Application

This kit can be used for detection of VE content in samples, such as animal/plant tissue, serum (plasma). 

Detection significance

VE is a kind of natural lipid-soluble antioxidant, which exist in cellular membrane structure (cell membrane, mitochondrial, microsomal membrane), lipid droplets of adipocytes and lipoproteins of plasma. It is scavenger of singlet oxygen and superoxide, blocking agent of lipid peroxidation and can protect protein sulfhydryl.

Detection principle

Fe3+ can be deoxidized to Fe2+ by VE with ferroin existing. Fe2+ can react with phenanthroline and form pink compound under certain condition. After colorimetric assay, VE content can be figured out according to the standard curve or calculated through formula.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, Centrifuge, Spectrophotometer (533 nm)

Operation steps

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

1.   Detection of VE content in serum (plasma)

(1) Extraction of n-heptane VE in serum (plasma) (n-heptane is self-prepared):

Vortex mixing (extract thoroughly) for 1 min, then centrifuge at 3500~4000 rpm for 5~10 min. Take the n-heptane VE extraction solution (the upper layer solution) for chromogenic reaction.

Note: Liquid in the tube will be divided into three layers, and the upper layer is n-heptane VE extraction solution, the middle layer is water and absolute ethyl alcohol and the lower layer is protein precipitate.

(2) Chromogenic reaction:

Mix fully and stand for 2 min. Measure the OD values of each tube at 533 nm wavelength with cuvette of 0.5 cm optical path, set to zero with absolute ethyl alcohol.

2.   Detection of VE content in tissue

(1) Sample pretreatment:

Preparation of 10% homogenate solution: weigh the tissue accurately. Add Reagent 4 (homogenate medium) in a weight (g): volume (mL) ratio of 1: 9, then use mechanical homogenization in ice water bath. Centrifuge the mixture solution at 2500 rpm for10 min and take the supernatant for measurement.

(2) Extraction of n-heptane VE in tissue homogenate (n-heptane is not provided in this kit):

Vortex mixing (extract thoroughly) for 1 min, then centrifuge at 3500~4000 rpm for 5~10 min. Take the n-heptane VE extraction solution (the upper layer solution) for chromogenic reaction.

Note: Liquid in the tube will be divided into three layers, and the upper layer is n-heptane VE extraction solution, the middle layer is water and absolute ethyl alcohol and the lower layer is protein precipitate.

(3)  Chromogenic reaction:

Mix fully and stand for 2 min. Measure the OD values of each tube at 533 nm wavelength with cuvette of 0.5 cm diameter, set to zero with absolute ethyl alcohol.

Technical parameter

1.  The sensitivity of the kit is 0.09 μg/mL.

2.  The intra CV is 4.4% and the inter CV is 4.9%.

3.  The recovery of the kit is 98%.

4.  The detection range of the kit is 0.09-40 μg/mL.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

4. Do not use components from different batches of kit.

5. Test tubes should be cleaned with cleaning agent or boiling water, then wash with running water for second washing and double-distilled water for third washing.

6. It is recommended to prepare needed amount of fresh Reagent 2 before use.

7. The chromogenic reaction should be accurately 5 min.

8. The extraction of VE should be properly1 min.

9. As this kit is a micro-determination method, the first absorbed liquid should be discarded each time changing a pipette. The pipette should be vertical when adding sample or        reagent and avoid of touching the tube wall.

10. Be careful when extracting the n-heptane extraction solution. Do not mix the second layer (water and absolute alcohol) into it, or the OD value will be influenced.

11. Tubes for chromogenic reaction should be dry.

12.  During the process of standing, the test tube must be sealed to reduce the volatilization of absolute ethanol and n-heptane in the system.