REPORTING GUIDE FOR THE ANALYSIS OF URIC ACID ESTIMATION
- DATE =?
- TITLE = Estimation of Uric acid with Uricase-POD enzymatic colorimetric method with 4-amino-antipyrine.
- AIM = To Analyse a given plasma or serum sample for the estimation of Uric acid
- MATERIALS =?
- PRINCIPLE:
Uric acid is oxidized by uricase to allantoin, carbon dioxide and hydrogen peroxide. The hydrogen peroxide generated then reacts with 4-aminoantipyrine (4-AAP) and a phenolic chromogen in the presence of peroxidase (POD) to form a red quinonimine dye.
Reaction 1: Uric Acid + O2 + 2H2O -> Allantoin + CO2 + H2O2 (Uricase)
Reaction 2: H2O2 + 4-AAP + Phenolic Compound -> Quinoneimine Dye + 2H2O (Peroxidase)
- TABLE OF PROTOCOLS
|
|
Blank |
Standard |
Sample |
|
Reagent |
1.0 ml |
1.0 ml |
1.0 ml |
|
Standard |
– |
20 μL |
– |
|
sample |
– |
– |
20 μL |
Mix and incubate for 5 minutes at 37oC or 10 minutes at 15 – 25 oC. Measure absorbance of Sample and standard against reagent blank within 30 minutes.
Calculation:
Serum uric acid concentration (mg/dL) = Abs of sample/ Abs of standard x 6
Concentration of uric acid in urine = Abs of sample/ Abs of standard x 6 x 10
NOTE
Serum blank: Extremely lipemic samples may give falsely elevated results and a serum blank must be run. Add 20 ml serum to 1 ml water. Measure Absorbance of specimen against water at the specified wavelength. Read and record absorbance and subtract reading from test absorbance. Why?
You can see more information about uric acid estimation in Learning guide for clinical Chemistry (Chemical Pathology)