Experimental detection of serum inorganic phosphorus by the direct colorimetric method of mitochondria

Summary

Using phosphorus reacts with ammonium molybdate in acidic solution to form molybdate phosphate complex, which can be reduced by N-methyl-p-aminophenol sulphate (mithril) to form blue, and Tween-80 is added to the reagent to inhibit the interference of proteins. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

Experimental detection of serum inorganic phosphorus by the direct colorimetric method of mitochondria

Principle

Using phosphorus in acidic solution reacts with ammonium molybdate to form a molybdate phosphate complex, which can be reduced by N-methyl-p-aminophenol sulfate (mithril) to form a blue color, Tween-80 is added to the reagent to inhibit protein interference.

Move

I. Experimental reagents:

l. Ammonium molybdate solution: in 50 ml of deionized water with concentrated H2SO43.3 ml, then add ammonium molybdate 0.2 grams of solubility plus Tween-80 5 ml, and finally add deionized water to 100 ml.

2. N-methyl-p-aminophenol sulfate (Mithril) solution: weigh p-cresol sulfate 2 g dissolved in 80 ml of deionized water, add anhydrous sodium sulfate 5 g; finally add deionized water to 100 m1.

3. Color application solution: take 1 liquid 10ml, 2 liquid l.lml mix can be applied.

4. Phosphorus standard liquid:

(l) inorganic phosphorus standard storage solution (lml = lmgP) weigh anhydrous potassium dihydrogen phosphate (KH2PO4) 4.39 grams of dissolved in the de-ionized into a 1L volumetric flask, and diluted to the scale, and then add 2ml of chloroform antiseptic, and stored in the refrigerator.

(2) Inorganic phosphorus standard application solution (lml = 0.04mgP or 1.29mmol/L): take 4ml of inorganic phosphorus standard storage solution, add into 100ml volumetric flask, dilute to scale with deionized water, add chloroform for preservation, and store in the refrigerator.

Second, the experimental operation: take 3 test tubes, labeled determination, standard and blank tube, and then operate according to the following table:

After mixing and placed in a 37 ℃ water bath for 10min, remove the spectrophotometer at 650nm wavelength, with a blank tube zero, 10mm light diameter colorimetric cup for colorimetry, read the absorbance of each tube.

III.Calculation:

Serum inorganic phosphorus mmol/L = absorbance of the measurement tube / absorbance of the standard tube × 1.29

Reference value: Adult: 0.96-1.62 mmol/L (3.0-5.0 mg/dL)

Children: 1.45-2.1 mmol/L (4.5-6.5 mg/dL)

Caveat

1. The serum specimen should not be hemolyzed, the serum should be separated promptly after blood collection, and the jaundice and lipemia specimens should be made specimen main white.

2. This method for serum white . Globulin ratio inversion of the specimen is easy to produce turbidity, the solution is to use 30g / L trichloroacetic acid 1.8ml respectively after mixing and centrifugation, take the supernatant 1.0ml, phosphorus standard solution similarly disposed of, and then mixed with the color solution 4.0ml colorimetric.

3. Mitochondrial reagent should be prepared in small quantities and not left for too long, otherwise normal serum may sometimes produce mild turbidity.

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