Estimation of Bilirubin in serum – liver function tests

Estimation of Bilirubin in serum
Introduction

• Bilirubin is an orange – yellow pigment , a waste product primarily produced by the normal breakdown of heme
• This form of Bilirubin is called unconjugated (indirect) bilirubin ,not soluble form ,to convert it to soluble form and eliminate it ,The unconjugated bilirubin is carried by Albumin to the liver , where it converted to conjugated (direct) form , soluble , through UDP – glucuronyl transferase with glucuronic acid
• Because the bilirubin is chemically different after it goes through the conjugation in the liver , lab tests can be differentiate between the unconjugated(indirect) or conjugated (direct) bilirubin
• Conjugate bilirubin is water soluble form and reacts directly when dyes are added to the blood sample so , it called also direct bilirubin
• Unconjugated bilirubin is non-water soluble , free form ,doesn’t react to the reagents until alcohol  is added to the solution so , it called indirect bilirubin
• Total bilirubin measures both direct and indirect bilirubin

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Principle

• Diazotization is a principle of bilirubin test
• Total bilirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an azodye
• Normal saline is used to determination of direct bilirubin instead of caffeine
• Sulfanilic acid + HNO₂ (NaNO₂ + dil Hcl) → diazotized – sulfanilic acid
• Bilirubin + diazo reagent + (caffeine + benzoate) → Acid – Azo bilirubin ( pink color)
• Acid – azo bilirubin + Alkaline tartarate soln → Alkaline – Azo bilirubin (blue green color) , read at 578 nm

Normal range

• Total Bilirubin : up to 1 mg % ( 0.3 – 1 ) mg %
• Direct Bilirubin : 0.1 – 0.4 mg %
• Indirect Bilirubin : 0.2 – 0.7 mg %

Reagents

• Sulfanilic acid
• Sodium nitrite (NaNO₂)
• Caffeine and sodium benzoate ( act as a catalyst)
• Alkaline tartarate
• Saline solution (used in determination of direct bilirubin instead of caffeine)

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Procedure with kits in lab

• Depend on colorimetric technique 

Reagents	Total bilirubin	Direct bilirubin	Reagents
Test	Blank test	Test	Blank test
R1	100µ	100µ	100µ	100µ	R1
R2	25µ	–	25µ	–	R2
R3	500µ	500µ	1000µ	1000µ	Saline
Serum	100µ	100µ	100µ	100µ	Serum
Mix , wait for 10 min at RT then add R4	Mix , wait for 5 min at RT , read at 545 nm
R4	500µ	500µ
Mix , wait for 5 min at RT , read at 545 nm

• R1 : Sulfanilic acid 
• R2 : NaNO₂
• R3 : Caffeine
• R4 : Tartarate solution

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Calculations

• Total bilirubin (mg %) =Asample X 10.8
• Direct bilirubin (mg %) =Asample X 14.4
• Indirect bilirubin (mg %) = Total – Direct

Direct bilirubin level increased in:

• Hepatocellular disease as cirrhosis and hepatitis
• Blockage of bile ducts due to gall stones or tumers
• Drug reaction

Indirect bilirubin level increased in:

• Hemolytic anemia
• Transfusion reaction
• Cirrhosis
• Gilbert syndrome (low levels of glucoronyl transferase)

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Q: Why make sample blank in bilirubin test?

• To avoid color interference where serum is yellow color and alkaline azo-bilirubin color from yellow to blue green so , make sample blank by adding all contents except NaNO₂

Q:The media must converted from acidic to basic = Role of alkaline tartarate solution?

• Pink color low in sensitivity and can’t measure low concentration
• Maybe due to presence of high turbidity , but blue green color have high sensitivity and measure low concentrations

• Pink color low in sensitivity and can’t measure low concentration
• Maybe due to presence of high turbidity , but blue green color have high sensitivity and measure low concentrations