The organism produces oxygen free radicals through enzyme system and non-enzyme system, attacks polyunsaturated fatty acids in biofilm, induces lipid peroxidation, and thus forms lipid peroxide. Malondialdehyde (MDA) is one of the common products of lipid peroxidation in organisms. In clinical science, MDA is a biomarker of lipid peroxidation, which can reflect the degree of lipid peroxidation in organism and indirectly reflect the degree of cell injury.

1.  The temperature should be controlled at 95~100℃ when incubate in water bath for 1 hour.

2.  In water bath reaction, do not cover the caps of tubes tightly. It is recommended to seal the test tube mouth with the preservative film and make a hole in the preservative film.

3.  Prevent the formulation of bubbles when the supernatant is transferred into the microplate.

1)      Standard tube: Take 0.1 mL of Standard solution with different concentrations into numbered 10 mL glass tubes.

   Sample tube: Take 0.1 mL of tested Sample into numbered 10 mL glass tubes.

2)      Add 0.1 mL of Reagent 1 into each tube of Step 1.

3)      Add 4 mL of Chromogenic agent into each tube of Step 2.

4)      Fasten the tube mouth with fresh-keeping film, mix fully, and make a small hole in the film. Then incubate the tubes at 100℃ for 1 hour.

5)      Take the tubes out and put them in an ice bath to stop the reaction. After incubation on ice for 10 min, centrifuge the tubes at 1600 × g for 10 min.

6)      Take 0.25 mL the supernatant to the Microplate with a micropipette (the precipitation cannot be added to the Microplate).

7)      Measure the fluorescence values at the excitation wavelength of 530 nm and the emission wavelength of 550 nm. 

Dilute 20 μmol/L Standard Solution with double distilled water to a serial concentration. The recommended dilution gradient is as follows: 10, 8, 6, 4, 2, 1, 0.5, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using fluorescence value (F) of standard and correspondent concentration as y-axis and x-axis respectively. Create the standard curve with graph software (or EXCEL). The concentration of the sample can be calculated according to the formula based on the F value of sample. The standard curve is: y= ax + b.

For mouse liver tissue, dilute the 10% mouse liver tissue homogenate with PBS (0.01 M, pH 7.4) for 10 times, take 0.1 mL of diluted sample, carry the assay according to the operation table.

The results are as follows:

Standard curve: y = 63.104 x + 13.471, the average fluorescence value of the sample well is 70.527, the average fluorescence value of the blank well is 33.423, the concentration of protein in sample is 16.56 gprot/L, and the calculation result is:

TBARS content (μmol/gprot)=(70.527-33.423-13.471)÷63.104×10÷16.56=0.23μmol/gprot

Detect rat serum (dilute for 10 times), 10% mouse liver tissue homogenate (the concentration of protein is 16.56 gprot/L, dilute for 10 times), 10% rat lung tissue homogenate (the concentration of protein is 5.04 gprot/L, dilute for 2 times) and HepG2 cells (the concentration of protein is 6.09 gprot/L) according to the protocol, the result is as follows: