1. Lab (3): Liver Function profile (LFT)
T.A Nouf Alshareef
KAU-Faculty of Science- Biochemistry department
Clinical biochemistry lab (Bioc 416) 2012

2. Liver Function profile (LFT)
Liver enzymes
Execratory function
Synthetic function
Clinical Lab tests
Bilirubin, ALP
Proteins
TP, Alb, A/G ratio
AST, ALT, GGT, ALP

3. What is Bilirubin ???
is the product of heme degradation (80% hemoglobin, 20% other hemo-protein as cytochrome, myoglobin).
Bilirubin is transferred to liver to undergo further metabolic process called (conjugation) to make it more water soluble.
Then conjugated bilirubin is excreted in bile to help in food digestion and the excess amount is excreted in urine and stool.
Elevated levels of bilirubin in blood and urine indicate certain diseases.

4. Structure
Bilirubin consists of four open chain pyrrols, unlike heme which consists of four rings pyrrols called (porphyrin).

5. Bilirubin metabolism:
Bilirubin is water in-soluble and is carried in plasma bound to albumin (as carrier). When it reaches to the liver, bilirubin is taken by specific carrier meachanism.
Pathway of Bilirubin:
Liver ……bile…….intestine ……re abosrbe in blood ( back to liver or go to kidney) or go to large intestine

6. UDP-glucuronyl transferase
In liver: Bilirubin is conjugated with Glucouronic acid to produce bilirubin diglucuronides, which is water soluble and readily transported to bile.
Blilirubin + Glucouronic acid bilirubin diglucuronides
Further metabolic processes are occurred in intestine and kidney (figure)
UDP-glucuronyl transferase
"water soluble" "Conjugated BIL"
"water in soluble"
Bile

8. Types of bilirubin in serum:
Indirect bilirubin: is un conjugated or water insoluble, it is called indirect because it reacts slowly, so it indicates indirect reaching to reagent.
Direct bilirubin: is conjugated or water soluble it is called direct because it reacts faster, so it indicates direct reaching to reagent.
Total bilirubin = D+ ID
Knowing the level of each type of bilirubin has diagnostic important.

9. Jaundice: Pre-hepatic jaundice
is a medical term describes the elevation of bilirubin in blood result in yellow color of skin and sclera.
Types of Jaundice:
according to the cause of jaundice
it is classified to three main types:
Pre-hepatic jaundice
Hepatic jaundice
Post-hepatic (most common type)

10. Post-hepatic jaundice
Pre-hepatic jaundice
Due to liver cell damage due to cancer or cirrhosis
Due to obstruction of hepato-biliary duct.
“obstructive jaundice”
D.Bil is formed in liver but can’t pass to bile, so it accumulates in liver and leaks to blood “instead of bile”
Increase in RBCs breakdown due to hemolytic anemia.
Rate of RBCs degradation and Hb production more than ability of liver to convert it to conjugated form
Cause
High ID.Bil, T.Bil
D.Bil high
ID.Bil > D.Bil
Type of Bil
AST, ALT
ALP (high)
K+ (high) Hematology: CBC, low Hb
Conformation test

11. Physiologic Jaundice of the newborn:
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 newborn’s liver is not fully mature, it is 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 ???

12. Usually newborn is treated by phototherapy which breakdown bilirubin (ID<<<<D) and convert it to the photo isomer form which is more soluble.
Very high bilirubin is danger and toxic it may cause brain damage and effect on muscles, eyes and leading to death.

13. Experiment: Measuring serum bilirubin level
Principle:
Sulphanalic acid + NaNO diazotized sulphanalic acid (DSA)
DSA + Bilirubin “D” Azobilirubin “purple”
Bilirubin + DSA + accelerator Total bil.
(methanol )

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

15. Use this to calculate D.bil Use this to calculate T.bil
Procedure:
Test
Test blank
1.4 ml
Sulfanilic acid
25ml -
NaNO3
dis. H2O
Mix, stand for 1 min
100 ml
Sample
- Read the Abs after 1 min at 540nm, (Blank dis. H2O)
Use this to calculate D.bil
1.5 ml
Methanol
- Mix by inversion, stand 5 min or more
- Read Abs. at 540nm (Blank dis. H2O)
Use this to calculate T.bil
- Pour Bilirubinequavelant standard in clean cuvette read Abs. at 540nm Read the Abs after 1 min at 540nm, (blank dis. H2O)

16. Calculations Abs (test) - Abs (test blank) X 2.5 mg/dl
Abs of Bilirubinequavelant is 5mg/dl T.bil mg/dl D.bil
D.Bil:
Abs (test) - Abs (test blank) X 2.5 mg/dl
T.Bil:
Abs (test) - Abs (test blank) X 5 mg/dl
To convert to mmol/L multiply by 17.1
Abs of Bilirubinequavelant
Abs of Bilirubinequavelant

17. D.Bil: 0.5 mg/dl or 8.6 mmol/L T.Bil: 1 mg/dl or 17.1 mmol/L
Normal range
D.Bil: 0.5 mg/dl or 8.6 mmol/L T.Bil: 1 mg/dl or 17.1 mmol/L