BIOCHEM Jaundice Final

7/21/2019 BIOCHEM Jaundice Final

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JAUNDICE



Hemoglobin Catabolism:

Hemoglobin

Globin + Heme

Iron + Porphyrin

Bilirubin

REC, of liver, spleen, BM cells



1. Formation of bilirubin:

STEPS:

• Catalyzed by microsomal heme oxygenase system of

reticuloendothelial cells.

• In the presence of NADPH and oxygen, the enzyme adds a

hydroxyl group to he methenyl bridge between the twopyrrole rings with a concomitant oxidation f ferrous iron to

ferric state.

• A second oxidation of the same enzyme cleaves he

porphyrin ring.

• Ferric iron and CO2

are released.

• Biliverdin is produced.

• Bilirubin reductase reduces biliverdin to bilirubin.



2. Uptake of bilirubin by the liver:

•Bilirubin is non-covalently bound to

albumin while being transported to

the liver.

•Bilirubin dissociates from albumin

and enters a hepatocyte where it

binds to ligandin (cytoplasmic anionbinding protein).



3. Conjugation of bilirubin

•diglucuronide / conjugation•Enzyme: bilirubin glucuronyl

transferase

•Glucuronate donor: UDP-

glucuronic acid.



4. Excretion of bilirubin into bile:

•Conjugated bilirubin is actively transported against aconcentration gradient into the bile canaliculi and then into

the bile.

5. Formation of urobilins in the intestines:

•Bilirubin diglucuronide is hydrolyzed and reduced by

bacteria in the gut to urobilinogen.

•Urobilinogen can be reabsorbed in the gut and transported

to the kidneys to form urobilin (yellow color of urine).

•Most of the urobilinogens are reabsorbed to the

enterohepatic pathway.



Question # 2

Differentiate conjugatedbilirubin from

unconjugated bilirubin.



Unconjugated bilirubin Conjugated Bilirubin

Present normally in plasma Present normally in bile

Attached non covalently to

albumin

Conjugated to glucuronic acid

Has high molecular weight and

cannot be filtered through the

kidney

Has small molecular weight and

if present in plasma can be

filtered through the kidney.

Non-polar, insoluble in plasma

and can cross blood brain barrier

in neonates

Polar, soluble in plasma and

cannot cross blood brain barrier

Gives indirect Van den Bergh

reaction

Gives direct Van den Bergh

reaction



Van den Bergh Reaction:

•This is a reaction between bilirubin and

Ehrlich diazo reagent giving a reddish

purple compound.

•Conjugated bilirubin reacts directly to the

reagent. Thus, it is called direct bilirubin.

•Unconjugated bilirubin does not directly

react with the compound except after theaddition of methyl alcohol. It may be called

indirect bilirubin.



Question #3:

Discuss the different phases of

bilirubin metabolism. What are the

roles of proteins (albumins, ligandins,

etc.) in bilirubin metabolism?



TWO PHASES OF BILIRUBIN METABOLISM:

A. HEPATIC PHASE

1. Hepatic Uptake of Bilirubin

2. Bilirubin Conjugation

3. Biliary Excretion

B. INTESTINAL PHASE



A. HEPATIC PHASE

• Liver has a central role

• Divided into 3 distincts processes:

1. Hepatic Uptake

2. Bilirubin Conjugaction

3. Biliary Excretion



A. HEPATIC PHASE: Hepatic Uptake

• Bilirubin is only sparingly soluble in water,

but its solubility in plasma is increased bynoncovalent binding to albumin.

• Each molecule of albumin appears to have

one ____ for bilirubin:• High-affinity site – bilirubin, drugs (antibiotics)

• Low-affinity site (loose) - bilirubin

• In 100 mL of plasma, approximately 25 mg of

bilirubin can be tightly bound to albumin at itshigh-affinity site.




Things involved in this process:

a.OATP2 – a family of organic anion-transporting

polypeptidase (OATPs)

b.Proteins (OATPs)

• Ligandin (glutathione S-transferase B)

• Protein Y and Protein Z




Steps:

1. B1-Albumin complex dissociates

2. B1 (nonpolar) enters the hepatocyte by diffusion via

OATP2; and albumin – goes back to circulation for

more binding of B1

3. In the cytosol, B1 binds to proteins like Ligandin andProtein Y& Z for transport to ER for conjugation.



A. HEPATIC PHASE: Bilirubin Conjugation

• Occurs in smooth endoplasmic reticulum

• Things involved in this process:

o 2 molecules of Uridine Diphosphate - Glucuronyl

Transferase (UDP-GT)

o UDP- Glucuronic acid (bilirubin-UGT) – glucuronate

donor



A. HEPATIC PHASE: Bilirubin Conjugation



A. HEPATIC PHASE: Biliary Excretion

Things involved in this process:• Energy (ATP)

• MRP2 (multidrug resistance-like protein 2) or

cMOAT (canalicular multispecific organic anion

transporter) – located in plasma membrane of thebiliary canaliculi; a member of the family of ATP-

binding cassette (ABC) transporters; for biliary

excretion of B1



A. HEPATIC PHASE: Biliary Excretion



A. HEPATIC PHASE: Summary



Determination of Bilirubin Concentration

A. Van den Bergh Reaction / Diazo Reaction

Reaction: Diazotized sulfanilic acid ------ red

+ Bilirubin azodipyrroles

Coupling Accelerator :Methanol – provides a solution in which both B1 and diazo

reagent are soluble



Determination of Bilirubin Concentration

B. High Performance Liquid Chromatography

• Most accurate

• Most sensitive

• Quantitative method



B. INTESTINAL PHASE

Conjugated Bilirubin (B2) in small amount

Biliary duct

Duodenum

and

Colon

Enzymatic Deconjugation

(ex. bacterial B-glucuronidase)

Nonenzymatic Deconjugation

(ex. alkaline hydrolysis)

Unconjugated Bilirubin



B. INTESTINAL PHASE

UROBILINOGEN(Soluble)

KidneyLiver

Intestine

STERCOBILINOGEN STERCOBILIN

bacteria O2

≤4 mg



Question #4:

What are the causes of jaundice?



JAUNDICE

• Yellowish discoloration of the skin and sclera

• Newborn: there is a decreased level of hepaticbilirubin glucuronyl tranferase.

• It is also known as icterus

a.skin

b.conjunctival membrane over the sclera

(scleral icterus but more properly called

conjunctival icterus)

c.mucous membranes• Jaundice itself is not a disease



JAUNDICE

Types of Jaundice:

1.Hemolytic Jaundice

2.Obstructive or Cholestatic Jaundice

3.Hepatocellular Jaundice



Causes of JAUNDICE

HEMOLYSIS

LIVER DAMAGE BILE DUCT

OBSTRUCTION

JAUNDICE

Increased Production of Bilirubin

Decreased Excretion of Bilirubin

JAUNDICE: 3 Categories



JAUNDICE: 3 Categories

PRE-HEPATIC HEPATIC POST-HEPATIC

Pathologic

location

Prior to liver Within the liver After conjugation in

the liver Mechanism Too much

(massive)

destruction of

RBC

Damage to liver cells (

caused by viruses, alcohol,

and parasites); impaired

cellular uptake, defective

conjugation, abnormal

secretion of bilirubin by the

hepatocytes

Obstruction of bile

duct (failure of bile to

flow to the intestine /

impaired bilirubin

excretion)

Comparison

a. B1 Increased Increased Normal

b. B2 Normal Increased Increased

c. Urobilinogen Normal Decreased Decreased

d. Urine Bilirubin Negative Positive Positive

e. Stool Color Normal Normal Pale

f. ALP / ALT / AST Normal Increase Increased

PRE HEPATIC



PRE-HEPATIC

HEPATIC

POST-HEPATIC

Location ?

Mechanism?

B1 ?

B2 ?

Urobilinogen ?

Urine Bilirubin ?

Stool Color ?

ALP ?

AST ?

ALT ?



• Question # 5

• Differentiate between the different

types of jaundice (conjugated vs

unconjugated hyperbilirubinemia);(choluric vs acholuric jaundice)



Unconjugated

Hyperbilirubinemia



I. Overproduction

• Hemolysis (intra- and extravascular)

• Ineffective Errythropoiesis

II. Decreased hepatic uptake

• prolonged fasting

• sepsis



III. Decreased bilirubin conjugation

(decreased hepatic

glucuronosyl transferase

activity)

A. Hereditary transferase deficiency

• Gilbert’s syndrome (mild transferase

deficiency)

• Crigler-Najjar type II (moderate

transferase deficiency)

• Crigler- Najjar type I (absence of

transferase)



B. Neonatal jaundice (transient

transferase deficiency; increased

intestinal absorption of unconjugated

bilirubin)

• Drug inhibition (e.g., chlorampenicol,

pregnanediol)

• Breast milk jaundice (transferase inhibition by

pregnanediol and fatty acids in breast milk)• hepatocellular disease (hepatitis, cirrhosis)

C. Acquired transferase deficiencyof

unconjugated bilirubin)



Conjugated

Hyperbilirubinemia



I. Impaired hepatic excretion

(intrahepatic defects)

• A. Familial or hereditary disorders

• Dubin-Johnson syndrome

• Rotor syndrome

• Recurrent (benign)intrahepatic cholestasis

• Cholestatic jaundice of

pregnancy



B. Acquired disorders

• Hepatocellular disease (e.g., viral or drug-induced hepatitis, cirrhosis)

• Drug-induced cholestasis (e.g., oral

contraceptives)

• Alcoholic liver disease• Sepsis

• Post operative state

• Parenteral nutrition

• Biliary cirrhosis (primary or secondary)



II. Extrahepatic biliary

obstruction

A. Intraductal obstruction

• Gallstones

• Biliary malformation (e.g., stricture,

atresia, choledochol cyst)• Infection (e.g., Clonorchis, Ascaris)

• Malignancy (cholangiocarcinoma,

ampullary carcinoma)

• Hemobilia (trauma, tumor)• Sclerosing cholangitis



B. Compression of biliary ducts

• Malignancy (e.g., pancreatic carcinoma,lymphoma, metastases to portal lymph

nodes

• Inflammation (e.g., pancreatitis)



Choluric Jaundice

• Conjugated bilirubin is water soluble

therefore can be excreted in urine

• To which imparts a yellow-brown

coloration

• This is called choluric jaundice



Acholuric jaundice

• Hemolytic jaundice

• Jaundice without bilirubinemia

• Associated with elevated unconjugated

bilirubin that is nnot excreted by the kidney



• Question # 6 Explain theoccurrence of jaundice in the

following conditions:



• Physiologic jaundice of the newborn

• Decreased hepatic bilirubin glucuronyl

transferase at birth

• Immature liver• Increase in unconjugated bilirubin

• Toxic encephalopathy/kernicterus

• Treated with blue flourescent light



• Hemolytic jaundice

• Important cause of unconjugatedhyperbilirubinemia

• High levels of hemoglobin released from

erythrocytes due to hemolysis

• Rate of bilirubin production exceeds the rate of theliver clearance



• Defects in conjugation

• Toxic hyperbilirubinemia• Toxin induced liver dysfunction

• (chloroform, arsphenamines, carbon

tetrachloride, acetaminophen, hepatitis virus,

cirrhosis and Ammantia mushroom poisoning)• Due to hepatic parenchymal cell damage (which impairs

conjugation)

• Dubin Johnson syndrome

• Rotor syndrome



• Breast milk jaundice

• Begins 1st

week after birth• Peaks within two weeks after birth

• Infant’s immature liver and intestines

• Milk of some mothers has non-esterified long chain

fatty acids or other substances which inhibitglucuronyl transferases conjugation activity

• Nursing may be discontinued until bilirubin levels fall

rapidly, then nursing may be resumed.



• Obstruction of the biliary tract

• most common cause of conjugated hyperbilirubinemia

• blockage of the hepatic or common bile ducts

• gallstone or cancer of the head of the pancreas

• bilirubin diglucuronide cannot be excreted

• regurgitates into the hepatic veins and lymphatics• conjugated bilirubin appears I the blood and urine



Phototherapy works through aprocess of isomerization that

changes trans-bilirubin into the

water-soluble cis-bilirubin isomer.

TREATMENT

Phenobarbital was used to treat neonatal

jaundice by increasing liver metabolism

and thus lowering bilirubin levels.

http://www.ask.com/wiki/Phototherapy?qsrc=3044

http://en.wikipedia.org/wiki/Neonatal_jaundice


http://en.wikipedia.org/wiki/Bilirubin

http://en.wikipedia.org/wiki/Bilirubin



http://www.ask.com/wiki/Phototherapy?qsrc=3044

BIOCHEM Jaundice Final

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