Serum uric acid (UA) assay by phosphotungstic acid reduction assay experiment

Summary

Uric acid in deproteinized blood filtrate is oxidized to allantoin and carbon dioxide by tungstic acid in alkaline solution, and phosphotungstic acid is reduced to tungstic acid in this reaction, and the amount of tungstic acid generated is proportional to the content of uric acid in the reaction solution, which can be measured colorimetrically. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

Serum uric acid (UA) assay by phosphotungstic acid reduction assay experiment

Principle

Uric acid in deproteinized hemofiltrate is oxidized to allantoin and carbon dioxide by tungstic acid in alkaline solution, while phosphotungstic acid is reduced to tungstolan in this reaction, and the amount of tungstolan produced is proportional to the amount of uric acid in the reaction solution, which can be measured colorimetrically.

Move

I. Experimental reagents:

1. Phosphotungstic acid storage solution: weigh 50g of sodium tungstate, dissolved in about 400ml of distilled water, add 40ml of concentrated phosphoric acid and a few grains of glass, boiling reflux water cooled to room temperature, diluted to 1 liter with distilled water, stored in a brown reagent bottle.

2. Phosphotungstic acid application solution: take 10ml of phosphotungstic acid storage solution, dilute to 100ml with distilled water.

3. 0.3 mol/L sodium tungstate solution: weigh 100g of sodium tungstate (Na2WO42H2O, MW329.36), dissolve with distilled water and dilute to 1 liter.

4. 0.33 mol/L sulfuric acid: take 18.5 ml of concentrated sulfuric acid, add it to 500 ml of distilled water, and then dilute it to 1 liter with distilled water.

5. tungstic acid reagent: add 50ml, 0.3mol/L sodium tungstate solution, 0.05ml concentrated phosphoric acid and 50ml, 0.33mol/L sulphuric acid to 800ml distilled water and mix well, it can be stabilized for several months at room temperature.

6. lmol/L sodium carbonate solution: weigh 10.6g anhydrous sodium phosphate solution in distilled water and dilute to 1 liter, place in a plastic flask, if turbid, can be filtered and used.

7. 6.0 mol/L uric acid standard storage solution: take 60mg of lithium carbonate (AR) dissolved in 40ml of distilled water heated to 60 ℃, so that it is completely dissolved, accurately weighed 100.9mg of uric acid (MW168.11), dissolved in lithium carbonate solution, cooled to room temperature, transferred to 1000ml volumetric flasks, diluted to the scale with distilled water, and stored in a brown bottle.

Second, the experimental operation: in three 16mm × l00mm test tubes (determination, standard and blank) each with 4.5ml tungstic acid reagent, respectively, add 0.5ml serum, 0.5ml standard application of liquid and 0.5ml distilled water, after mixing and static for a few minutes, determination of the tube centrifugal precipitation, and then operated in accordance with the following table:

Mixed, placed at room temperature for 20 minutes, with spectrophotometric in his length of 660nm, the colorimetric cup optical diameter of 1.0cm, with a blank tube to adjust the zero, read the absorbance of each tube.

Third, calculation:

Serum uric acid μmol/L = absorbance of measurement tube / absorbance of standard tube × 300

Reference value: men 149-461 μmol/L (2.57-7 mg/dL)

Female 89-357 umol/L (1.5-6mg/dL)

Caveat

1. A variety of non-specific reducing substances exist in red blood cells, so it is better to use serum or plasma than whole blood.

2. using potassium oxalate with sodium tungstate tends to form insoluble potassium phosphotungstate, resulting in turbidity of the color-developing solution; potassium oxalate anticoagulant cannot be used.

3. Uric acid in serum and urine specimen can be stabilized for 3 days at room temperature, and urine specimen can cause urate precipitation after refrigerated, at this time, PH can be adjusted to 7.5-8.0, and the specimen can be heated to 50℃, and then measured after the precipitate is dissolved.

4. Uric acid in water solubility is very low, but soluble in carbonate solution, the preparation of standard solution, add lithium carbonate and heating to help dissolve, such as no lithium carbonate, potassium carbonate or sodium carbonate can be used instead.

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