Khadija Balubaid

KAU-Faculty of Science- Biochemistry department

Clinical biochemistry lab (BIOC 416)
2013
Liver Function profile (LFT)
Excretion functions
(Bilirubin measurement)
Clinical Lab tests
Liver Function Test (LFT) profile
Synthetic function Execratory function Integrity of liver cells
Bilirubin, ALP
Proteins
TP, Alb, A/G ratio
Liver enzymes
AST, ALT, GGT, ALP
Definition of bilirubin
Bilirubin is the water insoluble breakdown product of normal
heme catabolism
Its a yellow pigment present in bile ( a fluid made by the liver) ,
urine and feces .
Heme is found in hemoglobin, a principal component of RBCs
[Heme: iron + organic compound porphyrin].
Heme source in body:
80% from hemoglobin
20% other hemo-protein: cytochrome, myoglobin)

Heme and bilirubin
Heme four pyrrols rings connected together to form
(porphyrin).
Bilirubin consists of open chain of four pyrrols-like rings


Hemoglobin degrading and bilirubin
formation

Spleen
Plasma
Protein and a.a
pool
Iron pool
Liver
globin
iron
Heme
Hemoglobin
Bilirubin Binds with
albumin
Conjugation
process
Bilirubin Metabolism
1. Unconjugation process :
RBCs are phagocytized in the spleen. Hemoglobin is
catabolized into amino acids, iron and heme.
Heme ring is broken open and converted to unconjugated (
indirect ) bilirubin.
This unconjugated bilirubin is not soluble in water, due to
intramolecular hydrogen bonding. It is then bound to albumin
and sent to the liver.
2. Congugation process:

In liver: Bilirubin is conjugated with Glucouronic acid to
produce bilirubin diglucuronides, which is water soluble and
readily transported to bile. and thus out into the small intestine.


Blilirubin + Glucouronic acid bilirubin diglucuronides




Then conjugated bilirubin is excreted in bile through bile duct
to help in food digestion (mainly fat).





UDP-glucuronyl
transferase


"water soluble"
"Conjugated BIL"



"water insoluble"

Bile
The excess amount transferred to intestine to be excreted in urine
and stool.
However 95% of the secreted bile is reabsorbed by the small
intestine. This bile is then resecreted by the liver into the small
intestine. This process is known as enterohepatic circulation
About half of the conjugated bilirubin remaining in the large
intestine (about 5% of what was originally secreted) is metabolised
by colonic bacteria to form urobilinogen , which may be further
oxidized to urobilin and stercobilin . Urobilin, stercobilin and their
degradation products give feces its brown color.
[


Elevated levels of bilirubin in blood and urine indicate certain
diseases.

Direct bilirubin: is conjugated (water soluble bilirubin) in
aqueous solution it reacts rapidly with reagent (direct reacting).

Indirect bilirubin: is unconjugated (water insoluble bilirubin)
because it is less soluble in it reacts more slowly with reagent
(reaction carried out in methanol).
- in this case both conjugated and unconjugated bilirubin are
measured given total bilirubin. Unconjugated will calculated by
subtracting direct from total and so called indirect.

Total bilirubin = D+ ID

Knowing the level of each type of bilirubin has diagnostic
important.

It is a medical term describes the elevation of bilirubin in
blood result in yellow color of skin and sclera.
Other symptoms include nausea, vomiting, dark-colored urine
andTypes of Jaundice:
fatigue.
according to the cause of jaundice
it is classified to three main types:

Pre-hepatic jaundice
Hepatic jaundice
Post-hepatic (most common type)
Post-hepatic jaundice Hepatic jaundice Pre-hepatic jaundice

Due to obstruction of bile
duct which prevents
passage of bilirubin into
intestine.

D.Bil will back to liver
and then to circulation
elevating its level in blood
and urine.
Occur in:
Biliary stricture
Cancer of the pancreas or
gallbladder
Gallstones




Due to liver cell damage
(cancer, cirrhosis or hepatitis)
Conjugation of bilirubin
decreased (ID.Bil. ).

Blilirubin that is conjugated is
not efficiently secreted into
bile but leaks to blood (D.Bil.
)
Occur in :
Cirrhosis (scarring of the liver)
Hepatitis
Gilbert's disease


Due to increase in RBCs
breakdown due to
hemolytic anemia.

The rate of RBCs lysis
and bilirubin production
more than ability of liver
to convert it to the
conjugated form
Occur in:
Erythroblastosis fetalis
Hemolytic anemia
Transfusion reaction
Causes
D.Bil (High) D.Bil, ID.Bil, T.Bil all (High) ID.Bil > D.Bil Type of Bil.
ALP ( High) ALT, AST (High)
K
+
( High)
Hematology:
CBC (low Hb)
Conformational
test
haemolytic jaundice hepato-cellular jaundice obstructive jaundice

High bilirubin levels is common
in newborns age (1-3 days).
After birth the newborns breaking down the excess RBCs they are born
with and, because the newborns liver is not fully mature, (unable to
process the extra bilirubin) leads to elevate its level in blood and other
body tissues.
This situation usually resolves itself within a few days
SO, WHAT TYPE OF JUNDUCE IS THIS ???
Note:Your child's doctor must consider the following when deciding
whether your baby's bilirubin levels are too high:
How fast the level has been rising
Whether the baby was born early
How old the baby is



New born jaundice treatment:

Usually newborn is treated by phototherapy which
breakdown bilirubin (IDD) and convert it to the photo
isomer form which is more soluble.


Bilirubin Toxicity :

Very high bilirubin is danger and toxic it may cause
brain damage effect on muscles, eyes
and Leading to death


Bilirubin measurement
How to Prepare for the Test
You should not eat or drink for at least 4 hours before
the test.
Your health care provider may instruct you to stop
taking drugs that affect the test.
Many drugs may change the bilirubin levels in your
blood. Make sure your doctor knows which
medications you are taking.
Tell your doctor if you have allergy

Why the Test is Performed
Large amounts of bilirubin in the blood can lead to jaundice.
Jaundice is a yellow color in the skin, mucus membranes, or
eyes.
Jaundice is the most common reason to check bilirubin levels.
Most newborns have some jaundice. The doctor or nurse will
often check the newborn's bilirubin level. See: Newborn
jaundice
The test may also be done in older infants, children, and adults
who develop jaundice.
A bilirubin test will also be done if your doctor thinks you may
have liver or gallbladder problems

Procedure
Measuring serum bilirubin level
Principle:




Kit components
Sulfanalic acid reagent
Sodium nitrate reagent
Methanol reagent
Bilirubinequavalent standard (5mg/dl T.bil; 2.5 mg/dl D.bil)


Sulphanalic acid + NaNO3 diazotized sulphanalic acid (DSA)

DSA + Bilirubin D Azobilirubin purple

Bilirubin ID+ DSA + accelerator Total bil.


(methanol )
Procedure:
Test Test blank Equivalent
Standard
1.4 ml 1.4 ml 1.4ml Sulfanilic
acid
25ml - 25ml NaNO
3
- 25ml dis. H
2
O
Mix, stand for 1 min
100 l
(serum)
100 l
(serum)
100 l
(Standard)
Sample
- Read the Abs after 1 min at 540nm, (Blank dis. H
2
O)
Use this to calculate D.bil
1.5 ml 1.5 ml 1.5 ml Methanol
- Mix by inversion, stand 5 min or more
- Read Abs. at 540nm (Blank dis. H
2
O)
Use this to calculate T.bil
Pour Bilirubinequavelant standard in clean cuvette read Abs. at
540nm, (blank dis. H
2
O)

Calculations
Conc. of Bilirubin equavelant is 5mg/dl for T.bil, and 2.5 mg/dl for D.bil
D.Bil:

Abs (test) - Abs (test blank) X 2.5 mg/dl

Abs of bilirubin equivalent
T.Bil:
Abs (test) - Abs (test blank) X 5 mg/dl


To convert to mmol/L multiply by 17.1
Abs of Bilirubin equivalent
Normal Results
It is normal to have some bilirubin in your blood.
Normal levels are:
Direct (also called conjugated) bilirubin: 0 to 0.5 mg/dL
Total bilirubin: 0.3 to 1.9 mg/dL
Note: mg/dL = milligrams per deciliter
Normal value ranges may vary slightly among different
laboratories.

Case Study
This 48-year-old man had complained of abdominal pain and intermittent fever for 3
months. The pain was usually felt in the right upper quadrant. His appetite was not
good and lost his body weight apparently. He once suffered from hepatitis ten years
ago. Physical examination revealed the patient who appeared chronically ill with
icteric sclera. The liver was enlarged to 2 cm below the costal margin with
tenderness, and the spleen was enlarged to 3 cm below costal margin
Laboratory data: Hb 9 mg/dL, WBC 8.0109/L, PC 90109/L, ALT 1020U/L, AST
800U/L,ALP 255U/L, TP 55g/L, A 25g/L, Y 40%, TBI 55umol/L, DBI 38umol/L,
URO (++), UBI (+)

Question:
What is your diagnosis for this patient?
Which kind of jaundice this patient has?
How to evaluate this patients liver function?