Determination of glucose in serum by o-toluidine assay

Summary

Through this experiment, students can master the principle of o-toluidine method of blood glucose measurement, the preparation of reagents and learn the method of o-toluidine method of blood glucose determination. This experiment is from the laboratory guide of Mudanjiang Medical College undergraduate 5-year testing program.

Operation method

Determination of glucose in serum by o-toluidine assay

Principle

Glucose and o-toluidine heated in a strong acid solution, glucose aldehyde group and o-toluidine condensed to glucosylamine the latter dehydrated to generate Schiff (Schiff) base, and then the path of the structural rearrangement of organic compounds, absorbance peak 630nm.

Move

I. Experimental reagents:

l. o-toluidine reagent: 940 ml of glacial acetic acid add thiourea 1.5 g o-toluidine 60 ml mixed. Until the thiourea completely dissolved, placed in a brown bottle.

2. 12mmol/L benzoic acid solution: add benzoic acid (MW=122.12) 1.46g in 900ml distilled water, heat to dissolve, cool and put in 1L volumetric flask, add distilled water to the scale.

3, glucose standard storage solution (100mmol / L): weigh anhydrous glucose (MW = 180.16) pre-80 ℃ oven drying to a constant, transferred to a desiccator for preservation) 1.802g, dissolved in 80ml of benzoic acid solution in a 100ml volumetric flask, and then add benzoic acid solution to the scale.

4, Glucose standard application solution (5mmol / L): glucose standard storage solution 50ml, placed in 100ml volumetric flask with benzoic acid solution to the scale.

Second, the experimental operation;

Take several 16mm × 150mm test tubes and operate according to the following table:

After mixing, placed in a boiling water bath heated for 12min, removed and placed in cold water to cool for 5min; spectrophotometer 630nm, colorimetric cup aperture 1.0cm blank tube to adjust the zero, read the standard tube and determine the absorbance of the tube.

V. Calculation:

Body fluid glucose mmol/L = absorbance of measuring tube / absorbance of standard tube × 5

Reference value: serum glucose is 3.89-6.11mmol/L [70-110mg/dL].

Caveat

1、This method does not need to remove protein, o-toluidine reagent only with aldose color development, other reducing substances in the blood basically has no effect.

2， glucose and o-toluidine reagent color shade and stability and reagent o-toluidine concentration of glacial acetic acid and heating time related. Therefore, the heating time and temperature of the measuring tube, standard tube and blank tube must be the same, the number of measurements in each batch should not be too many, and the amount of water in the water bath should be large, and the water surface should be more than the liquid surface of the reagent in the test tube in order to control the reaction conditions better.

3， the final reaction solution will occasionally produce turbidity, the most common cause is hyperlipidemia; at this time, you can add isopropyl alcohol to 3 ml of the color solution, fully mixed to dissolve lipids, can clear the turbidity, the measured absorbance multiplied by 1.

5.

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