BIOCHEMICAL PROFILE OF JAUNDICE DR MUHAMMAD MUSTANSAR
Introduction• 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.
Erythrocytes become “old” as they lose their flexibilityand become pikilocytes (spherical), increasingly rigidand fragile. Once the cell become fragile, they easilydestruct during passage through tight circulationspots, especially in spleen, where the intra-capillaryspace is about 3 micron as compared to 8 micron ofcell size RBCs useful life span is 100 to 120 days,After which they become trapped and fragment in smaller circulatory channels, particularly in those of the spleen. For this reason, the spleen is sometimes called the “red blood cell graveyard.”Dying erythrocytes are engulfed and destroyed bymacrophages.
Formation of Bilirubin• Primary site of synthesis:- SPLEEN: The Graveyard of Red Blood Cells• Secondary site of synthesis:- LIVER & BONE MARROW
An average person TOTAL BILIRUBIN produces about 4 mg/kg of bilirubin per day. 85% 15% HEMOGLOBIN RBC PRECURSORS The daily bilirubin FROM SENESCENT DESTROYED IN THE RBC’S DESTROYED IN BONE MARROW production from all RETICULOENDOTHELIAL sources in man CELLS OF averages from 250 LIVER, SPLEEN & CATABOLISM OF to 300 mg. BONE MARROW HEME-CONTAINING PROTEINS (MYOGLOBIN, CYTOCHROMES & PEROXIDASES)
Extravascular Pathway for RBC Destruction (Liver, Bone marrow, & Spleen) Phagocytosis & Lysis Hemoglobin Globin Heme Bilirubin Amino acids Fe2+ Amino acid pool Recycled Excreted
• 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 occurs on a specific carbon producing equimolar amounts of the biliverdin, iron , 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, abridging methylenegroup is cleaved byheme oxygenase toform Linear Biliverdinfrom Cyclic Hememolecule. Heme Oxygenase OxidationFe 2+ is released fromthe ring in this process. I III IV II
I III IV II• In the next reaction, a second bridging methylene (between rings III and IV) is reduced by biliverdin reductase, producing bilirubin. Reduction Biliverdin Reductase I III IV II
• 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.
Bilirubin is not very water-soluble, so most of it is carried to the liver bound toalbumin.
In cells of the liver, bilirubin undergoes modification to In cells of the liver, bilirubin undergoes modification toincrease its water solubility so that it can be excreted more increase its water solubility so that it can be excreted moreeasily. easily.a.Bilirubin is conjugated to twoa.Bilirubin is conjugated to twomolecules of glucuronic acid, creatingmolecules of glucuronic acid, creatingbilirubin diglucuronide.bilirubin diglucuronide.b. Bilirubin diglucuronide is transportedb. Bilirubin diglucuronide is transportedout of the hepatocytes into the bileout of the hepatocytes into the bilecanaliculi and is thus excreted in bile.canaliculi and is thus excreted in bile.
In Blood Unconjugated bilirubin• The bilirubin synthesized in – Lipid soluble spleen, liver & bone marrow – : limits excretion is unconjugated bilirubin. – 1 gm albumin binds 8.5 mg bilirubin• It is hydrophobic in nature so – Fatty acids & drugs can it is transported to the liver displace bilirubin as a complex with the – Indirect positive reaction plasma protein, albumin. in van den Bergh test
Role of Blood Proteins in the Metabolism of Bilirubin 1. Albumin Dissolved in Blood
BloodLiver Ligandin Ligandin (-) charge (-) charge Ligandin Prevents bilirubin from going back to plasma
In Endoplasmic ReticulumIn the microsomes of the endoplasmic reticulum,unconjugated bilirubin is converted to water solublemono- or di- conjugates by sequential covalentcoupling with glucuronic acid.
Bilirubin is conjugated ina two step process toform bilirubin mono- &di- glucuronide
Conjugation with Glucoronates BILIRUBIN DIGLUCORONIDE
In the Intestine• In the small intestine, conjugated bilirubins are poorly reabsorbed, but are partly hydrolyzed back to unconjugated bilirubin by catalytic action of bacterial ß-glucuronidases.• In the distal ileum and colon, anaerobic flora mediate further catabolism of bile pigments: a) hydrolysis of conjugated bilirubin to unconjugated bilirubin by bacterial β-glucuronidases; b) multistep hydrogenation (reduction) of unconjugated bilirubin to form colorless urobilinogens; and c) oxidation of unconjugated bilirubin to brown colored mesobilifuscins.
• Urobilinogens is a collective term for a group of 3 tetrapyrroles; – Stercobilinogen (6H) – Mesobilinogen (8H)&, – Urobilinogen (12H)• Upto 20 % of urobilinogen produced daily is reabsorbed from the intestine & enters the entero-hepatic circulation. Urobilinogen Structure
• Most of the reabsorbed urobilinogen is taken up by the liver & is re-excreted in the bile.• A small fraction (2 % - 5 %) enters the general circulation & appears in the urine.• In the lower intestinal tract, the 3 urobilinogens spontaneously oxidize to produce the corresponding bile pigments; – Stercobilin – Mesobilin & – Urobilin; which are orange-brown in color and are the major pigments of stool.
SYMPTOMSo Yellowing of the skin, scleras (white of the eye), and mucous membranes (jaundice)o Detectable when total plasma bilirubin levels exceed 2mg/100mL AHHH!!! I have symptoms of hyperbilirubinemia!!!
Clinical SignificanceHyperbilirubinemia & Types of Jaundice• Hyperbilirubinemia : Increased plasma concentrations of bilirubin (> 3 mg/dl) occurs when there is an imbalance between its production and excretion.• Recognized clinically as jaundice.• Also known as icterus, a yellow discoloration of the skin, sclerae and mucous membrane.
• Jaundice becomes clinically evident when the serum bilirubin level exceeds 2.5mg/dL.• Several types of Jaundice: – Hemolytic – Hepatocellular – Obstructive• Symptoms: – Yellow discoloration of the skin, sclerae and mucous membranes – Itching (pruritus) due to deposits of bile salts on the skin – Stool becomes light in color – Urine becomes deep orange and foamy
Different Causes of Jaundice • Excessive Production of Bilirubin • Reduced Hepatocyte Uptake • Impaired Bilirubin conjugation • Impaired Bile Flow
Prehepatic (hemolytic) jaundice • Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis • Excess RBC lysis is commonly the result of autoimmune disease; hemolytic disease of the newborn (Rh- or ABO- incompatibility); structurally abnormal RBCs (Sickle cell disease); or breakdown of extravasated blood • High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL)
Hepatic jaundice • Impaired uptake, conjugation, or secretion of bilirubin • Reflects a generalized liver (hepatocyte) dysfunction • In this case, hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function
• Hemolytic jaundice arises as a consequence of excessive destruction of RBCs.• – This overloads the capacity of the RE system to metabolize heme.• – Failure to conjugate bilirubin to glucuronic acid causes accumulation of bilirubin in the unconjugated form in the blood.
• Hepatocellular jaundice arises from liver disease, either inherited or acquired.• – Liver dysfunction impairs conjugation of bilirubin.• – Consequently, unconjugated bilirubin spills over into the blood.• –In addition, urobilinogen is elevated in the urine.
Ongoing liver damage with liver cell necrosisfollowed by fibrosis and hepatocyteregeneration results in cirrhosis. Thisproduces a nodular, firm liver. The nodulesseen here are larger than 3 mm and, hence,this is an example of "macronodular"
Obstructive jaundiceDefinition : Is a conditioncharacterized byYellow discolorationof the skin , sclera &mucous membrane asa result of an elevatedSr. Bilirubin conc. dueto an obstructive cause.
Posthepatic(Obstructive) jaundice • Caused by an obstruction of the biliary tree. • Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids accumulate in the plasma. • Characterized by pale colored stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin). • In a complete obstruction, urobilin is absent from the urine.
• • Obstructive jaundice, as the name implies, is caused by blockage of the bile duct by a gallstone or a• tumor (usually of the head of the pancreas).• – This prevents passage of bile into the intestine and consequently conjugated bilirubin builds up in the blood.• – Patients with this condition suffer severe abdominal pain associated with the obstruction (if due togallstone) and their feces are gray in color due to lack of stercobilin.
Pre-hepatic Hepatic Post hepaticcause Excessive break down Infective Bile Duct Obstruction Of RBC’s Liver Damage Malaria,HS Gilbert SyndromeSerum Bilirubin unconjugated Both conj+unconj. conjugatedUrine bilirubin Absent Bilirubinemia + As in hepatic jaundice Achloric jaundice Deep yellow urine ++Urine Increases Decreases Absent(-)urobilinogen Because of increased Because of decreased stercobilinogen stercobilinogenFecal Markedly increased Reduced Absentstercobilinogen Dark brown stool Pale coloured stool clay colored stool20-250mg/dayFecal fat 5-6% normal Increased 40-50% As hepatic jaundice Bulky,pale greasy foul smelling faecesLiver functions normal Impaired SGOT/SGPT Normal Alkaline phosphatase++Vonden burg test Indirect+ biphasic Direct+
Neonatal Jaundice• Common, particularly in premature infants.• Transient (resolves in the first 10 days).• Due to immaturity of the enzymes involved in bilirubin conjugation.• High levels of unconjugated bilirubin are toxic to the newborn – due to its hydrophobicity it can cross the blood-brain barrier and cause a type of mental retardation known as kernicterus• If bilirubin levels are judged to be too high, then phototherapy with UV light is used to convert it to a water soluble, non-toxic form.
• If necessary, exchange blood transfusion is used to remove excess bilirubin• Phenobarbital is oftentimes administered to Mom prior to an induced labor of a premature infant – crosses the placenta and induces the synthesis of UDP glucuronyl transferase• Jaundice within the first 24 hrs of life or which takes longer then 10 days to resolve is usually pathological and needs to be further investigated
CLINICAL FEATURES• Severe unconjugated hyperbilirubinemia at birthPrior to phototherapy:• Kernicterus• Death in infancy
Phototherapy•Phototherapy is usually not neededunless the bilirubin levels rise veryquickly or go above 16-20 mg/dl inhealthy, full term babies.• During phototherapy, thetreatment of choice forjaundice, babies are placedunder blue lights that convert the bilirubin into compoundsthat can be eliminated fromthe body.
Bilirubin Toxicity - Kernicterus• Kernicterus or brain encephalopathy refers to the yellow staining of the deep nuclei (i.e., the kernel) of the brain namely, the basal ganglia.• It is a form of permanent brain damage caused by excessive jaundice.• The concentration of bilirubin in serum is so high that it can move out of the blood into brain tissue by crossing the fetal blood-brain barrier.• This condition develops in newborns with prolonged jaundice due to: – Polycythemia – Rh incompatibility between mother & fetus
Algorithm for differentiating the familial causes of Hyperbilirubinemia Isolated increased serum bilirubin Ruling out of hemolysis, subsequent fractionation of the bilirubin Conjugated Unconjugated Possibility of following syndromesPossibility of the based on the bilirubin concentration:following syndromes: • Gilbert’s - <3 mg/dl• Dublin-Johnson • Crigler-Najjar (Type I) - >25 mg/dl• Rotor • Crigler-Najjar (Type II) - 5 to 20 mg/dl • Lucey-Driscoll - Transiently ~ 5 mg/dl
Crigler-Najjar Syndrome (Type I)• Crigler-Najjar Syndrome (Type I) is a rare genetic disorder caused by complete absence of UDP- glucuronyltransferase and manifested by very high levels of unconjugated bilirubin.• It is inherited as an autosomal recessive trait.• Most patients die of severe brain damage caused by kernicterus within the first year of life.• Early liver transplantation is the only effective therapy.
Crigler-Najjar Syndrome (Type II)• This is a rare autosomal dominant disorder.• It is characterized by partial deficiency of UDP- glucuronyltransferase.• Unconjugated bilirubin is usually 5 – 20 mg/dl.• Unlike Crigler-Najjar Type I, Type II responds dramatically to Phenobarbital & a normal life can be expected.
Gilbert’s Syndrome• Gilbert’s syndrome is also called as familial non-hemolytic non-obstructive jaundice.• mild unconjugated Hyperbilirubinemia.• It affects 3% – 5% of the population. It is often misdiagnosed as chronic Hepatitis.• The concentration of Bilirubin in serum fluctuates between 1.5 & 3 mg/dl.• In this condition the activity of hepatic glucuronyltransferase is low as a result of mutation in the bilirubin-UDP- glucuronyltransferase gene(UGT1A1).
Dubin-Johnson Syndrome• It is a benign, autosomal recessive condition characterized by jaundice with predominantly elevated conjugated bilirubin and a minor elevation of unconjugated bilirubin.• Excretion of various conjugated anions and bilirubin into bile is impaired, reflecting the underlying defect in canalicular excretion.• The Liver has a characteristic greenish black appearance and liver biopsy reveals a dark brown melanin- like pigment in hepatocytes and kupffer cells.
Rotor’s Syndrome• It is another form of conjugated hyperbilirubinemia.• It is similar to dubin-johnson syndrome but without pigmentation in liver.