Jaundice - BILIRUBIN
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Transcript of Jaundice - BILIRUBIN
A True Liver Function Test
Introduction History Structure Biochemistry
The Road Ahead
Function of Bilirubin Clinical Significance
Synthesis of Bilirubin Excretion of Bilirubin
Clinical estimation of Bilirubin Urine Bilirubin Treatment Methods Serum
Hyperbilirubinemia & Types of Jaundice Inherited disorders of bilirubin metabolism Bilirubin Toxicity Kernicterus
Bilirubin A true indicator of Liver function
Phototherapy Exchange Transfusion
[Latin blis, bile + ruber, red + IN.]
Bilirubin is the orange-yellow pigment derived from senescent red blood cells. It is a toxic waste product in the body. It is extracted and biotransformed mainly in the liver, and excreted in bile and urine. It is a bile pigment Elevations in serum and urine bilirubin levels are normally associated with Jaundice.
History1849 Virchow discovered bilirubin in blood extravasates; termed it as Hematoidin
coined the term Bilirubin
demonstrated the direct association of bile pigments to hemoglobin synthesized Bilirubin IX and proposed a structure for it which was accepted for more than 30 years
Fischer & Plieninger
Pyrrole, or pyrrol, is a heterocyclic aromatic organic compound, a five-membered ring, having an activated metal ion, with the formula C4H4NH. Substituted derivatives are also called pyrroles. Tetrapyrroles are compounds containing four pyrrole rings. Linear tetrapyrroles, using three one-carbon bridges, include: Bilanes (e.g. bilirubin, biliverdin, urobilinogen, urobilin) Phycobilins (found in cyanobacteria) Cyclic tetrapyrroles, using four one-carbon bridges, include: Porphyrins (e.g. heme) Chlorophylls
Linear Tetrapyrrole structure of Bilirubin
1 3 4
Methyl group Propionic Acid The four pyrrole rings are linked with methylidine bridges. Vinyl group Chemical Formula: C33H36N4O6
Points to ponder: A puzzling chemical property of bilirubin molecule is its insolubility in water and its solubility in variety of nonpolar solvents. The solubility of bilirubin in nonpolar lipid solvents is not predicted from its linear tetrapyrrolic structure as its 2 propionic acid side chains would be expected to make the molecule highly polar and therefore water soluble.
According to X-Ray Crystallography; Bilirubin assumes a ridge-tiled configuration stabilized by six intramolecular hydrogen bonds. Two additional important structural features have also been noted:1. A so called Z-Z (trans) conformation for the double bonds between carbons 4 & 5 and 15 & 16, and 3. An involuted hydrogen-bonded structure in which the propionic acid-carboxylic acid groups are hydrogen bonded to the nitrogen atoms of the pyrrole rings.
Bilirubin IX structureThe folded conformation showing extensive internal hydrogen bonding
Space-filling model of the preferred ridge-tile conformation of bilirubin IX
In this rigid structure, all the polar groups are tied up with each other, limiting interaction with water and the ionization of the COOH groups. The molecule is therefore virtually insoluble in water. Ionization of each COOH group removes one hydrogen bond, allowing interaction of the COO- group with water. The mono- and di-anion of Unconjugated Bilirubin are therefore water soluble. Conjugation of each COOH group with polar Glucuronic Acid groups breaks one H-bond and renders the conjugate water-soluble.
The hydrogen bonds stabilize the Z-Z configuration of bilirubin and prevent its interaction with polar groups in aqueous media. When exposed to light, the Z-Z configuration is converted to the E-E (cis) conformation and to other combinations, namely 4E-15Z and 4Z-15E. The E-E conformation and other E-containing isomers do not permit the degree of internal hydrogen bonding that occurs in the Z-Z and are therefore more water soluble than in the Z-Z conformation. Thus light exposed forms of bilirubin are more water soluble and readily excreted in the bile.
The bilirubin molecule in the crystalline state takes the form of a ridge tile rather than a linear tetrapyrrole. The ridge is along the line joining C8-C10-C12. In this configuration, a pair of pyrrole rings lie in each plane with an angle of 98 angle between the two rings.
Unconjugated bilirubin (diacid) consists of two planar dipyrrolic halves connected by an - HCHbridge, folded at a right angle along the dashed line (like a half closed book). The COOH group on each half interacts, via a trio of hydrogen bonds (||||), with the ring oxygen and nitrogen of the opposite dipyrrole.
Note: Shaded areas denote hydrophobic domains of the molecule.
Ring opening of heme to give four isomeric biliverdins (IX , IX , IX and IX ) and reduction of the IX and IX isomers to the corresponding bilirubins. By convention, the terms biliverdin and bilirubin used alone refer to the IX isomers with the Zdouble bond stereochemistry depicted in the figure.
Bilirubin Fractionation by HPLC
= unconjugated = singly conj. = doubly conj. = covalently bound to albumin
Unconjugated bilirubin Lipid soluble Water insoluble: limits excretion 1 gm albumin binds 8.5 mg bilirubin Fatty acids & drugs can displace bilirubin Indirect reagent in van den Bergh test
Conjugated bilirubin UDP-glucuronide transferase is rate-limiting step Water soluble Direct reaction to van den Bergh test In GI: urobilinogen & stercobilinogen
Delta bilirubin The fraction of bilirubin covalently bound to albumin; in conventional methods it is measured as part of conjugated bilirubin. Because of its covalent bond during the recovery phase of hepatocellular jaundice, it may persist in the blood for a week or more after urine clears. Delta bilirubin, which is covalently bound to albumin, has a longer half-life in the circulation than the other bilirubins and may cause bilirubin elevation for some time after the others have returned to normal Delta-bilirubin is nontoxic and excreted neither in urine nor in bile
Formation of Bilirubin
Primary site of synthesis:SPLEEN: The Graveyard of Red Blood Cells Secondary site of synthesis:LIVER & BONE MARROW
An average person produces about 4 mg/kg of bilirubin per day. The daily bilirubin production from all sources in man averages from 250 to 300 mg.
85%HEMOGLOBIN FROM SENESCENT RBCS DESTROYED IN RETICULOENDOTHELIAL CELLS OF LIVER, SPLEEN & BONE MARROW
15%RBC PRECURSORS DESTROYED IN THE BONE MARROW CATABOLISM OF HEME-CONTAINING PROTEINS (MYOGLOBIN, CYTOCHROMES & PEROXIDASES)
Heme Metabolism Fate Of Red Blood Cells The largest repository of heme in the human body is in red blood cells, which have a life span of about 120 days. There is thus a turnover of about 6 g/day of hemoglobin, which presents 2 problems: The porphyrin ring is hydrophobic and must be solubilized to be excreted. Iron must be conserved for subsequent new heme synthesis.
Normally, senescent red blood cells and heme from other sources are engulfed & lysed by cells of the reticuloendothelial system.
The globin is recycled or converted into amino acids, which in turn are recycled or catabolized as required. Heme is oxidized, with the heme porphyrin ring being opened by the endoplasmic reticulum enzyme, heme oxygenase. The oxidation step requires heme as a substrate, and any hemin (Fe3+) is reduced to heme (Fe2+) prior to oxidation by heme oxygenase. The oxidation occurs on a specific carbon producing equimolar amounts of the linear tetrapyrrole biliverdin, ferric iron (Fe3+), and carbon monoxide (CO). This is the only reaction in the body that is known to produce CO. Most of the CO is excreted through the lungs, with the result that the CO content of expired air is a direct measure of the activity of heme oxygenase in an individual.
In the first reaction, a bridging methylene group is cleaved by heme oxygenase to form Linear Biliverdin from Cyclic Heme molecule. Fe 2+ is released from the ring in this process. I
In the next reaction, a second bridging methylene (between rings III and IV) is reduced by biliverdin reductase, producing bilirubin.
Bilirubin is significantly less extensively conjugated than biliverdin causing a change in the color of the molecule from blue-green (biliverdin) to yellow-red (bilirubin). The latter catabolic changes in the structure of tetrapyrroles are responsible for the progressive changes in color of a hematoma, or bruise, in which the damaged tissue changes its color from an initial dark blue to a red-yellow and finally to a yellow color before all the pigment is transported out of the affected tissue. Peripherally arising bilirubin is transported to the liver in association with albumin, where the remaining catabolic reactions take place.
Extravascular Pathway for RBC Destruction(Liver, Bone marrow, & Spleen)Phagocytosis & Lysis
Amino acid pool
Excretion of BilirubinIn Blood The bilirubin synthesized in spleen, liver & bone marrow is unconjugated bilirubin. It is hydrophobic in nature so it is transported to the liver as a complex with the plasma protein, albumin. Bilirubin-albumin complex d