Estimation of Uric Acid

The quantitative determination of uric acid concentration in serum and urine by Enzymatic
colorimetric method (Uricase /PAP test)
Description
In the human body uric acid is the end-product of purine metabolism. It is excreted by the
kidney. Increases of uric acid in the serum plasma or urine can be due to the overproduction of
purine containing molecules or to insufficient excretion. The concentration is increased in
various renal diseases, with increased cell lysis in the presence of tumors, leukemia, toxemia of
pregnancy. Prolonged elevation of the concentration leads to gout.
Principle
Uricase transforms Uric acid in the sample into Allantoin, Carbon dioxide (CO2) and Hydrogen
peroxide (H2O2). By the action of Peroxidase (POD) and in the presence of phenol-derivative
DHBS and 4-Aminoantipyrine, Hydrogen peroxide gives a coloured indicator reaction
which can be measured at 520 nm. The increasing in absorbance correlates with is
proportional to the uric acid concentration of the sample.

Uricase
Uric acid+ 2H2O + O2 Allantoin+ CO2 + H2O2
POD
2H2O2 + 4-Aminoantipyrine + DHBS Red quinone+4H2O

Reagents
1. Reagent (R1)
 Phosphate buffer, pH=7.5, 50 mmol/l
 DHBS 4 mmol/l
2. Reagent (R2)
 Uricase 80 U/l
 4-Aminoantipyrine 1 mmol/l
 Peroxidase 660 U/l
Reagent (R3)
 Uric Acid Solution 6mg/dl
Samples
 Serum.
 Urine diluted in ratio of 1:10 with distilled water.
 If the urine sample is opalic then incubate at 60°C for ten minutes. The ascorbic acid
in the urine sample interferes with the test, so use diluted sample.
PROCEDURE
Preparation and stability of working reagent
 Dissolve one vial of R2 in appropriate amount of Rl.
 Stability: at: 20-25 °C: 7 days at 2- 8 °C: 4 weeks
 If the absorbance of working reagent is higher than 0.1 at 492 nm the reagent cannot be
used.

Assay Conditions

1 Type of reaction End point

2 Wavelength 546 nm
3 Temperature 37°C
4 Sample volume 20 µl
5 Reagent volume 2.0 ml
6 Incubation time 10 min at 37°C
7 Std. concentration 200 mg/dl
8 Light path 1.0 cm
9 Zero setting with Reagent blank
Procedure

1. Take three cuvettes and labelled them as test ‘T’,standard ‘S’ and blank ‘B’.
2. Added 2ml of enzyme reagent in all three cuvettes.
3. Added 20µl of standard solution in ‘S’ cuvette.
4. Added 20µl of serum in cuvette ‘T’.
5. Mixed well and incubated for 10 minutes at 37°C.
6. Measured absorbance of standard and sample at 546 nm against reagent blank.

blank standard sample

working reagent 2ml 2ml 2ml
Standard __ 20μl __
Sample __ __ 20μl
Water 20μl __ __

Calculations

Uric Acid Concentration (mg/dl) = Absorbance of test x concentration of Standard

Absorbance of standard
Uric Acid Reference Values:

Female: 2.5-6.0 mg/dl or 148-357 μmol/l

Male: 3.4-7.0 mg/dl or 200-416 μmol/l
Urine: 250-750 mg/24 h