Biochemistry

Biochemistry of Porphyrins and Bile Pigments

Novemeber
7 2015

Dr.Pacifico Eric E. Calderon

TRANS OUTLINE
I.
II.
III.
IV.

Heme Synthesis
Heme Catabolism
Fate of Bilirubin
Clinical Notes

I. Heme Synthesis
Biomedical Importance:
Heme: Synthesized from Porphyrins & Iron
Porphyrins Cyclic compounds formed by linkages
of 4 pyrrole rings through methyne bridges.
Heme: Produces Bile Pigments & Iron when degraded
Biosynthesis of Heme:
Occurs in the mitochondria & cytoplasm of RBC
precursors.
Begins with the condensation of Glycine & Succinyl
CoA.
Glycine is activated by Pyridoxal Phosphate.
st
1 Product of Glycine & Succinyl CoA is a-Amino-Bketoadipic acid this product is rapidly
decarboxylated to form a-Aminolevulainate (ALA).
Decarboxylation process of a-Amino-B-ketoapidic
acid is catalyzed by: Aminolevulinate Synthase
(ALAS)
ALA Dehydratase + ALA = Porphobilinogen
ALA Dehydratase is aka: Porphobilinogen Synthase.
ALA Dehydratase is a Zinc containing enzyme
sensitive to inhibition by Lead.
Porphobilinogen Deaminase (aka: hydroxymethylilane
synthase or Urophorphyrinogen 1 Synthase) +
porphobilinogen = Hydroxyl methylbilane or
Urophorphyrinogen 1
Urophorphyrinogen 1 is converted to Uriphorphyrinogen
III by Urophorphyrinogen III Synthase
Urophorphyrinogen III converted to Coporphyrinogen III
by acetate decarboxylation.
This acetate decarboxylation action will convert
acetate to Methyl substituents.
Urophorphyrinogen III conversion to
Coporphyrinogen III is catalyzed by
Urophorphyrinogen Decarboxylase.
Corpophyrinogen III enters the Mitochondria where it is
converted to Protophorphyrinogen III.
Coprophyrinogen Oxidase catalyzes the production
of Porphopyrinogen. (This enzyme only acts on
Coprophyrinogen III).
Protophorphyrinogen III converted to Protophyrin III.
An oxidation process catalyzed by
Protophophyrinogen Oxidase Enzyme.
FINAL STEP OF HEME SYNTHESIS:
Ferrous Iron is incorporated into the Protophyrin.
This reaction is catalyzed by: Ferrochelatase (aka:
Heme Synthase)- a mitochondrial enzyme.

Baquiran, Garcia, Imperial

** ALA Synthase is the key regulatory enzyme in hepatix

biosynthesis of heme.

II. HEME CATABOLISM

Heme is a ubiquitous molecule that is
Involved in many essential biological
processes:
oxygen transport
respiration
photosynthesis,
detoxification drugs (cytochrome ezymes)
signal transduction. (enzymes that use heme
proteins

Most of these reactions are carried out by redox

reactions of heme iron.

The heme biosynthetic and catabolic pathways

generate pro- and antioxidant compounds, and
consequently, influence cellular sensitivity to
oxidants.
Why regulate Heme?

Free heme is a potent pro-oxidant, leading to

the formation of reactive oxygen species that
can damage a variety of biological molecules

Heme precursors (-aminolevulinic acid,

porphyrins) generate reactive oxygen species
(ROS) from autoxidation and photochemical
reactions, respectively

Heme can associate with phospholipid

membranes, altering bilayer structure and
thus, causing cell disruption
For this reason, the cells strictly regulate
heme homeostasis

To regulate Heme, you need to break it.

Heme Catabolism

As the heme is not recycled, most cells

containing heme proteins have the
microsomal mixed function oxygenase, heme
oxygenase, which enzymatically degrades
heme to biliverdin, carbon monoxide, and
iron

The catabolism of heme from all heme proteins is

carried out in the microsomal fraction of cells by
heme oxygenase
Heme Catabolism

Heme oxygenase- is operational in various tissues such as

spleen, liver , kidney and macrophages.

Plays a role in heme catabolism

1 of 8

 Iron/ Ferritin (Fe)

Degrades heme to :

biliverdin
carbon monoxide
iron

The iron of the heme that reaches heme oxygenase

has been oxidized to its ferric form (hemin)

Biliverdin

Has biliverdin reductase that reduces the central

methylene bridge of biliverdin to a methyl group,
producing the yellow pigment Bilirubin.
Biliverdin + NADPH +H

Bilirubin + NADP

Effects of hemooxygenase activity

In mammals, CO, a gaseous messenger, has
anti- inflammatory and anti-apoptotic effects
Biliverdin and its reduced product bilirubin
may function as important antioxidants
Heme oxygenase
Humans harbor two distinct heme oxygenase
genes identified as HMOX1 and HMOX2
The heme oxygenase enzyme encoded by the:
HMOX1 gene- is the rate-limiting enzyme of
heme catabolism
Both HMOX1 and HMOX2 genes are
constitutively expressed, however, the activity of
Fig. 1. Heme Catabolism Products
Heme Catabolism- the only endogenous/ natural biological
reaction in the body that produces Carbon monoxide (CO)

3-

3+

The conversion of of one mole of heme Fe to

3+
bilivedin, CO, and Fe consumes three molecules
of O2 plus seven e provided by NADH and NADPH
cytochrome P450 reductase.

Fe Heme + 3 O2 + 7 e

bliverdin + CO+ Fe

3-

Why breakdown heme. then it has to be processed again?

Heme breakdown can alter physiological and
biochemical reactions of the cells.
Because each of the products of the heme
breakdown has its own importance. That why, we
regulated its breakdown as well as its synthesis.
Products of Heme Catabolism
Carbon monoxide (CO)
Because CO is the product of incomplete
combustion of hydrocarbons.
If so, how do release Carbon monoxide?

It is released through he breath

Clinical and phyiological significance of CO:

CO in the breath can be a marker for heme breakdown.
2-

Has high affinity for heme Fe

The CO that produced does not severley inhibit

heme oxygenase

Biochemistry: Baquiran, Garcia, Imperial

Heme Catabolism
the HMOX1 encoded enzyme is inducible by
heme, heavy metals, and conditions of stress
such as hypoxia

Meaning both HMOX1 and HMOX2 genes are

expressed continuously.

HMOX1 encoded enzyme is inducible expression

only occurs when need arises.
Factors for Heme Degration:
Heme
Heavy metals
Hypoxia
Over production of these factors can cause heme
degradation.
Heme catabolism

The red cell with the largest pool of heme

protein, hemoglobin, contains no heme
oxygenase
Enzymatic degradation of the red cell heme
occurs only after the senescent red cells are
removed by the reticuloendothelial system.
RBC is devoid of heme oxygenase because:
It carries its own oxidation

8 of 8

Gene is not expressed continuously because RBC

doesnt have genes and it is devoid of nucleus.
RBC Degradation:

Globin- degraded to Amino Acids

Iron- enters the iron pool and will be reused

Iron-free porphrin- degraded into the

reticuloendothelial cells of liver, spleen and bone
marrow.
Heme - breaks down into:
Bile
Iron
CO

Heme breakdown into Bilirubin

Within hepatic and splenic macrophages,
heme is first converted to bilirubin in a twostep
enzymatic process which employs
biliverdin as an intermediate (enzyme:
biliverdin reductase)

Fig.4. Oxidative metabolism of heme by HO and

biliverdin reductase, giving rise to CO, iron, biliverdin,
and bilirubin
Fig. 2. Degradition of RBC (Heme+globin)
Heme catabolism
The largest repository of heme in the human
body is in RBCs which have a life span of
about 120 days
There is 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 new heme synthesis

Heme is not recycled

Fe is recycled, for new heme synthesis to occur
So, Heme and Fe should part ways. L
Sources of Heme metabolites

Roughly 80% of heme destined for

degradation and excretion comes from
senescent erythrocytes
20% comes from premature erythrocytes in
the bone marrow which are destroyed prior
to release into the circulation and a minor
component is derived from other cell types

Biliverdin is insoluble in water so for it to be excreted, it has

to be converted to bilirubin through biliverdin reductase .
This results to the oxidation and opening of heme
ring.
Macrophage will excrete resulted bilirubin into the
plasma.
Heme breakdown into bilirubin
These steps result in oxidation and opening
of the heme ring
Macrophages then excrete the resultant
bilirubin into the plasma as unconjugated
bilirubin
Bilirubin redcutase
There are two biliverdin reductase genes in
humans identified as BLVRA and BLVRB
The enzyme encoded by the BLVRA gene is
pricipally responsible for the catabolism of
biliverdin
The enzyme encoded by the BLVRB gene
catalyzes the reduction of not only biliverdin but
also a variety of flavins, such as riboflavin, FAD
or FMN, and methemoglobin
aka NADPHdependent
flavin reductase

Sources:
Senescent RBC
Premature RBC Bone Marrow

Biochemistry: Baquiran, Garcia, Imperial

8 of 8

biliverdin reductase
genes

BLVRA

1.

BLVRB
NADPH dependent flavin
redcutase
2.

Catabolism of:
-Biliverdin

Catabolism of :
-Bilive-Biliverdin rdin
-Flavins
Eg. Riboflavin
FAD FMN
-MethHG

Uptake of bilirubin by liver parenchymal cells

Bilirubin is removed in the albumin, then it will be
taken up to at the sinusoidal surface of the
hepatocytes by Facilitated Transport System
(malaking molecule ang bilirubin), once inside it will
bind to cytosol proteins(glutathione S-transferase or
know as ligandin) to prevent reentering the blood
stream.
Conjugation of bilirubin with glucoronate in the
endoplasmic reticulum
Bilirubin is non polar, to make it more polar it will be
converted to glucuronic acid by conjugation.
Within the hepatocyte, the enzyme UGT1A1 (UDPglucuronyltransferase) covalently attaches one or
two molecules of glucuronic acid to bilirubin, either
bilirubin mono- or di-glucuronide is generated to
form acylglucoronate

Fig. 3. Bilirubin reductase genes

Fate of bilirubin
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)
Peripherally arising bilirubin is transported to
the liver in association with albumin, where
the remaining catabolic reactions take

Bilirubin is sparingly water souble (between being

soluble and insoluble) but when it is bounded by
albumin, it will readily transported to the liver.

Albumin both have a high affinity site and low

affinity site for bilirubin.

Since most of the bilirubin is loosely attached to

albumin, it will detached and diffuse into the tissues.

Liver- where HEPATIC CATABOLISM OF

BILIRUBIN takes place.

Fig. 4. Conjugation of Bilirubin

3 Stages of Bilirubin Metabolism:

- Uptake by the liver
- Conjugation with glucorinic acid,
- Secretion in the bile

Fig. 5. UGT1A1 in hepatocytes

Glucuronyltransferase
-
Enzyme for conjugation, uses UDP-glucuronic
acid as the glucuronosyl donor and referred to
as bilirubin-UGT.
Unconjugated bilirubin
-
Not yet process in the liver
Conjugated bilirubin
-
direct reacting bilirubin

Biochemistry: Baquiran, Garcia, Imperial

8 of 8

-
-
-

already processed in the liver

glucuronic acid-attached species of bilirubin
increased water solubility of the tetrapyrrole
facilitates its excretion with the remainder of the
bile as the bile pigment

Glucuronide
-
Increases polarity and solubility
Glucuronidation
-
Addition of glucuronide
-
Most important of conjugation reaction
-
Reaction does not proceed spontaneously
-
Requires the activated form of glucuronic acid
(glucuronic acid uridine diphosphate)
Glucuronidation and Grey Baby Syndrome

When Chloramphenicol is given without regard for

infants
diminished
capacities
for
hepatic
detoxification and renal elimination hypothermia,
vomiting, acidosis, cyanosis, grey discoloration
occur
-
Very versatile in baby
-
Immaturity of the liver

3.

When the senescent RBC goes into the liver or

spleen, the Hemoglobin will be degraded into Heme
and Globin.

Globin, since its a protein, will be broken down into

Amino acids recycled, for another synthesis of
Erythropoeisis.

Heme, will be broken down into Carbon Monoxide,

Fe and Biliverdin by the enzyme Heme
Oxygenase

Biliverdin, will be converted into Bilirubin by the

enzyme Bilirubin reductase

When bilirubin becomes conjugated, using

glucorinic acid, from being sparingly water soluble
(between being soluble and insoluble) it will now
become water-soluble and secreted in the bile.

Immature glucuronosyl transferase activity

-
Toxic to the cells
-
Immaturity of the enzyme (not yet fully
expressed)
Secretion of conjugated bilirubin into the bile
-
Occurs by active transport
-
Protein involved is MRP-2 (Multidrug
resistance like protein 2) or MOIT
(Multispecific organic Ion Transporter)
located in the plasma membrane of the
bile canalicular membrane.
As the conjugated bilirubin reaches the terminal
ileum and large intestine, the glucoronides are
removed by -glucuronidases (bacterial
enzymes). The pigment is then reduced to
Urobilinogen
(a
group
of
colorless
tetrapyrroles), urobilinogens is reabsorbed in
the terminal ileum and large intestine through
the liver via enterohepatic urobilinogen cycle.

Biochemistry: Baquiran, Garcia, Imperial

8 of 8

Fig. 5. General View of Bilirubin Metabolism

Uptake by
the Liver

Conjugation
with
Glucorinic
acid

Secretion of
Bilirubin
into the Bile

Bile

RBC will circulate the body for 120 days, when these RBC reached their life span, Old
RBC it will be
phagocytosed by macrophages in spleen and bonemarrow .
RBC now will release to its Hemoglobin molecule
Hemoglobin degraged into globin and heme
Globin is a protein and it will breakdown into AMINO ACIDS and be reuse for
Erythropoiesis
HEME will be broken down to unconjugated bilirubin and Iron
Iron will re-enter the circulation and re- used for Erythropoiesis like the Globin
Unconjugated bilirubin is not recycled and thus needs to be excreted because it is toxic. It
has a yellowish orange color
For the Unconjugated bilirubin to be Lipid soluble when it is in the blood, it requires
protein for it to be carried around. This protein is called Albumin
Albumin will carry the unconjugated bilirubin into the liver for further metabolism
Liver has own macrophage Kupffer cells which break donwn other old/senescent RBC
Unconjugated Bilirubin is lipid soluble
LIVER is the site for conjugation (lipid soluble to water soluble)
Unconjugated Bilirubin will form into conjugated by glucoronic acid

Conjugated Bilirubin will be water soluble for it to be excreted by bile contains which
contains Conjugated Bilirubin and Bile salt
Conjugated Bilirubin and Bile salt will be excreted into small intestine
Conjugated Bilirubin will go to the small intestine through the common bile duct
Conjugated Bilirubin travels in large intestine
Ileum where Conjugated Bilirubin is converted by intestinal bacteria to become
Urobilinogen by removing glucorinic acid
Urobilinogen lipid soluble 10-15 percent bound to albumin and reabsorbed
Stercobilin brown colored pigment
Conjugated Bilirubin excreted

NOTE: In general view or process only. Para lang

madaling intindihin J

Biochemistry: Baquiran, Garcia, Imperial

8 of 8

-
-
-

Due to excess bilirubin production or

liver failure
Obstruction of the excretory ducts of
the liver
When bilirubin reaches 2-2.5 mg/dL in
blood, it diffuses into tissues causing
Jaundice

Types of Hyperbilirubinemia
1. Retention hyperbilirubinemia due to over
production of bilirubin (unconjugated bilirubin)
2. Regurgitation hyperbilirubinemia due to reflux
into the bloodstream secondary to biliary
obstruction (conjugated bilirubin)

Fig.6.Heme Degradation
Clinical Notes
Two types of Clinical problems associated with heme
metabolism:
1. Porphyrias
-
Disorders that arise from defects in the
enzymes of heme biosynthesis
-
Both inherited and acquired
-
Classified as either erythroid or hepatic,
depending upon the principal site
expression of the enzyme defect
-
Cause elevation in serum and urine
Type

Deficient enzyme

Gene

ALADehydratase
Acute
Intermittent
Porphyria

ALA-Dehydratase

ALAD

Gene
locus
9q34

Hydroxymethylbila
ne
synthase
(Porphobilinogen
deaminase
Uroporphyrinogen
III synthase

HMBS

11q23

UROS

10q25-26

Uroporphyrinogen
decarboxylase

UROD

1p34

Uroporphyrinogen
decarboxylase

UROD

3q12

Hereditary
Coproporphia
(HCP)
Variegata
Porphyria (VP)

Coproporhyrinogen
oxidase

CPOX

1q22

Protoporphyrinoge
n oxidase

PPOX

18q21

Erythropietic
(EPP)
X-linked
Protoporphyria
(XLP)

Ferrochelatase

FECH

-Aminolevulinate
synthase 2

ALAS2

Congenital
Erythropoietic
Porphyria
(CEP)
Porphyria
Cutanea Tarda
(PCT), familial
form
Hepatoerythrop
oietic Porphyria
(HEP)

2.

Kernicterus
-
Because
of
its
hydrophobicity,
only
unconjugated bilirubin can cross the bloodbrain barrier into the CNS
-
Encephalopathy due to hyperbilirubinemia
(kernicterus) can occur in only with
unconjugated bilirubin, as found in retention
hyperbilirubinemia
Jaundice
-
Conjugated bilirubin can appear in urine

Choluric jaundice
-
Occurs only in regurgitation hyperbilirubinemia

Acholuric jaundice
-
Occurs only in the presence of an excess of
unconjugated bilirubin

Unconjugated Hyperbilirubinemia

Xp11.21

Bilirubinemias
-
Inherited disorders of bilirubin metabolism
Hyperbilirubunemia and Jaundice
-
Occurs when bilirubin in the blood exceeds
1mg/dL (17.1 mol)

Biochemistry: Baquiran, Garcia, Imperial

Neonatal Physiological Jaundice

-
Unconjugated hyperbilirubinemia of neonatal
physiological jaundice results from accelerated
hemolysis and an immature hepatic system for
the uptake, conjugation, and secretion of
bilirubin.
-
Decreased bilirubin-glucosyltransferase activity
and UDP-glucuronate synthesis
-
Plasma concentration of unconjugated bilirubin
exceeds that which can be tightly bound by
albumin (20-25 mg/dL) can penetrate the blood
brain barrier.
Crigler-Najjar Syndrome Type I
-
Congenital nonhemolytic jaundice
-
Autosomal recessive
-
Due to mutations in the gene encoding
bilirubin-UGT activity in hepatic tissues
-
Often fatal within the first 15 months
Crigler-Najjr Syndrome Type II
-
Like type I but more benign
Gilbert Syndrome
-
Also has mutations in genes encoding bilirubinUGT but retains 30% of enzyme activity
Toxic Hyperbilirubinemia
-
Caused by chloroform, arsphenamines, carbon
tetrachloride, acetaminophen, hepatitis virus,
cirrhosis, or Amanita mushroom poisoning.
-
Can result from toxin-induced liver dysfunction
-
Hepatic parenchymal cell damage which
impairs bilirubin conjugation.
Hemolytic Anemia

8 of 8

Conjugated Hyperbilirubinemia

Biliary obstruction
-
Regurgitation into hepatic veins and lymphatics
-
Conjugated bile in blood urine
Dubin-Johnson Syndrome
-
Benign autosomal recessive
-
Mutation in the gene encoding MRP-2 for thee
secretion of conjugated bilirubin into bile
Rotor Syndrome
-
Chronic conjugated hyperbilirubinemia and
normal liver
References

https://www.rarediseasesnetwork.org/porphyrias/patients/lear
nmore/index.htm
th
Harpers Illustrated Biochemistry 30 Edition
Dr. Pacifico Eric E. Calderons Lecture

Biochemistry: Baquiran, Garcia, Imperial

8 of 8