Experimental determination of serum creatinine (Cr) by the deproteinization endpoint method

Summary

Creatinine in serum (plasma) reacts with alkaline picric acid to form a yellowish-red picric acid-creatinine complex, which is measured colorimetrically at 510 nm. This experiment is derived from the experimental guide of Mudanjiang Medical College, undergraduate 5-year laboratory testing program.

Operation method

Experimental determination of serum creatinine (Cr) by the deproteinization endpoint method

Principle

Creatinine in serum (plasma) reacts with basic picric acid to form a yellowish-red complex of picric acid creatinine, which is determined colorimetrically at 510 nm.

Materials and Instruments

Picric acid solution Sodium hydroxide Tungstic acid solution Polyvinyl alcohol Thick sulfuric acid

Move

I. Experimental reagents:

1. 0.04mmol/L picric acid solution: 9.3g of picric acid (AR), dissolved in 500ml of distilled water cooled to room temperature. Add distilled water to 1 liter, titrate with 0.1 mol/L sodium hydroxide with phenolphthalein as indicator, dilute to 0.04 mol/L with distilled water according to the titration stored in a brown bottle.

2. 0.75mmol/L sodium hydroxide: 30g of sodium hydroxide (AR), add a little distilled water to dissolve it, cool and dilute to 1 liter with distilled water.

3. 35 mmol/L tungstic acid solution:

① Add 1g polyvinyl alcohol to 100ml distilled water, heat to aid dissolution (not boil) and cool.

Add 11.1g of sodium tungstate to 300ml of distilled water and dissolve completely.

③ Add 2.1ml of concentrated sulfuric acid to 300ml of distilled water and cool.

In a 1 liter volumetric flask, add ① solution to ② solution, and then mix with ③ solution. Add distilled water to the scale and store at room temperature for at least one year.

4. 10 mol/L creatinine standard storage solution: creatinine (MW, 13.12) 113mg dissolved in 0.1mmol/L hydrochloric acid and transferred to 100ml volumetric flask, then diluted with 0.1mol/L hydrochloric acid to the scale, and stored in the refrigerator for one year.

5. 10μmol/L creatinine standard application solution: accurately aspirate 1.0ml of 10mmol/L creatinine standard storage solution and add it into 1000ml volumetric flask, dilute to scale with 0.1mol/L hydrochloric acid and store in refrigerator.

II. Experimental operation:

In 16mm×100mm test tube, put 0.5ml of serum (or plasma), add 4.5ml of 35mmol/L tungstic acid solution, mix well. 30O0r/min, centrifugation for 10 minutes, take the supernatant, and then measure according to Table 3-6-4.

Urine specimen diluted with distilled water 1:2O0, also according to the table.

After mixing, place at room temperature for 15 minutes, spectrophotometer 510nm wavelength, colorimetric cup aperture 1.0cm with a blank tube to adjust the zero. Read the absorbance of each tube

Calculation:

Serum (plasma) creatinine μmol/L = Measurement tube polarization / standard tube absorbance × 100

Urine creatinine μmol/L = Measurement tube polarization / standard tube absorbance × 100 × 200 × 24-hour urine volume (L)

Reference value: Male 44-133μmol/L (0.5-1.5 mg/dL)

Female 70-106μmol/L (O.8-1.2 mg/dL)

Caveat

1. When the temperature rises, can make the alkaline picric acid solution color darkening, but the standard solution and the determination of the degree of proliferation is not proportional, so the determination of the temperature of each tube need to be raised to room temperature.

2. serum (plasma) specimens, such as not when the time is fixed, can be stored in the refrigerator for 3 days, can only be maintained for a longer period of time should be -20 ℃ storage, slight hemolysis specimen on the determination of creatinine impact, can make creatinine results are high.

3. The recovery rate of creatinine determination is affected by the PH of the protein-free solution, the recovery rate is 85-90% when the PH of the solution is 3-4.

5， and 100% when the PH2 is used.

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