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1、Experiment 10 Determination of uric acid in human serumObjective:After learning this course, students should grasp the principle for the quantitative determination of uric acid in blood serum and its significance for the clinically diagnostic use.PrincipleUric acid is the waste product produced from
2、 the degradation of purines. It is produced by the action of xanthine oxidase on xanthine and hypoxanthine which are products of nucleic acid degradation. When uric acid is present in abnormally high concentrations in the blood, it tends to crystallize out in the body joints, causing a very painful
3、inflammatory condition known as gout. Increased levels of uric acid are also associated with renal failure and large dietary intake of purines. Uric acid determination is thus important and useful for clinical diagnosis.The classic chemical method for the determination of uric acid is based upon the
4、 reduction of phosphotungstic acid by uric acid to a blue phosphotungstate complex. The method is not specific. In 1980 Fossati described a procedure for assaying uric acid using uricase. The procedure is more specific. The hydrogen peroxide formed by the action of uricase on uric acid reacts with N
5、-ethyL ?/-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) and 4-aminoantipyrine in the presence of peroxidase to form a red colored quinoneimine dye.Uric acid is virtually insoluble in water or common organic solvents, but it can be dissolved in basic solution such as aqueous solutions of Na2co3, N
6、aOH and ammonium hydroxide.UricaseUric acid + H2O + O2 Allantoin + CO2 + H2O2Peroxidase2 H2O2 + 4-aminoantipyrine + TOOS Quinoneimine + 4H2OThe intensity of the colored product is directly proportional to the concentration of uric acid in the sample. The concentration of uric acid is determinate by
7、the colorimeter with maximum absorbance at 550 nm. The uric acid assay is calibrated by referencing the absorbance of the unknown sample to the absorbance of the calibrator.EXPECTED VALUESMales: 208-428 : u mol/L (3.5-7.2 mg/dL)Females: 155-357 : u mol/L (2.6-6.0 mg/dL)Apparatus and Reagents1. Spect
8、rophotometer or colorimeter.Any instrument capable of reading absorbance accurately with a sensitivity of 0.001 absorbance at 550 nm may be used. 0.5 cm cuvettes or a flow cell capable of transmitting light at 550 nm.2. Reagents necessary for the determination of uric acid are included in the kit (1
9、0 X 5 ml)The working reagent, uric acid reagent (RI) contains, after reconstitution with the buffer (R2): peroxidase1800 U/L、uricase600 U/Lascorbate oxidase1000 U/L RITOOS1.4 mM4-aminoantipyrine0.3 mMBuffer (pH 7.8), preservatives, and stabilizersR2Procedures1. Prepare the required volume of working
10、 reagent.The working reagent is prepared by adding 5 ml of buffer (R2) to a bottle of uric acid reagent (RI), mix at once.2. Prepare 3 dry clean tubes, and do as follows:numberreagents(ml)blankStandard samplesample(1)(2)(3)Standard uric acid liquid (5 mg / dL)00.020Serum sample000.02Distilled water0
11、.0200Working reagent1.01.01.0Mix at once!3. Incubate for 10 minutes at 37 C and determine the absorbance of the calibrator (As) and of serum (A) at 550 nm using the distilled water sample as the reagent blank.4. The calculate the uric acid concentration of the sampleA550 of the tube for testingSerum uric acid concentration (mg/dL) = x concentration of standardA550 of the tube for standarduric acid (mg/dL)Question1. Whafs the principle for the quantitative determination of uric acid in blood serum?2. Whafs the clinical significance of uric acid determination?