Estimation of serum bilirubin (total and direct) Prepared by : Mona alharbi

Objective: To measured serum total bilirubin and direct then calculate indirect bilirubin ,to screen for or to monitor liver or gall bladder dysfunction Introduction: Bilirubin is a breakdown product of hemoglobin . Bilirubin metabolism is hem catabolism (It is begins on the death of the RBC) . The RBC contain hemoglobin ,which is broken down to heme and globin . Heme is converted to biliverden the it will be reduction to bilirubin (unconjugated or indirect bilirubin) *the unconjugated bilirubin is water insoluble so it need to carrier. Then the bilirubin carried by albumin in the blood to liver.

The fate of unconjugated bilirubin in liver: Most of the bilirubin is chemically attached to a glucuronide before it is excreated in the bile . This conjugated bilirubin is called direct bilirubin (water soluble). **The total bilirubin is equal direct bilirubin plus indirect bilirubin Conjecated bilirubin is excreated into blie by liver and stored in the gall bladder or transferred direct to small intestines. Bilirubin is further broken down by bacteria in intestine to urobilins ,,small amount of these compounds are reabsorbed and appear in urin , where they are referred to as urobilinogen.

The healthy liver conjugated glucuronic acid to the bilirubin by means of bilirubin-UDP glucuronsyl transferase

Normal range of direct bilirubin : 0 to 0.3 mg/dl Normal range of total bilirubin : 0.3 to 1.9 mg/dl Above 2mg/dl in the blood we get the condition known as Jaundice Jaundice: Is condition occurs when bilirubin accumulate in the blood at level,greater than approximately 2.5 mg/dl , lead to discoloration of skin and sclera of eye

The causes of jundice may be classified as (Types of jundice) 1- Pre-hepatide jundice 2- Hepatide jundice (classified into 3 types : non infection hepatitis jundice , infection Hepatitis jundice , Neonatal jundice 3- Post-hepatide jundice the liver function test is help in diagnosis to determine the type of jundice.. Explanation of each type; Pre-hepatide jundice : e.g. haemolytic disease .. The production of uncojugated bilirubin may exceed the conjugated capacity of the liver ,. So, the serum levels of indirect (and of total) bilirubin will be increased . But the direct is remain in upper normal range or just a little elevated. The liver function test will usually give normal result.

(2) Hepatic jundice: Non infection hepatite jundice e.g. Cirrhosis of liver Destruction of liver cells lead to a reduced conjugating capacity with increased serum level of indirect (and total ) bilirubin . [the direct bilirubin is low while indirect bilirubin ( and total) is abnormally high , leading to excreated in urin] *the liver function test give positive result , because the AST ,ALT and ALP is high in serum.

(B) Infection Hepatitis: The conjugative capacity of the liver is normal , but the ability there is inability to transport the direct bilirubin from liver to biliary system and it will be back to blood. So, the level of direct (total)bilirubin is abnormal high while the level of indirect bilirubin is normal. :(c) Neonatal jundice The liver function is normal but the conjugating enzyme in liver are often absent at birth . For that the level of indirect and total bilirubin is high while the level of direct bilirubin is low

(3) post-Hepatic jundice: e.g. Cholecytitis Here; the bile duct is blocked , the indirect bilirubin in serum is normal but the direct (and total) bilirubin is high because it will be back to blood . The liver function tests are normal :Principle **we will estimate the direct and total bilirubin *The direct bilirubin is water soluble conjugated bilirubin react easily with reagent such as diazotizes sulphanilic acid and is thus known as direct .. While the water insoluble unconjugated bilirubin requires a solubilizing reagent such as caffeine , in order to react with the diazotized . Direct bilirubin is estimated in the absent of solubilsing reagent and then further bilirubin is estimated in the presence of solubilizing agent will give the total bilirubin level (direct +indirect) Then the indirect bilurubin estimating by the difference (between direct and total bilirubin )

Mix, let stand for 30 min. at 20ºC-25ºC Method: (1)Determination the total bilirubin: Pipette into two dry clean test tubes Sample Blank 0.20 ml (200µl) Solution 1 1 drop (50µl) -- Solution 2 1 ml Solution 3 0.20 ml (200µl) Serum (sample) Mix, let stand for 30 min. at 20ºC-25ºC Solution 4 Mix , let stand for 15 min. at 20ºC-25ºC .. Then read the Ab of sample aginist sample blank at 578 nm

(2)Determination the direct bilirubin: Pipette into two dry clean test tubes Sample Blank 0.20 ml (200µl) Solution 1 1 drop (50µl) -- Solution 2 2 ml NaCL solution 0.9% 0.20 ml (200µl) Serum (sample) Mix , let stand for 5 min. at 20ºC-25ºC .. Then read the Ab of sample aginist sample blank at 546 nm

Calculation : :(1)Determine the total bilirubin Ab= Ab at 578 X 10.8 =………mg/dl (2)Determinate the direct bilirubin: Ab at 546 X 14.4 = ……… mg/dl (3) Determine the indirect bilirubin: Indirect = Total – Direct =……….mg/dl