Aim: – Determination of Serum Direct Bilirubin by DMSO method & Malloy and Evelyn method

Summary:-

Bilirubin is a breakdown product of hemoglobin, insoluble in water. It is transported from the spleen to the liver and excreted into bile. Hyperbilirubinemia results from the increase of bilirubin concentrations in plasma.

PRINCIPLE:-

Bilirubin is converted to colored azobilirubin by diazotized sulfanilic acid and measured photometrically. Of the two fractions presents in serum, bilirubin-glucuromide and free bilirubin loosely bound to albumin, only the former reacts directly in aqueous solution (bilirubin direct), while free bilirubin requires
solubilization with dimethylsulphoxide (DMSO) to react (bilirubin indirect). In the determination of indirect bilirubin, the direct is also determined; the results correspond to total bilirubin. The intensity of the color formed is proportional to the bilirubin concentration in the sample.

 REQUIREMENTS:-

*Two test-tube, 

*Colorimeter, Bilirubin

*Direct Reagents,

 *Incubator,

*Cuvette, 

*Pipette etc.

*Fresh Serum.

PROCEDURE-

1.      Adjust the instrument to zero with distilled water.

2.   Pipette into a cuvette or test tube:

Contents	Blank	Test
Direct Bilirubin Reagent (R2)	1.5 ml	1.5 ml
Working Reagent (R3)	–	50µl
Sample /Calibrator	50µl	50µl

 1.      Mix and incubate for exactly 5 minutes at room temperature.

       2.      Read the absorbance (A). at 546 nm wavelength.

Calculation:-

 With Factor:

Direct Bilirubin (mg/dl) =              (A) Sample – (A) Sample Blank x Factor

Theoretical Factor

Direct bilirubin = 14 

Direct Bilirubin (mg/dl)  =             (A) Sample – (A) Sample Blank x 14

After Testing,

 If, (A) Sample = 0.21

                                           (A)   Sample Blank = 0.16

                                                                 =             0.05 X 14

                                                                 =             0.7 mg/dl

   D. Bilirubin in mg/dl to µmol/L   =             mg/dL x 17.1     

=          0.7 X 17.1

=          11.97 µmol/L

Normal Value:-    Direct Bilirubin (mg/dl)           =         <0.3 mg/dl

Clinical Significance:- 

Causes of hyperbilirubinemia:-

Direct bilirubin: Hepatic cholestasis, genetic errors, hepatocellular damage.