Heme Degradation & Hyperbilirubinemias

FATE OF RED BLOOD CELLS

 Life span in blood stream is 60-120 days

 Senescent RBCs are phagocytosed and/or

lysed

Normally, lysis occurs extravascularly in the

reticuloendothelial system subsequent to RBC
phagocytosis

 Lysis can also occur intravascularly (in blood

stream)
Extravascular Pathway for RBC Destruction
(Liver, Bone marrow,
& Spleen)

Phagocytosis & Lysis

Hemoglobin

Globin Heme Bilirubin

Amino acids Fe2+

Amino acid pool Excreted

 Heme to Bilirubin
Heme
Heme Oxigenase (Oxidation)

Biliverdin

Biliverdin Reductase (Reduction)

Equimolar concn of CO &Fe3+ Released
Bilirubin
DEGRADATION OF HEME TO BILIRUBIN

 75% is derived from RBCs

 In normal adults this

results in a daily load of
P450 cytochrome
250-300 mg of bilirubin

 Normal plasma
concentrations are less then
1 mg/dL

 Hydrophobic – transported
by albumin to the liver for
further metabolism prior to
its excretion
“unconjugated” bilirubin
NORMAL BILIRUBIN  Uptake of bilirubin by the liver is mediated by
METABOLISM a carrier protein (receptor)

 Uptake may be competitively inhibited by

other organic anions

 On the smooth ER, bilirubin is conjugated with

glucoronic acid, xylose, or ribose

 Glucoronic acid is the major conjugate -

catalyzed by UDP glucuronyl tranferase

“Conjugated” bilirubin is water soluble and is

secreted by the hepatocytes into the biliary
canaliculi

 Converted to stercobilinogen (urobilinogen)

(colorless) by bacteria in the gut

 Oxidized to stercobilin which is colored

 Excreted in feces

 Some stercobilin may be re-adsorbed by the

gut and re-excreted by either the liver or
kidney
 HYPERBILIRUBINEMIA
 Increased plasma concentrations of bilirubin (> 3 mg/dL) occurs when
there is an imbalance between its production and excretion
 Recognized clinically as jaundice
Pathophysiologic classification of
Jaundice

 Hemolytic Jaundice

 Hepatic Jaundice

 Obstructive Jaundice(Cholestasis)

 Congenital Jaundice
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)
 Hemolytic Jaundice
Symptoms
weakness, Dark urine, anemia,
Icterus, splenomegaly
Lab
 UB without bilirubinuria

 fecal and urine urobilinogen

 hemolytic anemia

 hemoglobinuria (in acute intravascular hemolysis)

 Reticulocyte counts

urinary changes:
• bilirubin: absent
• urobilinogen: increased or normal
faecal changes: stercobilinogen: normal
 Obstructive Jaundice
Pathogenesis
It is due to intra- and extra hepatic obstruction of bile ducts
• Intrahepatic Jaundice: Hepatitis, PBC, Drugs
• Extra Hepatic Biliary Obstruction: Stones, Stricture, Inflammation,
Tumors, etc.
• Intrahepatic-Liver cell Damage/Blockage of Bile Canaliculi
Extrahepatic-Obstructive of bile Ducts
• Compression obstruction from tumors
• Gallstones
• Postoperative –Stenosis
• Cholestasis-pain (gall blader disease), enlarged liver etc.
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
 Obstructive Jaundice
Lab Findings
• Serum Bilirubin
• Feceal urobilinogen (incomplete obstruction)
• Feceal urobilinogen absence (complete obstruction)
• urobilinogenuria is absent in complete obstructive jaundice
• bilirubinuria 
• ALP 
• cholesterol 
urinary changes
• bilirubin: increased
• urobilinogen: reduced or absent
faecal changes
stercobilinogen: reduced or absent
 Hepatic Jaundice
Due to a disease affective hepatic
tissue either congenital or acquired
diffuse hepatocellular injury.
• Acquired disorders
 hepatocellular necrosis
 intrahepatic cholestasis
(Hepatitis, Cirrhosis, Drug-related)
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
 Hepatic Jaundice
Pathogenesis
• Impaired or absent hepatic conjugation of bilirubin
 decreased GT activity (Gilbert‘s syndrome)
 hereditary absence or deficiency of UDPGT (Grigler-Najjar
Syndrome)
• Familiar or hereditary disorders
 Dubin-Johnson Syndrome
 Rotor syndrome
• Acquired disorders
 hepatocellular necrosis
 intrahepatic cholestasis
(Hepatitis, Cirrhosis, Drug-related)
 Hepatic Jaundice

Symptoms
weakness, loss appetite, hepatomegaly, palmar
erythema, spider

Lab Findings
• liver function tests are abnormal
• both CB and UCB
• Bilirubinuria 
 Hepatic Jaundice

urinary changes
• bilirubin: normal or increased
• urobilinogen: normal or reduced
faecal changes
stercobilinogen: normal or
reduced
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
Causes of Hyperbilirubinemia
 Gilbert’s Syndrome
Benign liver disorder

 ½ of the affected individuals inherited it

 Characterized by mild, fluctuating increases in

unconjugated bilirubin caused by decreased ability
of the liver to conjugate bilirubin – often
correlated with fasting or illness

 Males more frequently affected then females

Onset of symptoms in teens, early 20’s or 30’s

 Can be treated with small doses of phenobarbital

to stimulate UDP glucuronyl transferase activity
 Crigler-Najjar Syndrome
 Autosomal recessive

 Extremely rare < 200 cases worldwide – gene frequency is < 1:1000

 High incidence in the “plain people of Pennsylvania” (Amish and

Mennonites)

 Characterized by a complete absence or marked reduction in

bilirubin conjugation

 Present with a severe unconjugated hyperbilirubinemia that

usually presents at birth

 Afflicted individuals are at a high risk for kernicterus

 Condition is fatal when the enzyme is completely absent

 Treated by phototherapy (10-12 hrs/day) and liver transplant by

age 5
Dubin-Johnson and Rotor’s Syndromes

 Characterized by impaired biliary

secretion of conjugated bilirubin

 Present with a conjugated

hyperbilirubinemia that is usually mild
 BILIRUBIN

• - The appearance of bilirubin in the urine is the

first indication of liver disease and is often
detected long before the development of jaundice.

• - Bilirubin provides early detection of hepatitis,

cirrhosis, gallbladder disease, and cancer, and
should be included in every routine urinalysis.
 Test for Bilirubin

• OXIDATION TESTS (FOUCHET’S TEST)

• - Urine containing bilirubin usually appears dark yellow or

amber and produces a yellow foam when shaken, this foam
test was actually the first test for bilirubin.

• - Oxidation tests utilize the ability of feric chloride

dissolved in trichloracetic acid (Fouchet's reagent) to
oxidize bilirubin to biliverdin, producing a green color.
 Bilirubin

• REAGENT STRIP (DIAZO) REACTIONS

- Routine testing for urinary bilirubin by reagent strip

utilizes the diazo reaction, in an acid medium to produce
colors ranging from increasing degrees of tan or pink to
violet, respectively.

- Questionable results should be retested using the Ictotest

which produces a more sharply colored diazo reaction.
-
• - Colors other than blue or purple appearing on the mat
are considered negative.
 UROBILINOGEN

• EHRLICH'S TUBE TEST

- The reagent used in all tests was

- p-dimethylaminobenzaldehyde (Ehrlich's
reagent), addition of Ehrlich's reagent to urine
containing urobilinogen produces a cherry red color.
-
• - Positive results in dilutions greater than 1 to 20
were considered significant.
 Gmelin’s test
• A qualitative test for the pigments in bile; test
solution is mixed with nitric acid containing
nitrous acid; reaction is positive if color
appears at the acid-solution junction.
• Hay’s test for bilirubin
• Urine sample + Sulphur powder (Sprinkle)
• If Sulphur powder sinks indicates presence of
bile salts
• If Sulphur powder remains on the top of the
urine indicates absence of bile pigments.
Van Den Berg Test for bilirubin
Van Den Berg Test – 3 steps give you both “free”
and “conjugated” bilirubin
Direct: 1) Mix serum sample w/ diazotised
sulphanilic acid a) Color: depth of which is
proportional to amount of “conjugated” soluble
bilirubin present in the serum.
b) At this stage, “free” bilirubin is undetectable
b/c bound to serum albumin

Indirect: Mix the serum sample w/ diazotised

sulphanilic acid a) Add some alcohol to solubilize the
“free” bilirubin, pulling it off the serum albumin so
it can react:
b) See more color which is now the “Total” Bilirubin
Step 3 is simple subtraction:
Total Bilirubin– Conjugated bilirubin = Free Bilirubin