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 flexibility and become pikilocytes (spherical), increasingly rigid and fragile. Once the cell become fragile, they easily destruct during passage through tight circulation spots, especially in spleen, where the intra-capillary space is about 3 micron as compared to 8 micron of cell 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 by macrophages.

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.

TOTAL BILIRUBIN

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)

Extravascular Pathway for RBC Destruction

(Liver, Bone marrow, & Spleen)
Phagocytosis & Lysis

Hemoglobin

Globin

Heme Fe2+

Bilirubin

Amino acids

Amino acid pool

Recycled

Excreted

Pathophysiology
RBCs Breakdown

Hemoglobin Produces & Breakdown

Heme Heme Oxygenase Biliverdin Biliverdin Reductase Bilirubin

 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, a b r i d g i n g m e t h yl e n e group is cleaved by heme oxygenase to form Linear Biliverdin from Cyclic Heme m o l e c u l e .
Fe 2+ is released from the ring in this process. I

Oxidation

Heme Oxygenase

III

IV

II

Heme Oxygenase

I IV
Fe2+

C
II

NADP H

III

O2

O2

IV

III

II

Biliverdin

H
NADP H

Bilirubin

I In the next reaction, a second bridging methylene (between rings III and IV) is reduced by biliverdin reductase, producing bilirubin.

III

IV

II

Reduction

Biliverdin Reductase

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 to albumin.

In cells of the liver, bilirubin undergoes modification to increase its water solubility so that it can be excreted more easily.

a.Bilirubin is conjugated to two molecules of glucuronic acid, creating bilirubin diglucuronide.

b. Bilirubin diglucuronide is transported out of the hepatocytes into the bile canaliculi and is thus excreted in bile.

In 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.

Unconjugated bilirubin
Lipid soluble : limits excretion 1 gm albumin binds 8.5 mg bilirubin Fatty acids & drugs can displace bilirubin Indirect positive reaction in van den Bergh test

Role of Blood Proteins in the Metabolism of Bilirubin

1. Albumin Dissolved in Blood

Blood

Liver
Ligandin Ligandin (-) charge

(-) charge

Ligandin Prevents bilirubin from going back to plasma

In Endoplasmic Reticulum In the microsomes of the endoplasmic reticulum, unconjugated bilirubin is converted to water soluble mono- or di- conjugates by sequential covalent coupling with glucuronic acid.

Bilirubin is conjugated in a two step process to form bilirubin mono- & di- glucuronide

Conjugation with Glucoronates

BILIRUBIN DIGLUCORONIDE

Heme

Heme oxygenase

Biliverdin

Biliverdin reductase

Bilirubin

BILIRUBIN PHYSIOLOGY

Excretion of Bilirubin

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 -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 -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.

JAUNDICE

SYMPTOMS
o 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 Significance
Hyperbilirubinemia & 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

Classification

Jaundice

Pre-hepatic

Hepatic

Post-Hepatic

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 ABOincompatibility); 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 necrosis followed by fibrosis and hepatocyte regeneration results in cirrhosis. This produces a nodular, firm liver. The nodules seen here are larger than 3 mm and, hence, this is an example of "macronodular"

Obstructive jaundice
Definition :
Is a condition characterized by Yellow discoloration of the skin , sclera & mucous membrane as a result of an elevated Sr. Bilirubin conc. due to 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
cause

Hepatic

Post hepatic

Excessive break down Of RBCs Malaria,HS Gilbert Syndrome

unconjugated Absent Achloric jaundice Because of increased stercobilinogen

Infective Liver Damage

Bile Duct Obstruction

Serum Bilirubin Urine bilirubin

Both conj+unconj. Bilirubinemia + Deep yellow urine Decreases Because of decreased stercobilinogen Reduced Pale coloured stool Increased 40-50% Bulky,pale greasy foul smelling faeces Impaired SGOT/SGPT

conjugated As in hepatic jaundice ++ Absent(-)

Urine urobilinogen Increases

Fecal stercobilinogen 20-250mg/day Fecal fat 5-6%

Markedly increased Dark brown stool normal

Absent clay colored stool As hepatic jaundice

Liver functions

normal

Normal Alkaline phosphatase++

Vonden burg test

Indirect+

biphasic

Direct+

Diagnoses of Jaundice

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 birth Prior to phototherapy: Kernicterus Death in infancy

Phototherapy
Phototherapy is usually not needed unless the bilirubin levels rise very quickly or go above 16-20 mg/dl in healthy, full term babies.

During phototherapy, the treatment of choice for jaundice, babies are placed under blue lights that convert the bilirubin into compounds that can be eliminated from the body.

Phototherpy for infants

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

Inherited Disorders of Bilirubin Metabolism

Gilberts Syndrome Crigler-Najjar (Type I) Crigler-Najjar (Type II) Lucey-Driscoll Dubin-Johnson Rotors Syndrome

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 syndromes based on the bilirubin concentration: Gilberts - <3 mg/dl Crigler-Najjar (Type I) - >25 mg/dl Crigler-Najjar (Type II) - 5 to 20 mg/dl Lucey-Driscoll - Transiently ~ 5 mg/dl

Possibility of the following syndromes:

Dublin-Johnson Rotor

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 UDPglucuronyltransferase.

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.

Gilberts Syndrome
Gilberts 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-UDPglucuronyltransferase 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 melaninlike pigment in hepatocytes and kupffer cells.

Rotors Syndrome
It is another form of conjugated hyperbilirubinemia.
It is similar to dubin-johnson syndrome but without pigmentation in liver.