic acid is the precursor of GABA (an inhibitory neurotransmitter). Lysine can get methylated and participate in the production of carnitine (a very long process). Ornithine (also a very long process) produces spermine (the molecule that gives semen its special odour). Indole 11 P age