Biochemistry

Sheet 7

University of Jordan
Faculty of Medicine

3rd Year

Done by : Yaqeen
Qudah

"If suffer we must, let's suffer on the heights … "

‫بسم الله الرحمن الرحيم‬

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In the previous lecture we discussed the synthesis of Hemoglobin.
.Today we'll continue with its degradation
Rbc's life span is 120 days. At the end of those days .. the
enzymatic efficiency, synthetic activity and ATP production in RBCs
will be diminished . They will look ' old & aged ' . Just then, the
Macrophages of the Reticular Endothelial System will recognize
them . Degradation will happen in the spleen and liver . The
hemoglobin will be separated into heme and globin. The globin is
degraded into amino acids that will be utilized in the body. Heme
.will eventually be Bilirubin. Today, we're concerned about that

(follow up with the slides '35' .. or Lippincott pg 280)

First of all, notice that this reaction is happening inside a
.Macrophage in the liver or spleen
Heme will be converted to Biliverdin by the microsomal (1
.enzyme: Heme Oxygenase

.O2, NADPH, H+ .. are needed for this reaction

NADP+ , Fe+3 , CO .. are liberated & iron will be reutilized
(explained in the
. (coming lecture

 The alpha methenyl bridge is broken , thus Carbon

Monoxide is released. So now we have a linear tetra
. pyrrole : Biliverdin which is a GREEN colored pigment

Biliverdin will be converted to the 'Red-Orange' Bilirubin by (2

another microsomal enzyme : Biliverdin Reductase. It
.utilizes NADPH as well

:NOTES
 The conversion of HEME to Biliverdin is the only reaction
in our body that produces CO endogenously . (recall that
we've learnt earlier that we have 1% of carboxyhemoglobin in
the blood even if we didn’t smoke any CO from the
(environment ..this percent is due to this reaction
 The conversion of Heme is clearly observed in injuries. If
you got a bruise from falling off the stairs for instance , this
bruise will be RED at first (hemoglobin) then it'll be
Green(Biliverdin) and with time it will be Yellow ( Bilirubin)
and finally it will end up being golden brown
(Hemosidern)..so basically you can tell the age of the bruise

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 .from those colors

Biliverdin is soluble , unlike Bilirubin - which needs

conjugation to glucuronic acid to become soluble. Some might
think: why not excrete Biliverdin as it is without going
through the trouble of making Bilirubin , then
!? conjugating it
Well , Bilirubin is a quite potent anti-oxidant compound. If
you were subjected to oxidizing substances , hemoglobin well
be converted to>> Biliverdin >>Bilirubin . So it is an
.endogenous anti-oxidant that supports the body
Uric acid is another endogenous anti-oxidant, but still
.Bilirubin is more potent

:EXTRA INFO
Some scientists believe that the increase of Bilirubin in
newborns is an attempt to fight the Reactive Oxygen Species
and bacterial infections, especially in premature babies.
Neonatal blood has low content of glutathione peroxidase,
thus Bilirubin will practice its anti-oxidant activity to protect
the baby. Research is being made to recognize to what extent
this information can be helpful without causing toxicity to the
.premature baby

After Bilirubin is made , it has to be delivered to the Liver(3

since conjugation happens there. But, since Bilirubin is
insoluble (hydrophobic), it can't wander alone in blood; we
need Albumin to do that. ( if the degradation of heme
.(happened in the liver, there is no need for this step

Upon reaching the liver, Bilirubin dissociates from Albumin (4

& undergoes conjugation to be excretable from the body. It will
get linked to two molecules of glucuronic acid, so it is called
.Bilirubin Diglucuronide

(Open slide '36' .. under the name Conjugation of Bilirubin)

 It is shown how glucuronic acid is formed, then attached to
Bilirubin making the conjugated Bilirubin. The rxn is catalyzed
by the enzyme : Bilirubin UDP-glucuronyltransferase.
((SO IMP

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 notice the two Propionates . Each is attached to one
Glucuronic acid by an ester bond. Also, notice that glucuronic
.acid in the reaction is UDP-glucuronate , then UDP is liberated

The conjugated Bilirubin travels against a concentration(5

gradient to reach the bile duct where it is secreted into the
bile. As u know , the bile is eventually emptied in the
.duodenum, then the intestine
 Notice that bile only contains conjugated Bilirubin. If it
.was unconjugated, it won't be able to solublize in the bile

The bacteria in the intestine will split the glucuronic acid by (6

an enzyme called B-glucuronidase .. leaving Bilirubin
.alone
. Only bacteria possess this enzyme to do this rxn

Bacterial flora metabolizes Bilirubin to Urobilinogen in the (7

large intestine, then the bulk of Urobilinogen is converted to
Stercobilinogen. Stercobilinogen is then excreted in feces as
the brown pigment "Stercobilin" upon air oxidation ,
.contributing to the feces color

Some Urobilinogen is reabsorbed from the intestine into the (9

blood .Then, it goes back to the Liver >> excreted in the bile
and so on.( enterohepatic cycle) From this Urobilinogen in the
Liver, a portion goes to the Kidney by blood where it's filtered
& excreted in urine .. then it is oxidized upon exposure to light
.to "yellow" Urobilin giving urine its color as well

 If there is an increase in Urobilinogen in urine then this

indicates hepato-cellular dysfunction (as if all the absorbed
Urobilinogen shifted to the kidney) or an increase in Bilirubin
.formation

 Stercobilinogen ,Urobilinogen are both colorless. They

become colored upon exposure to light (oxidation). That’s
why if there is a sample of urine or feces left outside, it
will be darker and darker in color with time because more
Urobilin and Stercobilin are formed

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Bilirubin in Practice
Bilirubin has a significant diagnostic value. It's one of the most
requested tests in clinical medicine. If its level rises in blood
, it will precipitate in : skin , sclera (white of the eye) and
other mucus membranes causing their yellowish color …
Jaundice or 'icterus'. Jaundice can be either acquired or
inherited .Usually the concentration of the total Bilirubin in
.blood is 0.1-1 mg\dl and more than that indicates a problem
Conjugated Bilirubin in blood is about 0.1-0.4 mg\dl
unConjugated Bilirubin in blood is about 0.2-0.7 mg\dl

SO BASICALLY, UNCONJUGATED BiLiRuBiN is HIGHER <<

>> IN OUR BLooD

 Pathological Jaundice can be categorized in three different

: groups according to its source
PRE-HEPATIC Jaundice :excessive hemolysis of RBCs. (1
Bilirubin is going to be way too much for the liver to handle
Note: the liver has the capacity to conjugate 300 mg of*
Bilirubin each day. It can exceed that and handle 3000 mg, 10
times more than it normally does. Even though, massive
.hemolysis is going to be much more than that
E.g : Hemolytic Anemia due to G6PD, Sickle Cell Disease ,
Malaria , Incompatible Blood Transfusion, Hemolytic Disease of
the New Born , Pyruvate Kinase Deficiency
LAB TESTs: associated with increased unconjugated
Bilirubin in blood. (Bilirubin didn't get the chance to reach the
(liver for conjugation

Hepatocellular Jaundice : liver damage , so hepatocytes (2

.can't fulfill their function
E.g: Cirrhosis , Hepatitis , Hepatocellular Carcinoma
LAB TESTs: associated with high concentration of
unconjugated Bilirubin in blood due to the impairment of
.conjugation
Also, high levels of conjugated Bilirubin .. how come? An
amount of Bilirubin manages to get conjugated, but since the
liver is damaged it can't excrete them in the bile to the feces,
so they will diffuse to the blood due to their congestion in the
liver and cause this elevation ( still the unconjugated levels are
(higher

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POST-HEPATIC Jaundice (Obstructive Jaundice) : (3
Bilirubin is conjugated properly but it is not allowed to come
out due to a block in the bile duct ! So it will diffuse to the
. blood
.E.g:Gall Stones, Cancer of the head of Pancreas…etc
.LAB TESTS: High amounts of conjugated Bilirubin

Neonatal Jaundice
Earlier , we talked about the enzyme : Bilirubin UDP-
glucuronyltransferase. Its activity in newborns isn't
sufficient until after two days or so of their birth. So, Bilirubin is
poorly excreted out of the body. Thus, infants develop
physiological jaundice; it is natural to happen to most
healthy infants .. but it is most observed in premature babies
!since their liver is immature too

(go to the last page of the slides, or Lippincott pg.283)

Here , we can see a curve showing the activity of UDPGT in full
.term and premature babies
The enzyme's activity in the full term baby rises until it
reaches the normal activity of an adult.The premature
baby's enzyme activity increases as well but still.. it remains
.low
 In the next curve it shows how Bilirubin levels are high due
to the low enzymatic activity . In full term babies normally
this increase reaches the top on the 1st week then it declines.
This isn’t dangerous since it doesn’t exceed 25 mg\dl . In
premature babies it may continue to rise and reach toxic
levels. If not treated , Bilirubin will reach the brain( the
blood-brain-barrier isn’t well developed yet) and cause toxic
. (encephalopathy (Kernicterus
So in order to prevent that, those babies will undergo
Phototherapy. They are treated with blue fluorescent
light that converts Bilirubin to a more polar soluble compound
that can be exerted out of the body without the need for the
conjugation. The baby is kept in an incubator until the enzyme
.activity is normal

 All the previous reasons cause Acquired jaundice .Now we

will move to the genetic defects that cause the Inherited
.jaundice

Gilbert Syndrome 2) Crigler-Najjar Syndrome. Type (1

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1 and 2
.Both are characterized by a reduced activity of UDPGT
Both lead to accumulation of unconjugated Bilirubin in
.blood
Gilbert Syndrome is mild whereas Crigler-Najjar is moderate to
.severe

Dubin –Johnson Syndrome 4) Rotor Syndrome (3

Both are due to defective secretion of the conjugated
Bilirubin
.No serious symptoms

 Conjugated Bilirubin is also called Direct Bilirubin. In fact,

this is the name commonly used in clinical medicine. So when
you shall order a Bilirubin test someday you got to say Direct
( : ! .. Bilirubin
The test is simple and easy. You measure the Total and
Direct Bilirubin in a reaction known as van den Bergh
.(reaction (a very important test
Diazotized sulfanilic acid (DSA) reacts with Bilirubin to form
RED azodipyrroles that are easily measured by a
colorimeter. Conjugated bilurbuin only acts rapidly with
DSA since it's soluble, so it is called DIRECT-reacting Bilirubin.
.This is done in Tube 1
Unconjugated Bilirubin reacts slowly for its less solubility
thus it is called INDIRECT-reacting Bilirubin. So we get Tube 2
and add DSA to Bilirubin but add Methanol as well .. so both
Conj and unConj Bilirubin will solublize together and form the
RED azodipyrroles. By now we have the Direct B from Tube 1
and Total B from tube 2 so we subtract them from each other
to get the indirect B ! T-D = iD
. Remember that iD Bilirubin is higher in our blood

,, Good Luck

Yaqeen
Qudah

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