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HEMOGLOBIN AND PORPHYRINS HEME-CONTAINING PROTEINS • Hemoglobin • Myoglobin • Cytochromes • Catalase • Some peroxidases STRUCTURE OF HEMOGLOBIN Globular proteins belong to hemeproteins Heme proteins Heme The prosthetic group function dictated by the protein part + Protein Hemoglobin (Hb) is the red blood pigment, found in ERYTHROCYTES (greek:- erythrose – red; kytose – a hollow vessel). Normal level of Hb in blood in males is 14-16g/dl and in females 13-15g/dl. The adult hemoglobin (HbA) has 2 α chains & 2β chains. Mol.wt of HbA is 67,000. Hb is a conjugated protein, containing GLOBIN- the apoprotein part & the HEME – the non – protein part (prosthetic group). Hb is a tetrameric allosteric protein, Each gram of Hb contains 3.4 mg of iron. STRUCTURE OF GLOBIN Globin consists of 4 polypeptide chains of two different monomeric units. 2 α chains & 2β chains. Each α –chain contains 141 a.a. Each β- chain contains 146 a.a. Thus HbA1 contain 574 a.a. The 4 subunits of Hb are held together by non-covalent interactions. ( hydrophobic, ionic & H-bonds) Each subunit contain a Heme group. α -chain gene is on chromosome 16, while β,γ &δ- chains are on chromosome 11. Structure of heme Characteristic red colour of blood HEME Organic component + central iron atom Protophorphyrin IX 4 pyrrole rings linked by methenyl bridges to form tetra pyrrole 4 methyl groups, 2 vinyl groups, 2 propionate side chains are attached Central iron atom Iron atom is in Ferrous oxidation state (Fe2+) Iron has 6 valencies 4 bonded to pyrrole N2 Other two 5th coordinated site linked to Imidazole N2 of histidine deoxy-Hb oxy Hb & Myoglobin 6th coordinated site deoxy-Hb un occupied oxy Hb & Myoglobin – O2 6th 5th Structure of HbA 38 histidine residues – buffering action 58th residue in α-chain- distal histidine 87th residue in α-chain- proximal histidine Each α and β sub units has stretches of α-helix and heme binding pocket Interaction of heme with goblin: 4 heme residues per Hb molecule, 1 for each subunit in Hb The heme is located in a cleft between the E and F helices of Hb chain Heme group accounts for 4% of whole mass of Hb Quaternary structure of Hb Hb tetramer can be viewed as being composed of two identical dimers (αβ)1, (αβ)2 2 polypeptide chains within the dimer are held primarily by strong hydrophobic interactions The two dimers move with respective each other, being held primarily by weaker ionic and Hydrogen bonds The movement produces two different structures of Hb namely a “T,” or taut, structure of deoxy-Hb and “R,” or relaxed, structure of Oxy-Hb “T” form Deoxy form of Hb Taut or tense form The two dimers interact through a network of ionic and hydrogen bonds that constrain the movement of the polypeptide chain Low O2 affinity form of Hb “R” form Oxygen bound form of hemoglobin Relaxed form of Hb Binding of O2 causes rupture of some of ionic and hydrogen bonds between the dimers and hence freedom of movement. High O2 affinity form of Hb “T” and “R” form interchange during loading and unloading of O2 with Hb Structure and function of myoglobin Myoglobin – present in heart and skeletal muscle Single polypeptide chain of M.wt 17000 153 A.A residues Single polypeptide chain structurally similar to individual subunit polypeptide chains of Hb Compact molecule, 80% of it folded to 8 stretches of alpha helix (A - H) Mb has higher affinity for O2 than Hb. Bohr effect, co-operative effect & 2,3-BPG effects are absent. Heme Arrangement of A.A residues Interior – non polar A.A stabilized by hydrophobic interaction Surface – charged A.A forming hydrogen bonds with each other and water the heme group of myoglobin sits in crevice lined by non polar A.A, except for two histidine residues 1, the proximal histidine bind directly to the 5th coordinated site 2nd, distal histidine does not interact directly with heme but helps to stabilize the binding of O2 to Fe ion Function of Myoglobin Reservoir for O2 O2 carrier red muscles increases the rate of transport of O2 with in the muscle cell Biomedical importance of Myoglobin Myoglobinuria follows massive crush injury Myocardial infarction FUNCTIONS OF Hb Transport of O2 from lungs to tissues . Transport of CO2and protons from tissues to lungs . Transport of O2 by Hb Each Hb molecule can bind 4 molecules of O2 one at each of its 4 heme groups the degree of saturation of all Hb molecules can vary from 0-100% O2 dissociation curve (ODC) Hb Plot of degree of saturation (Y-axis) measured at different PO2 (X-axis). ODC for Hb is sigmoid Explains the ability of Hb to load and unload O2 at physiological PO2 PO2 of inspired air 158mmHg, PO2 of alveolar air 100mmHg(Hb 97% sat) PO2 of tissue capillaries 40mmHg(Hb 60% sat) 40% of bound O2 released at the tissue level ODC-MYOGLOBIN Hyperbola At po2in lungs –myoglobin-100% saturated At po2in tissues-almost same so at physiological po2 – myoglobin does not deliver it bound oxygen However ,at po2of 5mmHg (exist during sternous physical exercise)-myoglobin readily release O2 ODC OF Hb 1.Sigmoid 2.Binds 4mol of O2 3.Delivers its O2 to tissues at physiological pO2 ODC OF MYOGLOBIN 1. Hyperbola 2.Binds only one mol of O2 3.Functions mainly in storage.Delivers its bound O2 only during sternous physical exercise Heme-Heme interaction and co-operativity ODC for Hb- sigmoidal Indicates that the subunits co-operate in binding O2 which is called co-operativity It means that binding of an O2 molecule at one heme increases the O2 affinity of remaining heme groups in the same Hb molecule this is called heme-heme interaction What is the advantage of Heme-Heme interaction ? 1. Higher affinity Although it is more difficult for first O2 molecule to bind to hemoglobin, subsequent binding of O2 occurs with higher affinity Net effect of this- Last O2 bound has approximately 300times > affinity than for the first 2. Easy loading and unloading of O2 Co-operative binding of O2 allow Hb to deliver more O2 to the tissues in response to relatively small changes in P O2 Conformational changes accompany Oxygenation of Hb 1.Rupture of salt bonds between the carboxyl terminal residues of all 4 subunits 2.One pair of rigid subunit dimer (αβ)2 rotates through 15 relative to the other rigid pair(αβ)1 (T-R change takes place) 3.Iron atom of deoxyhemoglobin which lies about 0.06nm beyond the plane of heme ring moves into the plane of heme ring this is transmitted to proximal histidine and also to the residues attached Factors affecting ODC 1. P O2 2. pH 3. PC O2 4. 2,3-bisphospho glycerate(BPG) 5. Temperature 6. Bohr effect 7. Chloride shift Allosteric effectors pH and pCO2 Tissues- formation of metabolic acids like lactate pH & pC O2 affinity of Hb for O2 (ODC shift to right) R-T change takes place in Hb O2 released to tissues Lungs: pO2, pCO2 & pH Affinity of Hb for O2(ODC shift to left) T-R change takes place in Hb Bohr effect The influence of pH and pCO2 to facilitate oxygenation of Hb in lungs and deoxygenation at the tissues is known as Bohr effect Bohr effect causes a shift in the ODC to the right. Bohr effect is responsible for the release of O2 from oxyHb to the tissues. Mechanism of bohr effect Bohr effect is caused by binding of H+ and CO2 to Hb. O CH2 C N + O----------HN Asp 94 HbO2 + H+ His 146 Hb H+ + O2 Effect of 2,3BPG 2,3BPG Intermediate in glycolytic pathway Most abundant organic phosphate in RBC One molecule of 2,3BPG binds to a pocket formed by 2 beta globin chains (pocket contains +vely charged A.A that form ionic bonds with –vely charged phosphate of 2,3BPG) 2,3BPG is expelled from Hb on oxygenation Normal level 15+1.5 mg/g Hb. High O2 affinity of HbF is due to inability of γ-chain to bind 2,3- BPG Binding of 2,3-BPG Shift of ODC 2,3-BPG preferentially binds to the deoxy Hb and not to oxyhemoglobin Stabilizes the taut conformation of deoxyhemoglobin Reduces the affinity of Hb for O2 Shift ODC to right O2 released from Hb efficiently at the partial pressure found in tissues CLINICAL SIGNIFICANCE Increased 2,3-BPG in RBC 1. Chronic hypoxia:- for high O2 supply. Include high altitude, obstructive pulmonary emphysema. Chronic anemia In these conditions 2,3-BPG lowers the O2 affinity of Hb permitting grater unloading of O2 in the capillaries of the tissues Decreased 2,3-BPG in RBC In transfused blood 2,3-BPG in RBCs results in abnormally high O2 affinity and fails to unload its bound O2 to tissues. This can be prevented by adding inosine Chloride Shift In Tissues: Pco2 Co2+H2o H2Co3 H+ OxyHb Hco3HHb+o2 released to cells Diffuses into plasma and Chloride Shifts from plasma into RBC to maintain equilibrium.This shift of Chloride is Chloride shift. *** Reversal of Chloride Shift In Lungs O2+Hb Hbo2 Inhaled H++Hco3 HCO3 H2co3 H2o+Co2 Exhaled As Hco3- levels falls inside the RBC’S more and more Hco3- gets in and chloride ion diffuses out and it is called Reversal of chloride Shift SHIFT OF ODC PO2 PH PCO2 2,3BPG Temperature RIGHT LEFT Transport of Co2 Most of CO2 is hydrated and is transported as bicarbonate ion. At rest, about 200ml of CO2 is produced per minute in tissues. In aerobic metabolism, for every molecule of O2 utilized, one molecule of CO2 is liberated. About 15% of CO2 carried in blood directly binds with Hb. Carriage as carbamino - hemoglobi Some CO2 is carried as carbamate bound to the uncharged α amino group of hemoglobin (Carbaminohemoglobin). Hb-NH2 + CO Hb-NH-COO- + H+ 2 Binding of CO2 stabilizes the T form of hemoglobin, decrease in its affinity for oxygen . In lungs, CO2 dissociates from hemoglobin and is released in the breath. Dissolved form About 10% of CO2 is transported as dissolved form. CO2 + H2O H2CO3 HCO3- + H+ The H+ thus generated, are buffered by the buffer system of plasma. Minor Hemoglobins HbA2 Fetal Hb (HbF) Embryonic Hb HbA1C MINOR HEMOGLOBINS TYPE % OF Hb Hb A2 COMPOSITION & SYMBOL α2δ2 Hb F α2γ2 <2% Hb A1c Α2β2-glucose <5% <5% HbA2 2% of total normal adult Hb Appears about 12 weeks after birth 2α & 2δ chains Moves slower on electrophoresis compared to the normal Hb As a compensation HbA2 levels are increased in β-thalassemia Fetal Hb Tetrameric protein consisting of 2α& 2γ chains Alpha chain is identical to those found in HbA γ chain has 146 A.A (36 A.A differ from those of β) Major Hb found in fetus and newborn Synthesis starts from 7th week of gestation. At birth about 80% of Hb is HbF. During the first 6months of life it decreases to about 5% of total Fetal Hb cont.. Differentiating features; Decreased interaction with 2,3-BPG Increased solubility of deoxyHbF Slow electrophoretic mobility Increased resistance of Hbf to alkali denaturation Embryonic Hemoglobin Normal during fetal life Hb Gower1 & Hb Gower 2 Synthesized during 3rd to 8th week of gestation Hb Gower2 - 2α & 2ε Hb Gower1 - 2ζ & 2ε Hb derivatives Formed by the combination of different ligands with the heme part or by the change in the oxidation state of iron 1. Carboxy hemoglobin 2. Met-hemoglobin 3. Sulf hemoglobin Carboxy hemoglobin: co combines with Hb Affinity of co to Hb is 200 times more than that of O2 Binding of co: co binds to one or more of 4 heme sites T-R changes takes place in Hb O2 binds to other heme sites ODC shift to left (sigmoid ODC to Hyperbola) Decreased O2 release of the affected Hb to tissues CO poisoning causes Major occupation hazards in mines Breathing the automobile exhausts in closed space- commonest cause Normal level <1% (0.16%) Smokers >4% Clinical symptoms ≥20% Nausea, vomiting, head ache, breathlessness, pain in chest 40-60% death Treatment 100% O2 Met-Hemoglobin Iron in heme is in ferric state (Fe3+) Causes Mutations in α or β globin chains Free radicals Congenital deficiency of enzymes NADH met-hemoglobin reductase NADPH met-hemoglobin reductase Glutathione met-Hemoglobin reductase Intake of certain drugs Nitrates, Acetaminophen, Phenacetin Normal level < 1% Clinical symptom Met-Hb – has got only 5 valencies does not bind O2 does not involve in O2 transport Manifested as cyanosis Chocolate color blood due to dark colored met-Hb called as chocolate cyanosis Treatment Oral administration of methylene blue or ascorbic acid decreases met-Hb levels & reverses cyanosis Sulfhemoglobin Sulfhemoglobin results from the union of hemoglobin with medications such as phenacetin or sulfonamides. This form of hemoglobin is unable to transport oxygen, and is untreatable. The only solution is to wait until the affected red blood cells are destroyed as part of their normal life cycle. Hemoglobinopathies Production of structurally abnormal Hb or synthesis of insufficient quantities of normal Hb or rarely both Includes 1. Sickle cell disease (HbS) 2. Hemoglobin C disease (HbC) 3. Hemoglobin SC disease(HbSC) 4. Thalassemias Sickle cell disease Genetic disorder of Hb caused by a point mutation in the β-globin gene Homozygous recessive disorder Pathology: Point mutation Glutamic acid replaced by valine in 6th position of β chain of HbA Sticky patch on surface of Hb Sequence of events leading to cell death Sickle cell disease cont.. Sticky patch- present on oxyHbS & deoxyHbs but not on HbA complement to sticky patch - present on the surface of deoxyHbA & deoxyHbS but not on oxyHbA & oxyHbS Harper slide Events leading to sickle cell crisis Sticky patch on deoxy Hbs bind to complement site on another deoxy Hbs Polymerization of deoxyHbS Long fibrous precipitates Intracellular fibers of HbS distort the erythrocyte Elongated erythrocytes occlude blood flow in capillaries Microinfarcts produce tissue anoxia, resulting severe pain Defective haemoglobins bind together, forming long rods that stretch the red blood cell into a crescent. These “sickled” red blood cells cannot fit through small blood vessels. ** Factors which increase sickling Decreased O2 tension Increased pCO2 Decreased pH Increased conc of 2,3-BPG in RBC Cause more deoxyHb Diagnosis of sickle-cell anemia normal Sickle-cell trait Sickle-cell anemia - origin Hb S Hb A + Electrophoresis of hemoglobin's Hemoglobin C Point mutation gene level changing the Amino acid at 6th position of β globin chain Glutamic acid replaced by Lysine (Basic) Heterozygous state- No Clinical symptoms Homozygous state- Chronic hemolytic anemia No infarction crises and no specific theraphy Hemoglobin SC Compound heterozygote state Some β globins have sickle cell mutation Some β globulins have Hbc mutation Painful crises beginning in childhood remain normal until they suffer an Crises. Hemoglobin D Caused by the substitution of glutamine in place of glutamate in the 121st position. On electrophoresis moves along with HbS Hemoglobin E Replacement of glutamate by lysine at 26th position. No clinical symptoms In India, it is prevalent in west Bengal. THALASSEMIAS Hereditary hemolytic disease due to an imbalance in the synthesis of globin chains Synthesis of either the alpha or beta chain is defective Caused by variety of mutations including gene deletions or gene substitutions or deletion of 1 to many neucleotides in the DNA α0 or β0 – no globin chains are produced α+ or β+ - some chains but at reduced rate α- Thalassemia Silent carrier:- loss of one α-globin gene 2. α- Thalassemia trait:- loss of two genes 3. Hemoglobin H disease:- loss of three genes 4. Hydrops fetalis:- loss of all 4 genes. Most sever form. 1. β - Thalassemia Commonest Synthesis of β – globin chains is decreased or absent. α - globin chains synthesized normally α - globin chains precipitate forming intracellular precipitates or inclussion bodies lead to membrane damage and destruction of RBC. As a compensation γ or δ chain synthesis is increased. Cont ….. Homozygous state Clinical manifestations are severe and they are called Thalassemia major includes - Anemia - Hyper splenism - Hepato Splenomegaly Cont …. Heterozygous State Clinical Signs and symptoms are minimal Called as Thalassemia minor BIOSYNTHESIS OF HEME Structure of Porphyrins most common porphyrin in humans is heme - cyclic, with 4 pyrrole rings attached by methenyl bridges - side chains may vary - uroporphyrin has acetate and propionate chains - coproporphyrin has methyl and propionate chains - order of chains define subgroups of porphyrins: only Type III porphyrins are normally important for humans Synthesis takes place in liver & erythrocyte-producing cells of bone marrow. The pathway is partly cytoplasmic & partly mitochondrian. CH3 CH3 S HC CH2 protein N H3C CH3 N − OOC CH2 CH2 Fe N CH N S CH2 protein CH3 CH2 CH3 CH2 COO− Heme c O − OOC CH2 CH2 C S-CoA + succinyl-CoA δ-Aminolevulinic Acid Synthase − OOC CH2 NH3+ glycine H+ CoA-SH CO2 O − OOC CH2 CH2 C CH2 NH3+ δ-aminolevulinate (ALA) Heme synthesis begins with condensation of glycine & succinyl-CoA, with decarboxylation, to form δ-aminolevulinic acid (ALA). ALA Synthase is the committed step of the heme synthesis pathway, & is usually rate-limiting for the overall pathway. Regulation occurs through control of gene transcription. Heme functions as a feedback inhibitor, repressing transcription of the ALA Synthase gene in most cells. A variant of ALA Synthase expressed only in developing erythrocytes is regulated instead by availability of iron in the form of iron-sulfur clusters. COO− COO− CH2 CH2 CH2 CH2 C + O CH2 NH3 C PBG Synthase 2 H2O O CH2 + NH3+ 2 δ-aminolevulinate (ALA) H2C NH3 COO− COO− CH2 CH2 CH2 C C C CH + N H porphobilinogen (PBG) PBG Synthase (Porphobilinogen Synthase), also called ALA Dehydratase, catalyzes condensation of two molecules of δ-aminolevulinate to form the pyrrole ring of porphobilinogen (PBG). 4 PBG Urophorphyrinogen I synthase & Urophorphyrinogen III cosynthase 4NH4 hydroxymethylbilane - OOC COO - COO - CH 2 COO - CH 2 COO CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH HN NH HN COO - CH 2 - OOC uroporphyrinogen III - CH 2 NH HN NH HN CH 2 CH 2 CH 2 COO - HO C C CH 2 C COO - - OOC CH 2 C CH 2 CH 2 CH 2 CH 2 COO -COO - CH 2 COO - Uroporphyrinogen III Synthase CH 2 CH 2 CH 2 CH 2 COO - COO - Urophorphyrinogen III COO - 4 CO2 Urophorphyrinogen decarboxylation Coproporphyrinogen III 2 CO2 Copropophyrinogen oxidase Protoporphyrinogen IX 4 H+ Protoporphyrinogen oxidase Porphyrias caused by hereditary or acquired defects in heme synthesis - accumulation and increased excretion of metabolic precursors (each unique) - all porphyrias are autosomal dominant, except congenital erythropoietic porphyria, which is recessive can be hepatic or erythropoietic - hepatic can be acute or chronic Not a ‘vampire’s’ disease •Extreme sensitivity to sunlight •Anemia This idea has been discarded both for scientific reasons: •Porphyrias do not cause a craving for blood. •Drinking blood would not help a victim of porphyria. And for compasionate reasons:Porphyria is a rare, but frightening condition: hard to diagnose and there is no cure. 1. Acute intermittent porphyria (AIP) ENZYME DEFECT:Urophorphyrinogen I synthase INHERITNACE:- autosomal dominant ALA & PBG levels are elevated in blood & urine Urine gets darkened on exposure to air due to photo-oxidation phorphobilinogen to porphobilin and porphyrin. It is usually expressed after puberty. King George III Mad King George He had been Suffered with AIP SYMPTOMS:• Acute abdominal pain • Vomiting • Cardio vascular abnormalities • Neurological manifestations include, sensory & motor disturbances, confusion and agitation ( due to reduced activity tryptophan pyrrolase) • Symptoms are more severe after administration of drugs barbiturates . • Patients are not photosensitive TREATMEMT :- hematin which inhibit the enzyme ALA synthase. 2. congenital erythropoietic porphyria ENZYME DEFECT:-uroporphyrinogen III cosynthase INHERITNACE:- autosomal recessive The individual excrete uroporphyrinogen I & coproporphyrinogen I which oxidize to uroporphyrin I & coproporphyrin I (red pigments) Appearance of urine is port wine colour. Patients are PHOTOSENSITIVE. ( itching & burning of skin when exposed to sun light) Increased hemolysis. Repeated attacks of dermatitis & scarring lead to a typical facial deformity often referred to as ‘monkey face’. Repeated ulceration & scarring may cause mutilation of nose, ear & cartilage. This may mimic leprosy. When UV light reflected on to teeth a red fluorescence is seen, called ERYTHRODONTIA. 3.porphyria cutanea tarda Also called as CUTANEOUS HEPATIC PORPHYRIA. Most common porphyria. Occurs in alcoholics & iron overload. ENZYME DEFECT:- uroporphyrinogen decarboxylase Photosensitivity is seen Liver exhibits fiuoresence. 4. Hereditary coproporphyria ENZYME DEFECT:- coproporphyrinogen oxidase Coproporphyrinogen III, ALA & PBG are excreted in urine and feces. Patients are PHOTOSENSITIVE Clinical manifestations are similar to AIP Treatment:hematin(inhibits ALA synthase) 5.Variegate porphyria ENZYME DEFECT:- protoporphyrinogen oxidase Protoporphyrin IX synthesis is blocked. All intermediates of Heme synthesis accumulate in the body and excreted in urine and feces. Colored urine & photosensitivity is seen. Plasma shows red fluorescence due to coproporphyrinogen. 6. Protoporphyria (erythropoietic protoporphyria) ENZYME DEFECT:- ferrochelatase Protoporphyrin IX accumulates in tissues and excreted in urine and feces RBC and skin biopsy exhibit red fluorescence. The porphyrias. Type Enzyme Involved Major Symptoms Laboratory tests Acute intermittent porphyria Uroporphyrinogen synthase Abdominal pain Neuropsychiatric urinary porphobilinogen ⇑ Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Photosensitivity urinary uroporphyrin porphobilinogen ⇑ ⇔ Porphyria cutanea tarda Decarboxylase Photosensitivity urinary uroporphyrin porphobilinogen ⇑ ⇔ Variegate porphyria Oxidase Photosensitivity Abdominal pain Neuropsychiatric urinary uroporphyrin fecal coproporphyrin fecal protoporphyrin ⇑ ⇑ ⇑ Photosensitivity fecal protoporphyrin red cell protoporphyrin ⇑ ⇑ Erythropoietic protoporphyria Ferrochelatase CATABOLISM OF HEME The end products of heme catabolism are bile pigments(blirubin & biliverdin) Catabolism takes place in macrophages of reticuloendothelial system of spleen and liver. 6g of Hb is broken down per day, from which 250mg of bilirubin is formed. BLOOD CELLS Stercobilin excreted in feces Urobilin excreted in urine Hemoglobin Globin Heme O2 Heme oxygenase Urobilinogen formed by bacteria INTESTINE KIDNEY reabsorbed into blood CO Biliverdin IXα via bile duct to intestines NADPH Bilirubin diglucuronide (water-soluble) Biliverdin reductase NADP+ Bilirubin (water-insoluble) 2 UDP-glucuronic acid via blood to the liver Bilirubin (water-insoluble) LIVER Figure 2. Catabolism of hemoglobin HYPERBILIRUBINEMIAS Jaundice (icterus) hyperbilirubinemia - causes yellow color of skin, nail beds and sclerae - not a disease, but symptom of underlying disorders Types of Jaundice hemolytic jaundice - liver can handle 3000 mg bilirubin/day - normal is 300 - massive hemolysis causes more than can be processed - can’t be conjugated - increased bilirubin excreted into bile, urobilinogen is increased in blood, urine - unconjugated bilirubin in blood increases = jaundice obstructive jaundice - obstruction of the bile duct - tumor or bile stones - gastrointestinal pain - nausea - pale, clay-colored stools - can lead to liver damage and increased unconjugated bilirubin Types of Jaundice Hepatocellular Jaundice - liver damage (cirrhosis or hepatitis) cause increased bilirubin levels in blood due to decreased conjugation - conjugated bilirubin not efficiently exported to bile so diffuses into blood - increased urobilinogen in enterohepatic circulation - so urine is darker and stool is pale, clay-colored - AST and ALT levels are elevated - nausea and anorexia Jaundice in Newborns premature babies often accumulate bilirubin due to late onset of expression of bilirubin glucuronyltransferase - maximum expression (adult level) at ~ 4 weeks - excess bilirubin can cause toxic encephalopathy (kernicterus) - treated with blue fluorescent light - converts bilirubin to more polar compound - can be excreted in bile without conjugation - Crigler - Najjar syndrome is deficiency in bilirubin glucuronyltransferase Determination of bilirubin concentration van den Bergh reaction (aqueous) - conjugated bilirubin reacts readily - direct reaction - unconjugated, hydrophobic, reacts slowly - both conjuaged and unconjugated react same in methanol - gives total bilirubin value - subtraction of direct from total gives indirect in normal serum - only 4% is conjugated - SGOT, SGPT levels elevated in hepatic jaundice. ALP levels elevated in obstructive jaundice γ-GT levels elevated in chronic alcoholics CONGENITAL HYPERBILIRUBINEMIAS 1. CRIGLER-NAJJAR SYNDROME TYPE-I DEFECT IN CONJUGATION ENZYME DEFECT:UDP-glucuronyltransferase Jaundice appears within 24hr. Of life Unconjugated bilirubin increases to more than 20mg/dl. Children die first two years of life. 2. CRIGLER-NAJJAR SYNDROME TYPE-II Less sever than type-I Defect in bilirubin conjugation Bilirubin levels will be below 20mg/dl 3. GILBERT’S DISEASE It is inherited as an autosomal dominant trait. defect is in the uptake of bilirubin, impairment in conjugation, decreased hepatic clearance of bilirubin Bilirubin level around 3mg/dl It is asymptomatic condition. 4. DUBIN JOHNSON’S SYNDROME It is an autosomal recessive trait Defect in excretion of conjugated bilirubin & increased conjugated bilirubin in blood. Bilirubin deposited in liver & appears block (black liver jaundice) 5. Rotor syndrome Bilirubin excretion is defective, but no deposition in liver. ACQUIRED HYPERBILIRUBINEMIAS NEONATAL-PHYSIOLOGICAL JAUNDICE: It is caused by increased hemolysis. UDP-glucuronyl transferase activity is low Bilirubin level less than 5mg/dl It disappears by 2nd week of life. Table 2- Genetic Disorders of Bilirubin Metabolism Condition Defect Bilirubin Clinical Findings CriglerNajjar syndrome severely defective UDPglucuronyltransferase Unconjugated bilirubin ⇑⇑⇑ Profound jaundice Gilberts syndrome reduced activity of UDPglucuronyltransferase Unconjugated bilirubin ⇑ Very mild jaundice during illnesses DubinJohnson syndrome abnormal transport of conjugated bilirubin into the biliary system Conjugated bilirubin ⇑⇑ Moderate jaundice THAN Q