Metabolism of heme

Dr. Sandeep Appunni

 Heme
• Structure consist of ferrous ion complexed with protoporphyrin IX

• Prosthetic group in specialized proteins:

- Hemoglobin
- Myoglobin
- Cytochrome oxidase
- Heme peroxidase
- Catalase
- Endothelial nitric oxide synthase
Ogun AS, Valentine M. Biochemistry, Heme Synthesis. [Updated 2019 Jan 31]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing;
2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537329/
Structure of heme

I II

III IV
 Forms of heme

• Heme b: Present in hemoglobin

• Heme a & heme c: Present in mitochondria and required for

oxidative phosphorylation

• Heme a in cytochrome a and heme c in cytochrome c

Heme synthesis
Describe any pathway under the following
subheadings (For 5 & 10 marks)
• Background/purpose in brief
• Site (organ specific/ subcellular)
• Preparatory steps (if any)
• Main steps of synthesis or degradation. Explain each step with
necessary reactions. Draw complete cycle to summarize (Specifically
mention the organelle involved both in reactions as well as cycle)
• Regulation of key enzymes
• Energetics (related pathways)
• Biochemical and functional significance of the pathway or byproducts
• Clinical significance of the pathway
 Site

• Organ and tissue: Erythrocytes in bone marrow and

hepatocytes in liver

• Subcellular level: Mitochondria and cytosol

• Heme is directly absorbed from the intestine

 Step 1: Synthesis of ALA
• This step occurs in the mitochondria
• Rate limiting step in the pathway

Pyridoxal
Succinyl CoA + Glycine phosphate
δ-aminolevulinic acid (ALA)
ALAS

TCA
cycle
ALAS - aminolevulinic acid synthase
Step 2: Synthesis of porphobilinogen (PBG)

CYTOSOL

ALA + ALA ALA dehydratase PORPHOBILINOGEN

(PBG) (Zn2+)

• ALA dehydratase is a zinc requiring enzyme

• Also known as porphobilinogen synthase
Step 3: Synthesis of HMB
Step 4: Synthesis of UBG III
CYTOSOL

4 PBG HMB Synthase

HYDROXYMETHYLBILANE (HMB)
Linear
4NH3 UPG III Synthase
Asymmetric pyrrole
UROPORPHYRINOGEN III (UPG
 HMB synthase or PBG deaminase

Porphobilinogen (PBG) Hydroxymethylbilane (Linear tetrapyrrole)

 UPG III
synthase

Hydroxymethylbilane (Linear tetrapyrrole) Uroporphyrinogen III

Uroporphyrinogen III
 Step 5: Synthesis of CPG III

CYTOSOL

2 ALA ALA PORPHOBILINOGEN

(PBG) dehydratase
HMB Synthase

HYDROXYMETHYLBILANE
(HMB)
(Linear)
UPG III Synthase

UPG
COPROPORPHYRINOGEN III UROPORPHYRINOGEN III (UPG
decarboxylase (Asymmetric pyrrole)
(CPG III)
 M
A

UPG III
M
A decarboxylase
A
4 CO2 M

A M

Uroporphyrinogen III Coproporphyrinogen III

CPG III transported into mitochondria

CYTOSOL

2 ALA ALA PORPHOBILINOGEN

(PBG) dehydratase
HMB Synthase
HYDROXYMETHYLBILANE
(HMB)
(Linear)
UPG III Synthase

UROPORPHYRINOGEN III (UPG

(Asymmetric pyrrole) UPG
decarboxylase
COPROPORPHYRINOGEN III
(CPG III)
Step 6: Formation of colorless PPG IX

PROTOPORPHYRINOGEN IX (PPG)
Coproporphyrinogen
CPG III oxidase

2CO2
Step 7: Formation of protoporphyrin IX
(PP IX)

PROTOPORPHYRIN PPG
IX IX PPG IX CPG III
CPG III oxidase oxidase

CO2
Final step: Synthesis of HEME

HE
FERROCHELATASE PPG IX CPG III
PP IX PPG IX oxidase
CPG III
oxidase

ME Fe2+ CO2
 Ferrochelatase

PROTOPORPHYRIN IX HEME
Heme synthesis: Summary
 HMB synthase or UBG I synthase

PORPHOBILINOGEN

UPG I synthase or HMB synthase

HYDROXYMETHYLBILANE Spontaneous
UPG III synthase

Uroporphyrinogen III (UPG III) Uroporphyrinogen I (UPG I) L

I
UPG decarboxylase Uroporph G
yrin I
Coproporph H
T
Coproporphyrinogen I (CPG I) yrin II (CPG I)
Coproporphyrinogen
HEME NO HEME
Porphyrins are colored and fluoresce

• Porphyrinogens are colorless

• Porphyrins are colored substances and fluoresce

• Du to the presence of conjugated double bond in the pyrrole

ring and methylene bridges

• A characteristic feature of porphyrins is a sharp absorption

spectrum near 400 nm – Soret band
 Heme molecule
• Heme is ferrous protoporphyrin IX- four pyrrole rings
linked via methenyl bridges
α
• Iron is in ferrous (Fe2+) state
δ β

γ
Regulated enzyme in heme synthesis

• 5'-Aminolevulinic acid synthase (ALA-S) is the regulatory enzyme for

heme synthesis both in the liver and erythroid cells

• ALA Synthase exists in two forms: ALAS1 and ALAS2

• All cells express ALAS1 while only the liver and bone marrow
expresses ALAS2

• The gene for ALAS2 is on the X-chromosome

 Functions of heme as a co-factor
• Facilitates the transport of oxygen in hemoglobin
• Storage of oxygen in myoglobin
• As a prosthetic group for activity of cytochrome p450 enzymes
• Reservoir of iron
• Electron shuttle of enzymes in the electron transport chain
• Cellular respiration
• Signal transduction-heme regulates the antioxidant response to
circadian rhythms, microRNA processing
• Cellular differentiation and proliferation
 Heme synthesis in erythroid cells
• Heme induces the synthesis of globin polypeptide chains in
reticulocytes

• Erythropoietin stimulates heme synthesis

• Erythropoietin stimulates both RBC and hemoglobin synthesis

• Mature RBC can no longer synthesize heme and hemoglobin

• Availability of intracellular iron is essential for heme synthesis

 Heme synthesis in hepatocytes
• Tightly regulated as non-protein bound heme is toxic intracellularly
• In hepatocytes heme is required for synthesis of cytochrome P-450
enzymes
• Cytochrome P-450 is required for xenobiotic metabolism (drugs and
toxins)
• Heme synthesis stops after incorporation into proteins or when hemin
accumulates
• ALAS1 regulates the synthesis of heme in hepatocytes
• ALAS1 can be induced by drugs and low heme concentration
Hemin
• Salt of heme

• Iron containing porphyrin with

chloride ion

• Iron is in ferric state

 How hemin inhibits heme synthesis

• Reduces transcription of ALAS1 mRNA

• Destabilizes ALAS1 mRNA
• Impairs the mitochondrial transport of ALAS1 from the cytosol
Therapeutic use:
• Hemin is given intravenously for treating porphyria by reducing
the synthesis of ALA and PBG. Subsequently other porphyrins.
• Hemin also induces the catabolism of heme by inducing heme
oxygenase 1
Hematin

• Iron is in ferric (Fe3+)state

• Product of heme oxidation

• Suppress heme synthesis

• Reduces porphyrin synthesis in

porphyria
 Cancer phototherapy

• Cancer cells uptake large amounts of porphyrins than normal

tissue

• Hematoporphyrins are administered in certain tumors

• The porphyrin is excited using an argon laser that results in

cytotoxicity
Clinical significance
Clinical scenario resulting from deficient or
dysfunctional enzymes of heme synthesis

• Porphyria

• Drug induced porphyria

• X-linked sideroblastic anemia

 Porphyria

• Group of disorder resulting from retention of intermediates of heme

synthesis
• Intermediates degrade into porphyrins which accumulate in blood,
tissue and urine

• Porphyrins progressively damage the nervous and cutaneious tissue

• Occurs due to deficiency or defect in one of the enzymes involved

in heme synthesis
 Porphyria (classification)

• Porphyria's are hepatic or erythropoietic based on the organ affected

• Depending upon the duration of illness: acute or chronic

• Acute porphyria: Acute intermittent porphyria, porphyria cutanea tarda,

hereditary coproporphyria
• Chronic porphyria: Congenital erythropoietic porphyria, protoporphyria
 Porphyrias (clinical features)

• Common manifestation include: neurologic dysfunction, mental

disturbance (psychiatric manifestations) or photosensitivity

• Accumulation of intermediates which are formed after synthesis of

hydroxymethylbilane (HMB) results in photosensitivity

• Other manifestations: Change in urine colour, abdominal pain,

abdominal colic, highly agitated state, tachycardia, respiratory
problems, nausea, confusion, weakness of lower extremities
 Porphyrias
PORPHYRIA ENZYME DEFECT TYPE
HYDROXYMETHYLBILANE SYNTHASE OR
ACUTE INTERMITTENT PORPHYRIA PORPHOBILINOGEN DEAMINASE OR HEPATIC
UROPORPHYRINOGEN I SYNTHASE
CONGENITAL ERYTHROPOEITIC
UPG III SYNTHASE ERYTHOPOETIC
PORPHYRIA
PORPHYRIA CUTANEA TARDA UPG DECARBOXYLASE HEPATIC

HEREDITARY COPROPORPHYRIA CPG OXIDASE HEPATIC

VARIEGATE PORPHYRIA PPG OXIDASE HEPATIC

PROTOPORPHYRIA FERROCHELATASE ERYTHROPOETIC

 Acute intermittent porphyria

• Defect in hydroxymethylbilane synthase or porphobilinogen

deaminase or UPG I synthase (50% activity)
• Autosomal Dominant inheritance

• Accumulating intermediate is Porphobilinogen (PBG)

• Manifestations are those of hepatic porphyria

• Does not involve erythroblasts
AIP (Biochemical defect)
 Acute intermittent porphyria
Manifestations:
• Severe abdominal pain
• Vomiting
• Constipation
• Abdominal distention
• Behavioral changes (Irritability, insomnia, emotional lability)
• Hypertension (High blood pressure >140/90 mm Hg)
• Tachycardia (Resting heart beat >100 beats per minute)
• NO Photosensitivity (Why? Because photosensitivity is caused
by accumulation of intermediates which are formed after HMB)
 AIP predisposing factor

Acute attacks are provoked by:

• Drugs
• alcoholic beverages
• endocrine factors
• calorie restriction
• Stress
• Infections
Usually resolve within two weeks
 Laboratory testing and diagnosis
Biochemical:
• Increased excretion of ALA and PBG in urine
• Absence of porphyrins (uroporphyrin and coproporphyrin) in
urine and stools
• Hyponatremia
• Watson-Schwartz test (detect PBG)
Molecular:
• Molecular genetic testing for pathological variants
• Assay of erythrocyte HMBS enzyme activity
 AIP (urine)
• During acute attack the urine is
colorless

• On prolonged light exposure color

changes to pink due to photo-
oxidation of PBG to porphobilin

• Urine should be collected freshly

and transported in dark bottles
 Watson-Schwartz test
• Positive only when PBG is 5 times more than usual
• Step 1: Erlich’s reagent (para-dimetylaminobenzaldehyde) and sodium acetate
reacts with PBG and urobilinogen to form a pink colored complex
• Low specificity for Erlich’s test as it is answered pink by a normal urinary
pigment urobilinogen
• Step2: Pink colored urine is then transferred to chloroform and mixed well.
Chloroform is denser than urine and will settle to the bottom. PBG if present
will remain in the top aqueous layer. Urobilinogen will settle with chloroform
• Step 3: Transfer the pink layer to butanol. Butanol settles over the aqueous
layer. PBG will remain in the aqueous layer in the bottom.
 Porphyrins and UV radiation

• Most porphyrins have a sharp absorption spectrum at 400 nm

• So large wavelength UV rays (like UVA) can cause in vivo excitation of

porphyrins that can result in photosensitivity and cytotoxicity

• Therefore sun exposure should be preferably avoided to reduce

symptoms
Congenital erythropoietic porphyria
(CEP)
• Inheritance: Autosomal recessive, Biallelic defect resulting in
low enzyme activity
• Manifest at an early age

• Enzyme defect: UPG III synthase

• Chronic type of erythropoietic porphyria

 Biochemical alteration in CEP
• UPG III synthase deficiency (AR, Biallelic, 0-10% enzyme activity)
• UPG III synthesis is reduced
• HMB accumulates
• HMB spontaneously gets converted to UPG I isomer
• UPG decarboxylase converts UPG 1 to CPG 1 isomer
• CPG I isomer cannot further take part in heme synthesis
• Both UPG I and CPG I undergo photooxidation to form uroporphyrin I and
Coproporphyrin I
• Both Uroporphyrin I and Coproporphyrin I contribute to photosensitivity
• No heme synthesis contributes to no feedback inhibition
CEP (Biochemical defect)
 Clinical features (CEP)

• Photosensitivity: Increased blood porphyrins

• Urinary excretion of porphyrin: dark red in color (portwine appearance)
• Skin manifestations: Photosensitization due to porphyrins in the capillaries
• Reactive oxygen species (free radicals) are the cause for cell destruction
• Repeated attacks of dermatitis and scarring lead to a typical facial deformity
often referred to as ‘monkey face’
• Repeated ulceration and scarring: mutilation of nose, ear and cartilage
• UV light reflected on teeth: red fluorescence is seen called erythrodontia
• Hemolytic anemia is common due to porphyrin induced cytotoxicity
 Lab diagnosis (CEP)
Molecular diagnosis:
• Biallelic pathological variant in UROS (UPG III synthase) gene
• Reduced UPG III synthase enzyme activity in erythrocytes

Blood, urine and stool:

• Blood erythrocytic and urinary levels of uroporphyrin I and coproporphyrin I are
elevated
• Stool levels of coproporphyrin I is elevated
 Treatment

• Strict avoidance of sunlight and other long-wave UV light

exposure to reduce photosensitivity

• Absolute treatment is bone marrow transplantation

 Porphyria cutanea tarda (PCT)

• Genetic defect: UPG III decarboxylase (UROD)

• Inheritance: Autosomal dominant

• Elevated porphyrins (Uroporphyrin and Heptacarboxylporphyrins)

• Severe cutaneous photosensitivity

• Hepatic porphyria
 Clinical features

• Photosensitivity: Dorsal aspect of hands, sun-exposed region

• Skin friability to minor trauma
• Facial hypertrichosis
• Hyperpigmentation
• Sclerosis of hands resembling scleroderma (pseudoscleroderma)
• Increased risk for hepatocellular carcinoma (HCC)
 Lab diagnosis (PCT)

Molecular testing:
Heterozygous pathological variant of UROD (UPG III decarboxylase)
UROD enzyme activity is less than 20%

Blood, urine and stool:

• Uroporphyrin I isomer and heptacarboxylporphyrins are elevated in
urine and serum: Urine fluoresce pink with long wave UV rays
• ALA and PBG levels are normal
• Heptacarboxylporphyrin present in stool
 Hereditary coproporphyria (HCP)

• Enzyme defect: CPG oxidase (coproporphyrinogen-III oxidase, CPOX)

• Inheritance: Autosomal dominant

• Acute hepatic porphyria and photosensitivity

• Abdominal pain, motor neuropathy, seizures, hyponatremia
• Uroporphyrin and coproporphyrin can contribute to photosensitivity

• Predisposing factor: Medications, changes in female reproductive

hormones and calorie deprivation
 Lab diagnosis HCP
Molecular testing
• Heterozygous pathological variant in CPOX gene

Blood, urine and stool

• Blood PBG levels are elevated
• High fecal coproporphyrin III
• High coproporphyrin III/I ratio (Normal <0.5)
• More coproporphyrin III over Protoporphyrin
• In asymptomatic cases: PBG and Coproporphyrin III levels are normal
 Variegate porphyria

• Enzyme defect: PPG oxidase (Protoporphyrinogen oxidase, PPOX)

• Autosomal dominant
• Hepatic porphyria

• Cutaneous photosensitivity like blistering, scarring, hypertrichosis and

hyperpigmentation
• Acute porphyria (neurovisceral symptoms): Abdominal pain,
neuropathy, psychiatric disturbances
 Laboratory diagnosis

Molecular testing
Pathological variant in PPOX gene

Blood, urine and stool

Urinary PBG and total porphyrins (esp coproporphyrin III) are elevated
Coproporphyrin III and Protoporphyrin elevated in stool in equal (not
like HCP)
Asymptomatic levels will be normal
 Erythropoietic porphyria

• Ferrochelatase (FECH) deficiency

• AR, Biallelic pathological variant
• Enzyme activity 10-30 % of normal
• Erythrocytic porphyria
• Elevated erythrocytic protoporphyrin levels
• Sever photosensitivity and cutaneous changes
• Free protoporphyrin levels are elevated, stool protoporphyrin is
elevated
• Plasma porphyrins of III isomers are elevated
Lead poisoning (Acquired porphyria)

• ALA dehydratase (>80%) and ferrochelatase activity is reduced

• Mimic acute porphyria

• Serum and urinary Lead levels are elevated
• ALA levels are elevated
• ALA/PBG ratio is high
• Zinc chelated protoporphyrin levels are elevated
Hemoglobin
 Hemoglobin
• Quaternary structure
• Four subunits: 2 alpha and 2 beta (Adult hemoglobin)
• Each subunit or polypeptide has a heme (Fe2+-protoporphyrin IX)
prosthetic group bound
• Each Hb has four heme
• Each heme Fe2+ binds to single oxygen molecule
• Therefore each Hb can carry four oxygen molecules
• Alpha subunit: 141 amino acid, chromosome 16
• Beta, gamma and delta subunit: 146 amino acid, chromosome 11
 Hemoglobin

• Normal level of Hemoglobin (Hb) in blood in males is 14–16 g/dL and in

females, 13–15 g/dL
• The adult Hb (HbA) has 2 alpha chains and 2 beta chains. Molecular
weight of HbA is 67,000 Daltons.
• HbF (foetal Hb) is made up of 2 alpha and 2 gamma chains
• HbA2 has 2 alpha and 2 delta chains
• Normal adult blood contains
- 97% HbA
- 2% HbA2
- 1% HbF.
 Histidine residues in Hb structure
• 36 histidine residues in Hb molecule
• Important in buffering action
• 58th residue in alpha chain is called distal histidine because it is
far away from the iron atom
• 87th residue in alpha chain is called proximal histidine because
it lies nearer to the iron atom
• Fe2+ forms coordination complex directly with the proximal
histidine
• The oxygen bound to the ferrous iron forms hydrogen bonding
with the distal histidine
 Structures bound by each Fe2+

• Bound to the nitrogen of each pyrrole ring. So four coordination

complexes formed Fe2+-N

• The fifth coordination site of Fe2+ is bound to imidazole group of

histidine residue of the polypeptide subunit

• The final sixth coordination site of Fe2+ binds to oxygen molecule

• In oxidized Fe3+ state, it cannot bind to oxygen molecule

 Hemoglobin cooperativity
• Four subunits of haemoglobin actually bind to oxygen cooperatively

• Binding of oxygen to one site of the four subunits will increase the
likelihood of the remaining sites to bind with oxygen
Hemoglobin cooperativity
 Myoglobin
• Oxygen carrying pigment of the muscle tissue
• Localized in the cytoplasm
• Single polypeptide, single heme, 154 amino acids and forms a tertiary structure
• First protein to have its structure delineated by means of X-ray crystallography
• Oxy and deoxy forms of Mb are present
• Ferrous ion is bound to the oxygen molecules as in Hb
• Release oxygen following intense physical activity
• Absent in blood. Presence in blood is pathological as in rhabdomyolysis
• Blood Mb filtered into the renal tubules can produce acute kidney injury
Difference between hemoglobin and myoglobin

Hemoglobin Myoglobin
• Quaternary structure • Tertiary structure
• Larger molecule with more amino • Smaller molecule with less amino
acids acids
• Four polypeptide chain • Single polypeptide chain
• Follows co-operativity • No co-operativity instead, high affinity
for oxygen at low pO2
• Utilizes 90% of its oxygen carrying • Uses only 7% of oxygen carrying
capacity capacity
• Oxygen binding capacity modulated • Oxygen binding capacity not
by H+ and CO2 modulated by H+ and CO2
Hemoglobin has all the requirements of an ideal
respiratory pigment

• It can transport large quantities of oxygen

• It has great solubility
• It can take up and release oxygen at appropriate partial pressures
• It is a powerful buffer
 Function of hemoglobin

• Transport of oxygen
• Transport of carbon dioxide
• Intracellular buffer in RBC
• Role in decreasing the growth of malarial parasite (For eg: HbS in
Sickle cell anemia prevents intracellular growth of plasmodium)
 Transport of oxygen by Hb
• The ability to transport oxygen to different
tissues depends on the factors affecting
the oxygen dissociation curve
Factors are:
• Co-operativity
• Effect of pH and CO2
• Bohr effect (CO2 forces O2 release)
• Temperature
• Effect of 2,3-BPG
Bohr effect
 Transport of CO2

• Dissolved form (10%)

• Isohydric transport of carbon dioxide (75%)

• Carried as carbamino-hemoglobin (15%)

Isohydric transport of CO2

Haldane effect:
H+ buffered by
deoxy-Hb

Chloride effect:
HCO3- exchange
for Cl-
 Buffer function of Hb
• Executed by terminal –NH2 group and histidine residues
 Fetal hemoglobin
• 2 alpha and 2 gamma subunits (α2γ2)
• Tertiary structure is same but primary structure is different from adult Hb
Physicochemical properties of HbF:
1. Increased solubility of deoxy HbF
2. Slower electrophoretic mobility for HbF
3. Increased resistance of HbF to alkali denaturation
4. HbF has decreased interaction with 2,3-BPG
 Fetal hemoglobin
• Synthesis of HbF starts by 7th week of gestation; it becomes the
predominant Hb by 28th week.
• At birth, 80% of Hb is HbF

• During the first 6 months of life, it decreases to about 5% of total

• There is a rapid postnatal rise in 2,3-BPG content of RBC

• HbF level may remain elevated in children with anemia and beta
thalassemia as a compensatory measure
HbA2
• It is a normal hemoglobin; it is about 2% of total Hb
• 2 alpha chains and 2 delta chains
• The delta chain has sequence homology with beta chain.
• In beta thalassemia HbA2 is increased as a compensatory mechanism
• Isoeletric pH of HbA2 is 7.4 while HbA has the pI value of 6.85 thus
HbA2 has slower electrophoretic mobility
 Heme catabolism
or
Degradation of heme
 Hemoglobin in RBC
• RBC has a life span of 120 days

• Followed by which it reaches senescence and is destroyed in the

reticuloendothelial system (RES)

• Hemoglobin disintegrates into heme and globin

• Globin is a polypeptide chain which is further hydrolyzed into individual

amino acids
• Free heme is toxic and after removal of iron, the iron-free protoporphyrin is
immediately metabolized to non-toxic metabolites
 Heme catabolism
• Takes place in RES of spleen, liver and bone marrow in two steps
Step 1:
• First heme (ferric) is degraded to biliverdin
• Enzyme is heme oxygenase, present in the microsomal section of the cell
• Heme iron is already oxidized to the ferric state before reaching enzyme
• This reaction consumes 3 molecules of O2 and 7e- provided by NADH and
NADPH-cytochrome p450 reductase
• Only step in the body where carbon monoxide is produced
• Heme is the substrate as well as co-factor for the reaction
• CO does not interfere with heme-oxygenase activity
• Biliverdin is a green tetrapyrrolic colored pigment
 Step I:
Heme catabolism

FERRIC HEME

HEME
OXYGENASE

BILIVERDIN
 Step II: Heme catabolism

• Biliverdin reductase further reduces biliverdin to bilirubin

• Bilirubin is a yellow colored pigment

• The methylene bridges of biliverdin is converted to methyl group

• NADPH is utilized as the cofactor

 Step II:
Heme catabolism

BILIRUBIN
 Facts and figures
• Nearly 6 grams of hemoglobin is degraded every day

• Each gram of Hb produces 35 mg of bilirubin

• Each day nearly 250-350 mg of bilirubin is produced

(Sources: Hb, ineffective erythropoiesis and other heme proteins)
 Transport of bilirubin to liver
• The bilirubin formed is unconjugated and minimally soluble in plasma

• It is transported from the RES to the hepatocytes of the liver via blood

• Bound to albumin it can be quickly transported to liver

• Albumin has high affinity and low affinity binding sites for bilirubin

• High affinity sites can bind 35 mg/100 mL of bilirubin

• Loosely bound bilirubin detaches into the tissue
Further metabolism of bilirubin
 Bilirubin metabolism in liver

• Bilirubin reaching liver from the RES is unconjugated

• Conjugation of bilirubin detoxifies it as well as makes it water soluble

• Facilitated by conjugation of glucuronic acid to bilirubin

Three main steps in liver:
• Uptake of bilirubin by liver
• Conjugation of bilirubin
• Secretion into bile
 Uptake of bilirubin by liver

• Saturable facilitated transport system

• Albumin-bilirubin complex releases loosely bound unconjugated bilirubin

(UCB) into the sinusoids

• UCB is then taken up by hepatocytes through a membrane bound facilitated

transport mechanism
• Following uptake UCB binds to ligandin (Glutathione S transferase)

• Bilirubin uptake depends on subsequent bilirubin metabolism

 Conjugation

• Unconjugated bilirubin is non polar

• It is made polar by conjugation
• Conjugation is facilitated with glucuronic acid
• Enzyme: UDP-glucuronosyltransferase (UGT1A1)
• The enzyme catalyzes two step glucuronidation of bilirubin as follows:
Secretion of bilirubin diglucuronide
into the bile duct
• Conjugated bilirubin secretion into the bile is an active
transport mechanism
• Also the rate-limiting step in hepatic bilirubin metabolism
• Protein involved is a multi-specific organic anion transporter
(MOAT) located in the plasma membrane of the bile canaliculi
• MOAT belongs to the family of ATP binding cassette proteins
• Hepatic bilirubin conjugation and biliary secretion is inducible
• Drug phenobarbital induces the expression of UDP-
glucuronosyltransferase
• Most of the bilirubin excreted in the bile of mammals is bilirubin
diglucuronide

• In obstructive jaundice bilirubin conjugates predominantly exist as

monoglucuronides in plasma
Intestinal Bacteria Reduce Conjugated
Bilirubin to Urobilinogen
• Bacterial β-glucuronidases removes glucuronosyl moieties from the
conjugated bilirubin
• Occurs in terminal ileum and large intestine
• Fecal flora further reduces bilirubin to colorless tetrapyrroles called
urobilinogen
• Small amount of urobilinogen is absorbed and re-excreted via the
enterohepatic urobilinogen cycle
• In excess bile pigment formation or diseases of the biliary system,
urobilinogen's are present in the urine
• Urobilinogen is further oxidized to colored urobilins and excreted in feces
kidneys
kidneys Urine
 Measurement of bilirubin

• Measured calorimetrically based on the reddish-purple color formed

when bilirubin reacts with diazotized sulfanilic acid

• Total bilirubin = conjugated bilirubin + unconjugated Bilirubin

• Conjugated bilirubin = Direct bilirubin
• Unconjugated bilirubin = Indirect bilirubin

• Total bilirubin = Direct bilirubin + Indirect bilirubin

 Hyperbilirubinemia
• Excess circulating or serum bilirubin levels
Classification:
• Nature of illness: Congenital or acquired
• Based on the type of bilirubin elevated: Conjugated or unconjugated
hyperbilirubinemia
• Based on the site of defective bilirubin metabolism (congenital or acquired)
which can be differentiated based on laboratory investigation:
> Pre-hepatic
> Hepatic
> Post-hepatic
 Jaundice

Yellowish discoloration of the skin, sclera,

conjunctiva and mucus membrane
Bilirubin deposits in elastin of sclera
 Normal bilirubin levels
• Total bilirubin: 0.6-0.8 mg/dl
• Conjugated (Direct) bilirubin: 0.2-0.4 mg/dl
• Unconjugated (Indirect) bilirubin: 0.2-0.6 mg/dl

• Unconjugated hyperbilirubinemia: Elevated unconjugated

bilirubin(UCB) levels compared to (CB)
• Conjugated bilirubin: elevated levels of CB compared to UCB
 Congenital hyperbilirubinemia

• Inherited defects
• Abnormal uptake, conjugation or excretion of bilirubin
Four defects:
• Gilbert’s syndrome
• Crigler-Najjar syndrome
• Dubin-Johnson syndrome
• Rotor syndrome
 Acquired hyperbilirubinemia

• Not inherited
• Physiological jaundice
• Breast milk jaundice
• Acquired causes for pre-hepatic, hepatic and post-hepatic jaundice
 Gilbert syndrome
• Very common
• Mild unconjugated hyperbilirubinemia
• Autosomal dominant or recessive
Combination of defects:
• A defect in the uptake of bilirubin by liver cells
• Impairment in conjugation due to reduced activity of UDP-
glucuronosyl transferase (UGT1A1)
Features:
• Mild unconjugated hyperbilirubinemia
• Total bilirubin not more than 6 mg/dl
 Crigler-Najjar syndrome

• Inherited moderate to severe deficiency of UGT1A1

• Defect is in conjugation
Type 1 Crigler-Najjar syndrome:
• Severe deficiency of UDP glucuronosyl transferase
• Often fatal and the children die before the age of 2
• Jaundice usually appears within the first 24 hours of life.
• Unconjugated bilirubin level increases to more than 20 mg/dL
• Results in kernicterus
 Crigler-Najjar syndrome

Type 2 Crigler-Najjar syndrome:

• Less severe form than type 1
• Defect in UGT1A1 that results in defective second stage of
conjugation of bilirubin
• Administration of phenobarbital (barbiturates) results in
improvement of jaundice (by inducing the expression of the enzyme
UGT1A1)
• Serum bilirubin levels are < 20 mg/dl
 Dubin-Johnson syndrome

• Benign
• Defective clearance of conjugated bilirubin from the liver
• Genetic defect in MRP2 protein (Multi-specific organic anion transporter)
• Conjugated bilirubin accumulates in the hepatocytes which regurgitates back
into the blood
• Results in black colour of liver (intra-hepatocytic bilirubin accumulation):
Black liver jaundice
• Conjugated hyperbilirubinemia
 Rotor syndrome

• Defective excretion of conjugated bilirubin

• Benign
• Conjugated hyperbilirubinemia
• Deficiency of the major hepatic drug uptake transporters (OATP1B1 and
OATP1B3)
 Physiological jaundice
• Neonatal hyperbilirubinemia
• Appearance of mild jaundice in all new born infants after the 2nd day
of life
• Transient unconjugated hyperbilirubinemia
Cause:
• Accelerated rate of destruction of RBCs
• Immature hepatic system of conjugation of bilirubin
Outcome:
• Serum total bilirubin does not increase above 5 mg/dL
• Disappears by the second week of life
 Breast Milk Jaundice

• Breast-fed infants
• Prolonged jaundice due to high level of maternal estrogen which is
excreted through the milk
• Maternal estrogen inhibits UDP glucuronosyl transferase enzyme
• Elevated unconjugated fraction
 Kernicterus
• Neonates with jaundice
• Unconjugated bilirubin induced brain damage
• Bilirubin level exceeds 20 mg/dl. Beyond 20 mg/dl albumin bilirubin
binding sites get saturated and no further bind to it.
• Newborns blood brain barrier (BBB) is immature
• High circulating unconjugated bilirubin can cross the BBB and enter brain
• Affected neonates or infants develop bilirubin encephalopathy and is fatal
• Preventable by providing phototherapy
• Phototherapy causes photoisomerization of hydrophobic unconjugated
bilirubin into hydrophilic unconjugated lumirubin
Hyperbilirubinemia based on site of defective
bilirubin metabolism that can be differentiated
by laboratory investigation

• Pre-hepatic
• Hepatic
• Post-hepatic
 Pre-hepatic jaundice
• Hemolytic diseases are the main cause for pre-hepatic jaundice
• Only unconjugated bilirubin is elevated, conjugated bilirubin normal
• Hepatic and biliary systems are normally functioning
• So markers of hepato-biliary damage will be under normal limits
• Liver enzyme markers (Alanine amino transferase and aspartate amino
transferase are within normal limits)
• Obstructive biliary marker (Alkaline phosphatase) will be under normal limit
• Hemolytic disease cause increased release of hemoglobin
 Hemolytic diseases
Hemolytic disease of newborn
Rh incompatibility: Rh-ve mother conceives Rh+ve fetus. Antibodies against
Rhesus D antigen generated in mother crosses the placenta and causes
hemolysis of fetal RBC expressing Rh D antigen. Commonly occurs in
subsequent pregnancy following first pregnancy with similar Rh+ve fetus.

Severe hemolytic disease results in erythroblastosis fetalis

Kernicterus can develop if severe unconjugated hyperbilirubinemia prevails

Treatment is to give phenobarbital and phototherapy

 Hemolytic diseases
Hemolytic disease of adults
Unconjugated bilirubin in blood
Absence of bilirubinuria
Excessive excretion of UBG in urine and SBG in feces

Common causes:
i. Congenital spherocytosis
ii. GPD deficiency
iii. Autoimmune hemolytic anemias
iv. Toxins like carbon tetrachloride.
 Hepatic jaundice
• Hepatocellular jaundice
• Causes: viral hepatitis A, B,C, D or E
• RBC destruction rate is normal (No hemolytic disease)
• Hepatocellular damage is present due to inflammation or infection
• Obstruction of the biliary system (bile canaliculi) can occur
(obstructive phase of viral hepatitis)
• Both conjugated and unconjugated bilirubin is elevated
• Markers of liver damage are elevated (AST, ALT elevated)
• Biliary obstruction markers are elevated (ALP elevated)
 Hepatic jaundice
Hepatocellular phase:
• Conjugation is reduced so elevation of serum unconjugated bilirubin (UCB)
• Hepatocellular damage results in elevation of hepatic enzymes (ALT and AST)
• No obstruction of biliary canaliculi so no signs of obstructive jaundice
• No bile pigments in urine and urinary UBG is normal
Obstructive phase:
• Inflammatory edema of hepatocytes causes compression of the biliary canaliculi
• Features of obstructive jaundice begins
• Markers of biliary obstruction increases (elevated ALP)
• Urine shows presence of bile salts and bile pigments
• Reduced urobilinogen in urine
 Post-hepatic jaundice
• Obstructive jaundice
• Rate of RBC destruction as well as hepatocellular function is normal
• Hepatocellular conjugation is normal
• Conjugated bilirubin cannot be expelled via the hepatic duct into intestine
• Due to obstruction of the bile duct resulting in conjugated hyperbilirubinemia
• Markers of biliary obstruction like Alkaline phosphatase (ALP) is elevated
• UBG is decreased or absent
• Bile salts and bile pigments are present in urine
 PRE-HEPATIC HEPATIC POST-HEPATIC

BLOODInvestigations for evaluating jaundice

1. Total bilirubin Elevated Elevated Elevated
Conjugated (Direct) bilirubin Normal +/- +++
Unconjugated (indirect) bilirubin +++ ++ Normal
2. Enzyme panel
Aspartate aminotransferase (AST) Normal +++ Normal
Alanine aminotransferase (ALT) Normal +++ Normal
Alkaline phosphatase (ALP) Normal +/- +++
URINE
1. Bile salts - +/- +++
2. Bile pigments - +/- +++
3. Urobilinogen +++ Normal/- -
FECES
1. Stercobilinogen +++ +/- -
 Phototherapy

• Bilirubin maximally absorbs blue light (420–470 nm)

• Phototherapy deals with the exposure of the jaundiced neonates to blue light
• By photoisomerization, the toxic native unconjugated bilirubin gets
converted into a non-toxic isomer namely lumirubin
• Lumirubin can be easily excreted by the kidneys in the unconjugated form (in
contrast to bilirubin which cannot be excreted)
• Serum bilirubin is monitored every 12–24 hours, and phototherapy is
continuously carried out till the serum bilirubin becomes normal (< 1 mg/dl).
 Drugs for jaundice management

• Phenobarbital: Induces the expression of UDP-glucoronosyltransferase

(UGT1A1, UDP-glucuronosyltransferase Family 1 Member A1)
THANK YOU
 UGT1A1
Bilirubin + UDP-glucuronate Bilirubin monoglucuronide + UDP
UGT1A1
Bilirubin monoglucuronide + UDP-glucuronate Bilirubin diglucuronide + UDP

↑unconjugated ↑hepatic conj.

↑Hemoglobin ↑Heme
bilirubin (UCB) to CB
↑ UBG in urine ↑CB reaching ↑CB reaching
↑Urobilinogen
No UCB in urine terminal ileum
terminal ileum
 CYTOSOL

2 ALA ALA PORPHOBILINOGEN

(PBG) dehydratase
HMB Synthase
Aporepres 3 PBG
PBG deaminase
sor UBG I synthase
HYDROXYMETHYLBILANE
(HMB)
(Linear)
UPG III Synthase

UROPORPHYRINOGEN III (UPG

(Asymmetric pyrrole)
UPG
decarboxylase
COPROPORPHYRINOGEN III
(CPG III)
 Alb

- Bilirubin - Albumin