Determination of serum bilirubin

Name: Ruba Ijraiwi

The course name: Biochemistry lab 1

Number: 21710820

Aim:  determination of serum bilirubin in the blood.

Introduction : Bilirubin is a yellow substance that results from the
breakdown or breakdown of red blood cells from hemoglobin,
which gives red blood cells their color.
Bilirubin is the product of hemoglobin breakdown. After the red
blood cells age, they break down and are eaten by phagocytes.
Then the hemoglobin protein is divided into two parts: heme and
globin. The heme molecule is converted to bilirubin and is
transported to the liver. In the liver, most of the amount of bilirubin
is bound to It is called a glucuronide before it is excreted through
the gallbladder.

Materials : sulphanilic acid \HCl \caffeine \sodium benzoate\

sodium nitrite
Procedure :
1.    We put two tubes, plank and sample, for each of the
direct and direct bilirubin
2.     for total bilirubin:
 
Sample blank sample
Sulphanilic acid 0.2ml 0.2ml
Sodium nitrite - 1drop
caffeine 1.0 1.0
Sample plasma 1.0 1.0
Mix and incubate for 10min
tartale 1ml 1ml
3. Mix and stand for 5min measure at546nm
4.   for direct bilirubin:
 blank sample
Sulphanilic acid 0.2 0.2
Sodium nitrite - 1drop
saline 2ml 2ml
Sample serum 0.2ml 0.2ml
5. Mix and stand for 5min measure at 546nm

Results:
total bilirubin 1.749
direct bilirubin 1.3104
Indirect bilirubin 0.071
To find indirect bilirubin = total-direct
Total=abs*10.8                  0.162*10.8=1.749mg \dl
Direct=abs*14.4                   0.091*14.4=1.3104
Abs indirect =0.162-0.091=0.071

:Discussion

A simple and rapid method for the estimation of bilirubin in serum

is described. The method is suitable for routine use in laboratories
using photoelectric colorimeters. A number of observations are
made on the conditions necessary for satisfactory reaction
.between bilirubin solutions and diazotized sulphanilic acid

During our search for a good bilirubin standard

we carried out many preliminary experiments with

a view to determining the conditions governing

satisfactory coupling of bilirubin with diazotized

.sulphanilic acid

It was found desirable to have a buffer present in

order to regulate the rate of coupling. The

reaction proceeds only slowly in strongly acid

solution, but is very rapid in neutral or alkaline

solution. Further, the colour of the azobilirubin

formed varies with reaction, being violet in the

presence of mineral acid, the usual red or pink at

pH 4-7, and greenish blue in alkaline solution. In

the case of the ordinary van den Bergh test the

serum itself is a sufficient buffer to reduce the

acidity of the diazo solution to the useful pH

.range

The stability of the azobilirubin formed is

dependent on the reaction of the medium (it

. )decomposes rapidly in alkaline solution