MDA in the catabolite of lipid peroxide can react with thiobarbituric acid (TBA) and produce red compound, which has a maximum absorption peak at 532 nm.

1.  In the incubation of 100℃ water bath, the EP tube should not be closed directly. It is recommended to fasten the tube mouth with fresh-keeping film and make a small hole in the film.

2.  The temperature of water-bath and the time of incubation should be stabilized (95-100℃, 40 min).

                                                                               [Note]: A-H, standard wells; S1-S80, sample wells.

1)   Standard tube: Take 0.01 mL of Standard solution with different concentrations into numbered 1.5 mL EP tubes.

Sample tube: Take 0.01 mL of tested Sample into numbered 1.5 mL EP tubes.

Control tube: Take 0.01 mL of tested Sample into numbered 1.5 mL EP tubes.

2)   Add 0.01 mL of Reagent 1 into each tube of Step 1.

3)   Add 0.6 mL of Reagent 2 application solution into each tube of Step 2.

4)   Add 0.2 mL of Reagent 3 application solution into standard tubes and sample tubes, add 0.2 mL of 50% glacial acetic acid to the control tubes.

5)   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 40 min.

6)   Cool the tubes to room temperature with running water, centrifuge the tubes at 9569 g for 10 min.

7)   Take 0.25 mL the supernatant of each tube to the Microplate with a micropipette (the precipitation cannot be added to the Microplate).

8)   Measure the OD value at 532 nm with Microplate Reader.

Note: In general, the serum (plasma) samples are no hemolysis or lipidemia, control tube can be remove, just need to establish blank (the concentration of standard is 0 μmol/L) tube. 

Dilute 200 μmol/L Standard Solution with absolute ethyl alcohol to a serial concentration. The recommended dilution gradient is as follows: 200, 150, 100, 60, 40, 20, 10, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using OD value 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 OD value of sample. The standard curve is: y= ax + b.

For Rat liver tissue, take10% rat liver tissue homogenate, carry the assay according to the operation table.

The results are as follows:

standard curve: y = 0.0022 x - 0.0019, the average OD value of the sample tube is 0.057, the average OD value of the blank tube is 0.044, the concentration of protein in sample is 11.287 gprot/L, and the calculation result is:

MDA content (μmol/gprot)=(0.057-0.044+0.0019)÷0.0022÷11.287=0.60μmol/gprot