Determination Of Uric Acid Levels

MATERIAL V DETERMINATION OF URIC ACID LEVELS (QUANTITATIVE) Basic Theory Uric acid is the final product or waste product resulting from the metabolism/ breakdown of purines. Uric acid is actually an antioxidant from humans and animals, but if it is in excessive amounts in the blood it will crystallize and can cause gout. Uric acid has a role as an antioxidant if the levels are not excessive in the blood, but if the levels are excessive, uric acid will act as a prooxidant. The formation of uric acid in the blood can also increase due to external factors, especially food and drink which stimulate the formation of uric acid. Any disturbance in the excretion process in the body will cause a buildup of uric acid in the kidneys and joints. The complex pathway for uric acid formation starts from ribose 5-phosphate, a pentose derived from glycidic metabolism, converted into PRPP (phosphoribosyl pyrophosphate) and then phosphoribosylamine, then transformed into inosine monophosphate (IMP). From intermediate compounds derived from adenosine monophosphate (AMP) and guanosine monophosphate (GMP), purinic nucleotides are used for DNA and RNA synthesis, as well as inosine which will then be degraded into hypoxanthine, xanthine and finally into uric acid. Uric acid levels can be determined through the results of blood and urine tests. The reference value for normal uric acid blood levels in men is 3.6 - 8.2 mg/dl while in women it is 2.3 - 6.1 mg/d Principles for Determining Uric Acid Levels Determination of uric acid levels using the enzymatic photometric test method using TBHBA (2,4,6-tribromo-3-hydroxybenzoate) with the working principle of this enzymatic photometric test is that uric acid is oxidized to allatoin by uricase. Geberated hydrogen peroxide reacts with 4 aminoantripyrine and 2,4,6-tribromo-3 hydroxybenzoic acid (TBHBA) to quinoneimine uric acid + H2O+OuricaseAllantoin + Co2+ H2O2 TBHBA + 4-Aminoantipyrine + 2H2OPODQuinoeimine + 3H2O2 Procedure Wavelength : Hg 546 nm (500 -550 nm Spectrophotometer : 520 nm Incubation temperature : 20 – 25°C or 37° Material : Blood Sample 10-15 ml Reactor : Reagent 1 - Phosphate buffer pH 7.0 100 mmol/L - TBHBA (2,4,6-tribromo-3 hydroxybenzoic acid) 1.25 mmol/L Reagent 2 - Phosphate buffer pH 7.0 100 mmol/L - 4-aminoantryptine 1.5 mmol/L - K4[Fe(CN)6] 50 µmol/L - Peroxidase (POD) ≥ 10 kU/L - Uricase ≥ 150 kU/L Specimen (biological sample) Serum, plasma heparin or plasma EDTA Stability: • 3 days at a temperature of 20-250C, • 7 days at temperature 4-80C and • 6 months at temperature -200C Tool : 1.Reaction tube 2.EDTA 3.Centrifuge 4. Kits 5. Spectrophotometer 6. Myropipette Implementation: Preparation of working reagents Pipette in cuvette Blank Standard Sample Sample, std - 20 µl aquabides 20 µl - 20 µl reagent 1 1000 µl 1000 µl 1000 µl Mix, measure the absorbance of A1 against the reagent blank after 1-5 minutes at temperature of 20-25°C /37°C and add Reagent 2 250 µl 250 µl 250 µl Mix, incubate for 5 minutes at temperature 20-250C /370C. and measure the absorbance of A2 against the reagent blank within 60 minutes. (∆A) Sample Pipette in cuvette Blank Standard Sample Sample, std - 20 µl aquabides 20 µl - 20 µl reagent 1 1000 µl 1000 µl 1000 µl Mix, standards and samples against blank coreagent within 60 minutes. (∆A) incubation5 minutes at temperature 37°C. Measure absorbance