Serum triglyceride determination by acetone acetate assay experiment

Summary

This method uses isopropanol to extract serum TG, alumina adsorption of phospholipids and free glycerol saponification with potassium hydroxide, sodium periodate oxidation to produce formaldehyde, formaldehyde and acetone acetate in the presence of ammonia ions in the heating to produce a yellow 3,5 diacetyl-1,4 dimethyl pyridine [Hantgsch stop reaction) the degree of color development is directly proportional to the concentration of TG in the specimen.

This experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory tests.

Operation method

Serum triglyceride determination by acetone acetate assay experiment

Principle

This method uses isopropanol to extract serum TG, alumina adsorption of phospholipids and free glycerol saponification with potassium hydroxide, sodium periodate oxidation to produce formaldehyde, formaldehyde and acetone acetate in the presence of ammonia ions in the heating to produce a yellow 3,5 diacetyl-1,4 dimethyl pyridine [Hantgsch stop reaction) the degree of color development is directly proportional to the concentration of TG in the specimen.

Move

I. Experimental reagents:

1. Isopropyl alcohol (analytical reagent)

2. alumina (neutral, layer separation): wash several times with distilled water, pour off the fine particles that are not easy to sink, suction filter dry in 110 ℃ oven activation of at least 8h, stored in closed containers or desiccators

3. 50g/L potassium hydroxide, containing 40% isopropanol

4. oxidizing agent: each liter contains anhydrous ammonium acetate 77g glacial acetic acid 60ml, sodium periodate 650ml

5. Color developer: acetylacetone 0.75ml, isopropyl alcohol 20ml, diluted to 100ml with distilled water, stored in a brown bottle.

6, glyceryl trioleate standard solution (2.26mmol / l that is 200g / dl) accurately weighed 200mg of pure glyceryl trioleate, with isopropyl alcohol to 100ml, tightly stoppered storage 4 ℃ refrigerator

Second, the experimental operation:

1. extraction: the same method as cholesterol

2. Saponification, oxidation, color development, determination of the tube and the standard tube were added to the corresponding extraction solution 1.0ml, the empty

White tube with isopropyl alcohol lml, each tube with 5Og / LKOH solution O.2ml saponification, placed in a 60 ℃ water bath for 15min, cooled to add oxidant 0.5ml, mixing and add colorant O.25ml, then mixed, placed in a 60 ℃ water bath for 20min, removed and cooled.

3. Colorimetric: wavelength 410nm 1cm aperture colorimetric cup, with a blank tube to adjust zero, read the optical density of each tube.

III. Calculation:

Serum triglycerides = absorbance of measurement tube / absorbance of standard tube × standard concentration

Caveat

1. The saponification used in this method. Oxidation. The reagents used in this method are very stable, and can be stored at room temperature for half a year, but it is best to use a small amount of packaging to reduce reagent contamination caused by the deepening of the color of the blank tube.

2. saponification. Saponification, oxidation and color development of the temperature and time of the absorbance after color development have an impact. Therefore, each batch of specimens are required to make a good standard tube, and it is not appropriate to check the results from the standard curve, the absorbance after color development is basically stable, color development is placed at room temperature 20 ℃ without light conditions, the absorbance will be slightly higher, but the results of the determination of the impact is not significant.

3. Formaldehyde to acetone acetate reaction product absorption peak at 415nm, with 721-type spectrophotometer, 420nm colorimetric results are satisfactory.

4. This method has good recovery test and repeatability, and the accuracy can meet the clinical requirements.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/