Biochemistry Lab

College of Pharmacy

Estimation of Serum uric acid

C5H4N4O3
Introduction:

The breakdown product of purines in man is uric acid .The catabolism of purine nucleotides is
accomplished as given below. Adenosine is first deaminated to form inosine. this reaction is catalyzed
by adenosine deaminase which is found in liver and other tissues inosine is converted to hypoxanthine
by the action of nucleoside phosphorylase
Guanosine is converted to guanine which is then deaminated to form xanthine . this conversion is
catalyzed by guanase which is found in liver , spleen , pancreas and kidneys. hypoxanthine is
oxidized to form xanthine by xanthine oxidase which also catalyzes the formation of uric acid from
. xanthine .In man ,purine metabolism ends as uric acid which is excreted in urin
In mammals other than primates , however , uric acid is further metabolized to form allantoin by the
nezyme uricase

Clinical Significance

In clinical conditions wherein , the uric acid concentration exceed 8 mg/dl is normally referred to as
hyperuriciemia . Various diseased states such as
- renal failure
- nephrolithiasis
-polycythemia
-chronic nephritic
-leukemia
Msc pure Biochemistry-Ahmed Kareem Thamer
 -Gout
-influence the clinical condition of hyperuriciemia
Gout is a multi-factor syndrome wherein
1-there is an increase in serum urate concentration
2-accumulation of monosodium urate monohydrate crystals in leukocytes of synovial fluid
3-aggregation and deposition of monosodium urate monohydrate crystals in and around joints (referred
as tophi) resulting in arthritic condition.

In acute case of gout plasma urate level increase to a maximum of 15 mg /dl

liver disorders such as cirrhosis lead to hypouricimia wherein the blood uric acid level of <2 mg/dl
has been recorded ,as liver is the major site for uric acid synthesis

Normal plasma uric acid level in men (2-7 mg/dl) in higher than woman ( 2-6 mg/dl)

Principle:

Serum uric acid (2,6,8-trioxypurine) is an end product of purine catabolism in humans .Uric acid
reduces colourless phosphotungstic acid to a blue coloured -tungsten blue at alkaline pH .the colour
formed is colorimetrically determined at 700 nm.

Reagents:

1-sodium tungstate
2-Sulphuric acid
3-sodium carbonate

Procedure:

1-Transfer 1 ml of test serum into a clean graduated centrifuge tube (15 mL capacity ).Add 7mL of
distilled water , followed by addition of 1 mL each of sodium tungstan (10%) and sulpharic asid (0.67
N). mix the contents gently and allow it to stand for 10-15 min . centrifuge the contents for 15 min 2500
.rpm. Use 5 mL of the clear supernatant for uric acid analysis

2-Add 1mL each of soduim carbonate (10%) and phosphotungstic acid to

a)Blank and (b) standard and test ,as given below .Mix the contents and measurs the colour (
photometrically a t 700 nm.

e S.No Experiment sodium carbonate Phosphotungstic acid Absorbance

Reagent at 700 nm
1 Blank ( 5 ml distilled 1,0 1.0 1.0 -
Water)
2 Standard uric acid 1.0 1.0
(5 mL )
3 Test( 5ml of deprote- 1.0
inated serum supern- 1.0
Atant)

Msc pure Biochemistry-Ahmed Kareem Thamer

 Calculation:
x 100x25
Serum uric acid (mg/dl) = X
y 0.5x1000

x = Absorbance value of test sample

y = Absorbance value of test standard

Msc pure Biochemistry-Ahmed Kareem Thamer