BilirubiBilirubinnA True Liver Function

Test

Presented By:Sharon Isaac

• Introduction• History• Structure• Biochemistry

› Synthesis of Bilirubin› Excretion of Bilirubin

• Function of Bilirubin• Clinical Significance

› Hyperbilirubinemia & Types of Jaundice

› Inherited disorders of bilirubin metabolism

› Bilirubin Toxicity – Kernicterus• Clinical estimation of Bilirubin

› Serum• Urine Bilirubin• Treatment Methods

• Phototherapy• Exchange Transfusion

• Bilirubin – A true indicator of Liver function

The Road Ahead

Introduction• [Latin bīlis, 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.  

History

1849 Virchowdiscovered bilirubin in blood extravasates; termed it as “Hematoidin”

1864 Stadeler coined the term “Bilirubin”

1874 Tarchanoffdemonstrated the direct association of bile pigments to hemoglobin

1942Fischer & Plieninger

synthesized Bilirubin IXα and proposed a structure for it which was accepted for more than 30 years

StructureTetrapyrrolic structure

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

N

Pyrrole

Linear Tetrapyrrole structure of Bilirubin

Propionic Acid

12

3 4

Chemical Formula: C33H36N4O6

Methyl group

Vinyl group

The four pyrrole rings are linked with methylidine bridges.

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

2. 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 IXα structure

The 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 - HCH-bridge, 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.

GA

GA

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 Z-double bond stereochemistry depicted in the figure.

= unconjugated = singly conj. = doubly conj. = covalently bound to

albumin

Bilirubin Fractionation by HPLC

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.

TOTAL BILIRUBIN

HEMOGLOBIN FROM SENESCENT

RBC’S DESTROYED IN RETICULOENDOTHELIAL

CELLS OF LIVER, SPLEEN & BONE MARROW

RBC PRECURSORS DESTROYED IN THE

BONE MARROW

85% 15%

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.

III IVI II

Oxidation Heme Oxygenase

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.

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

I

I III

III IV

IV

II

II

Reduction Biliverdin Reductase

• 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)

Hemoglobin

Globin

Amino acids

Amino acid pool

Heme Bilirubin

Fe2+

Excreted

Phagocytosis & Lysis

Recycled

Excretion of Bilirubin

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.

• Bilirubin-albumin complex diffuses between endothelial cells in the liver sinusoids into the space of dissé.

In Liver

• After uptake by hepatocytes, bilirubin is reversibly bound to soluble proteins called “Y” Proteins or “Ligandins”.

• Ligandins are cytosolic proteins & constitute 5% of total protein of liver cytosol.

• Ligandins play an important role in the processing of bilirubin by limiting the passive reflux of bilirubin back into the plasma.

• It also promotes the transfer of bilirubin through the cytosol to the smooth endoplasmic reticulum where it is further processed.

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.

• The microsomal enzyme bilirubin uridine diphosphate (UDP)-glucuronyltransferase catalyzes the formation of bilirubin mono glucuronide.It is not yet clear whether the same enzyme also catalyzes the conversion of bilirubin mono glucuronide to bilirubin di-glucuronide.

• The sugar acid, Glucuronic acid is first activated by enzymatic formation of UDP-glucuronic acid and then transferred to bilirubin by the enzyme glucuronyltransferase

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

In Biliary Tract

• The excretion of bilirubin into bile is against a marked concentration gradient is thought to be an energy-dependent, active-transport process.

• Bilirubin conjugates pass with the bile successively from the canaliculi, through the bile ductules and intrahepatic ducts of progressively increasing caliber, to the extrahepatic bile ducts.

• Between meals, bile bilirubins are temporarily stored in the gallbladder, from which they are emptied into the duodenum during feeding.

The Biliary System

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.

Bilirubin Metabolism

HemeBiliverdinBilirubinBilirubin DiglucoronideUrobilinogenUrobilinStercobilin

Heme = planar; Others = not anymore

globin globin globin globin

Hemoglobin

heme heme heme heme

I

II

III

IV Fe2+

NADPHC

O2O O2

Heme Oxygenase

O

IIIIIIIV

Biliverdin

NADPH

H

Bilirubin

Excretion of Bilirubin

3 Steps of Bilirubin Metabolism

• Hepatic Uptake

-Unconjugated bilirubin is presented in the Unconjugated bilirubin is presented in the liver cellliver cell

-The albumin associated with it is dissociatedThe albumin associated with it is dissociated

-Ligandin is delivered to prevent efflux of Ligandin is delivered to prevent efflux of bilirubin back to plasmabilirubin back to plasma

• Conjugation

• Excretion

3 Steps of Biliverdin Metabolism

• Hepatic Uptake

-Unconjugated bilirubin is presented in the Unconjugated bilirubin is presented in the liver cellliver cell

-The albumin associated with it is dissociatedThe albumin associated with it is dissociated

-Ligandin is delivered to prevent efflux of Ligandin is delivered to prevent efflux of bilirubin back to plasmabilirubin back to plasma

• Conjugation

-Unconjugated bilirubin (water insoluble) is Unconjugated bilirubin (water insoluble) is converted to bilirubin diglucoronide (water converted to bilirubin diglucoronide (water soluble)soluble)

-Takes place in the smooth endoplasmic Takes place in the smooth endoplasmic reticulum of the liverreticulum of the liver

-Catalyzed by glucoronyl transferaseCatalyzed by glucoronyl transferase

• Excretion-Bilirubin which is now water soluble can now Bilirubin which is now water soluble can now be excreted from the liver cell to the biliary be excreted from the liver cell to the biliary system.system.

Conjugation with Glucoronates

BILIRUBIN DIGLUCORONIDE

Role of Blood Proteins in the Metabolism of Bilirubin

1. Albumin

Dissolved in Blood

Sparingly soluble in Blood

Blood

Liver

Ligandin

(-) charge

Ligandin

(-) charge

Ligandin Prevents bilirubin from going back to plasma

Function of Bilirubin

• Bilirubin is a powerful lipophilic antioxidant that protects membranes from lipid peroxidation and protects membrane proteins from oxidation.

• Much of the power of bilirubin as an antioxidant comes from the extreme rapidity with which biliverdin (oxidized bilirubin) is converted to bilirubin by biliverdin reductase.

• Bilirubin accounts for most of the antioxidant activity of human serum and is particularly potent against superoxide and peroxyl radicals.

• As early as 1959, it was suggested that bilirubin might be an antioxidant.

• Bilirubin can suppress oxidation of lysosomes at

oxygen concentrations that are physiologically relevant.

• Bilirubin can act as an important cytoprotector of tissues that are poorly equipped with antioxidant defense systems, including myocardium and nervous tissue.

• At very low concentrations , Bilirubin can protect against 10,000-fold greater concentrations of H2O2.

• Under physiologic conditions, bilirubin provides more potent protection against lipid peroxidation than α-tocopherol, formerly known to be most effective in preventing lipid peroxidation.

• Recent research indicates that bilirubin may be the most abundant endogenous antioxidant in mammalian tissues.

• Risk of atherosclerosis has been shown to vary inversely with

serum levels of bilirubin

• The Framingham Heart Study found high serum bilirubin correlated with low cardiovascular risk for men 

• A ten-year Belgian study of nearly 10,000 people found high serum bilirubin correlated with low cancer mortality (especially for men), but found no association with cardiovascular disease 

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

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)

Intrahepatic 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

Posthepatic 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.

Diagnoses of Jaundice

Hemolytic vs. Hepatocellular vs. Obstructive

Hemolytic Hepatocellular Obstructive

Urine/Fecal Urobilinogen

Increased DecreasedAbsent/

fluctuates

Bilirubin Absent Present Present

Conjugated No Yes Yes

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

Evaluation of jaundiceBhutani normogram

Inherited Disorders of Bilirubin Metabolism

• Gilbert’s Syndrome• Crigler-Najjar (Type I)• Crigler-Najjar (Type II)• Lucey-Driscoll• Dubin-Johnson• Rotor’s Syndrome

Isolated increased serum bilirubin

Ruling out of hemolysis, subsequent fractionation of the bilirubin

Possibility of the following syndromes:

• Dublin-Johnson

• Rotor

Possibility of following syndromes based on the bilirubin concentration:

• Gilbert’s - <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

Algorithm for differentiating the familial causes of Hyperbilirubinemia

Conjugated Unconjugated

Gilbert’s Syndrome• Gilbert’s syndrome is also called as familial non-hemolytic

non-obstructive jaundice.

• It is a benign condition manifested by 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).

• It is easily distinguished from chronic hepatitis by the absence of anemia & bilirubin in urine, & also by normal liver function tests

• Special diagnostic tests are occasionally necessary & include demonstrating a rise in bilirubin on fasting and a fall in bilirubin on taking Phenobarbital.

• Gilbert’s syndrome is inherited as an autosomal recessive trait.

• Males are more frequently affected then females. Onset of symptoms is seen in teens, early 20’s or 30’s.

• Gilbert’s syndrome does not require any treatment as it does not interfere with the normal lifestyle of a person.

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.

Lucey-Driscoll Syndrome

• Lucey-Driscoll Syndrome is a familial form of unconjugated hyperbilirubinemia caused by a circulating inhibitor of bilirubin conjugation.

• The hyperbilirubinemia is mild and lasts for the first 2 to 3 weeks of life.

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.

Clinical Conditions Related to Increased Conjugated

HyperbilirubinemiaDubin-Johnson

SyndromeRotor Syndrome

Defect (hepatocytes)

Secretory Transport

Presence of Pigmentation

Yes No

MetabolismAbnormal Porphyrin

MetabolismNone

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

• 3 major features:– Movement disorder– Gaze abnormality, esp. upward gaze limitation– Auditory abnormalities

• Concentrations of bilirubin in the blood serum of affected infants with hemolytic disease should be monitored so that treatment can begin before dangerous concentrations are reached. Treatment is with blood transfusion, and can be administered before birth.

Clinical Estimation of Bilirubin

Serum Bilirubin Estimation: The methods for determination of bilirubin concentration in

plasma (serum) are divided into the methods using:

1) Diazo Methods 2) Enzymatic Methods, and

3) Direct Spectrophotometric measurement of bilirubin.

4) Transcutaneous Measurement

1. Diazo Methods

• The reaction of bilirubin with diazotized sulphanilic acid, known as diazo reaction, was discovered by Erlich in 1883

• The above reaction was applied to measurement of bilirubin in serum & bile by Van den Berg & Muller in 1916 & is the most widely used methods for measuring bilirubin.

• Methods have been developed for the determination of total bilirubin (conjugated and unconjugated) or for conjugated bilirubin alone.

• The proportion of unconjugated bilirubin is calculated as difference between total and conjugated bilirubin.

• Unconjugated bilirubin should first be separated from albumin by use of an accelerator.

• The different Accelerators used are;– Methanol (Malloy & Evelyn)– sodium acetate – dyphylline – sodium benzoate – sodium acetate – caffeine (Jendrassik & Grof) or – Detergents

• Conjugated bilirubin reacts directly in the reaction of diazotization.

• Bilirubin diazotization is generally performed by use of a mixture of sulfanilic acid and sodium nitrite, however, diazonium salts of 2,5-dichlorophenyl or 2,4-dichloroaniline have recently also been introduced.

• The determination of bilirubin concentration by use of these methods is based on the formation of azobilirubin, which acts as an indicator.

• It is pink in acidic or neutral medium, and blue-green in alkaline medium.

2. Enzymatic Method Methods with bilirubin oxidase

• Enzymatic methods for total & direct bilirubin and for bilirubin conjugates with glucuronic acid are based on the oxidation of bilirubin with Bilirubin Oxidase to Biliverdin with molecular oxygen.

• Near pH 8 & in the presence of sodium cholate & sodium dodecyl sulfate (SDS), all 4 bilirubin fractions are oxidised to biliverdin, which is further oxidised to purple & finally colorless products.

• The decrease in absorbance, at 425 or 460 nm is proportional to the concentration of total bilirubin.

• Results by the bilirubin oxidase method were in good agreement with those obtained by the Jendrassik-Grof procedure.

• The intensity of violet color is measured at 405-460 nm. The concentration of bilirubin is proportional to the measured absorbance.

• The concentration of total bilirubin is determined by the addition of sodium dodecyl sulfate (SDS) or sodium cholate (separation of unconjugated bilirubin from albumin and precipitation of reaction).

3. Direct spectrophotometric determination of bilirubin

• This method uses a two component system i.e. measuring absorbance at two wavelengths & solving a system of 2 simultaneous equations.

• The concentration of bilirubin is measured directly at 454 nm and 540 nm.

• The measurement at two wavelengths is used to diminish the hemoglobin interference.

• The method is only used to determine bilirubin concentration in serum of neonates and in amniotic fluid.

4. Transcutaneous Measurement of Bilirubin

• This non-invasive approach was introduced by Yamanouchi et. al.

• The first bilirubinometer was a reflectance photometer, which used 2 filters to correct for the color of Hb & required measurements at 8 body sites.

• Transcutaneous bilirubinometers provide instantaneous information.

• They are useful to determine that whether it is necessary to go for treatment like phototherapy & Exchange Transfusion.

• Another application is predicting those babies that require follow up according to hour specific serum bilirubin nomogram by Bhutani et. al.

Urine Examination

Since conjugated bilirubin is excreted in the urine, its presence indicates conjugated hyperbilirubinemia.

• The methods for detecting Urine Bilirubin are:

– Foam Test method• Normal Urine – foam is absolutely white• Hyperbilirubinemia – foam is yellow

– Fouchet’s Test/ Harrison Spot Test• Bilirubin is oxidized to Biliverdin on reacting with Ferric chloride

dissolved in Trichloroacetic acid (Fouchet’s reagent). This reaction produces green color.

• The Harrison spot test is a modified version of the Fouchet’s test. In this test urine mixed with barium sulphate is filtered through heavy filter paper. Bilirubin attached to barium sulphate collects on the filter paper, & addition of Fouchet’s reagent to the paper produces a green color.

– Either Dipstick or Ictotest method

• Dipstick method is used for routine testing & utilizes the diazo reaction.

• Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to produce colors ranging from increasing degrees of tan or pink to violet, respectively.

• Ictotest produces a more sharply colored diazo reaction & is more sensitive than the dipstick method.

• Ictotest kit contains testing mats & tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, sulfosalicylic acid, sodium carbonate, & boric acid.

• 10 drops of urine are added to the mat, urine filters & bilirubin remains on the mat. The reagent tablet is placed on the mat & 1 water drop is added to he tablet, after 5 sec another water drop is added; Blue or purple color on the surface of the mat indicates a positive reaction.

Phototherapy

• First paper on phototherapy published in 1958 Cremer based on in vitro observation of falling values of bilirubin when serum samples were exposed to light.

• Several clinical trials in the ’60s confirmed the efficacy of phototherapy leading to its use as standard care.

• Trials also showed more efficacy among low birth weight infants.

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

• Phototherapy is the use of visible light for the treatment of hyperbilirubinemia, or jaundice, in the newborn.

• It is one of the most common non-routine therapy applied in the newborn population & is a treatment for all types of jaundice.

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

• Phototherapy is used at much lower levels of jaundice in premature or sick infants.

• Phototherapy does not have to be continuous to be useful.

• Breastfeeding can be continued through phototherapy.Phototherapy can be done at home and in the hospital.

• Phototherapy converts bilirubin that is present in the superficial capillaries and interstitial spaces to water-soluble isomers that are excretable without further metabolism by the liver

• Bilirubin molecules in skin exposed to light undergo relatively quick photochemical reactions on absorbing photons of light-– configurational isomerization, – structural isomerization, and – photo-oxidation-

to form nontoxic, excretable isomers.

• These bilirubin isomers have different shapes from the native isomer, are more polar, and can be excreted from the liver into the bile without undergoing conjugation or requiring special transport for their excretion.

• Urinary and gastrointestinal elimination are both important in reducing the bilirubin load.

The mechanism of phototherapy• When bilirubin molecules absorb light, 2 main photochemical reactions

occur:

• Native 4Z,15Z-bilirubin converts to 4Z,15E bilirubin (also known as photobilirubin) and to lumirubin.

• Unlike 4Z,15Z bilirubin, photobilirubin can be excreted via the liver without conjugation, but its clearance is very slow, and its conversion is reversible. In the bowel (away from the light), photobilirubin is converted back to native bilirubin.

• Lumirubin is not reversible. Although much less lumirubin than photobilirubin is formed, lumirubin is cleared from the serum much more rapidly, and it is likely that lumirubin formation is primarily responsible for the decline in serum bilirubin that results from phototherapy.

• Small amounts of native bilirubin are also oxidized to monopyrroles and dipyrroles that can be excreted in the urine. This is a slow process and only a minor contributor to the elimination of bilirubin during phototherapy

reversible

irreversible

• A strong relationship exists between the dose of phototherapy and the rate of decline in serum bilirubin level. Dose, in turn, is determined by several key factors:

– Spectral qualities of the light source used (wavelength range and peak);

• The most effective light sources for degrading bilirubin are those that emit light in a relatively narrow wavelength range (400 to 520 nanometers [nm]), with a peak of 460 ± 10 nm.

• At these wavelengths, light penetrates the skin well and is maximally absorbed by bilirubin.

• Blue, green, and turquoise light (the blue-green spectrum) are considered the most effective

– Intensity of the light (irradiance)

– Distance between the light and the infant's skin

– Body surface area exposed.

When Is Phototherapy Prescribed?• The aim of phototherapy is to curtail rising serum bilirubin and prevent its

toxic accumulation in the brain, where it can cause the serious, permanent neurological complication - kernicterus.

• Phototherapy has greatly reduced the need for exchange transfusion to

treat hyperbilirubinemia.

• Phototherapy is used in 2 main ways: prophylactically and therapeutically.– In preterm infants or those with a known hemolytic process, it is often used

prophylactically, to prevent a significant rapid rise in serum bilirubin.– In late-preterm and full-term infants, it is administered at therapeutic doses

to reduce excessive bilirubin levels and avoid development of kernicterus.

• The photoisomerization of bilirubin begins almost instantaneously when the skin is exposed to light.

• Unlike unconjugated bilirubin, the photoproducts of these processes are not neurotoxic. Therefore, when faced with a severely hyperbilirubinemic infant, it is important to begin phototherapy without delay.

Exchange Transfusion• Exchange transfusion is a potentially life-

saving procedure performed to counteract the effects of serious jaundice or changes in the blood (from, for example, sickle cell anemia).

• The procedure involves the incremental removal of the patient's blood and replacement with fresh donor blood or plasma.

Description    • In order to perform an exchange

transfusion, it is essential to have the ability to both remove and replace blood. In most cases, this involves the insertion of more than one intravenous (or arterial) catheter.

• The exchange transfusion proceeds in cycles, each generally of a few minutes duration.

• The patient’s blood is slowly withdrawn, and an equal amount of fresh, pre-warmed blood or plasma is transfused. This cycle is repeated until a predetermined volume of blood has been replaced.

• The blood is withdrawn usually in increments of 5 to 20 ml depending on the patient’s size and the severity of illness

• After the exchange transfusion, catheters may be left in place in case the procedure needs to be repeated.

• The exchange transfusion process is riddled with complications in process & post the procedure.

• So exchange therapy is generally never used until after intensive phototherapy has been attempted & exhausted.

Bilirubin – A True Indicator of Liver Function

• Liver function tests, also referred to as "LFTs" are blood tests that assess the state of the liver and the biliary system.

• LFTs are divided into

– Indicators of liver function, and – Indicators of liver injury or biliary tract disease.

• The Liver Function Tests are;– AST (SGOT)– ALT (SGPT)– ALP– GGT– TOTAL PROTEIN– ALBUMIN– BILIRUBIN– PROTHROMBIN TIME

• All the enzyme tests are indicators of hepatocellular damage or biliary flow obstruction.

• Tests like serum bilirubin, albumin & Prothrombin time are considered true indicators of Liver function

• Serum bilirubin is generally considered a true test of liver function

(LFT), since it reflects the liver’s ability to take up, process and secrete

bilirubin into the bile.