1.   The preparation of sample supernatant

      Take 0.1 mL serum (plasma) or 10% tissue homogenate sample, then add 0.4 mL reagent 4, mix fully. Centrifuge at 1100 × g for 10 min and take the supernatant for detection.

2.   The measurement of samples

1)   Blank tube: Take 0.2 mL of double distilled water to an EP tube.

Standard tube: Take 0.2 mL of 0.5 mmol/L standard solution to an EP tube.

Sample tube: Take 0.2 mL of sample supernatant to the EP tubes.

2)   Add 2.0 mL of Chromogenic agent into each tube of Step 1 and mix fully.

3)   Incubate the tubes at 37℃ for 30 min, then cooling to room temperature.

4)   Set the spectrophotometer to zero with double distilled water, and measure the OD at 660 nm with 1 cm optical path quartz cuvette.

Dilute 10 mmol/L standard stock solution with double distilled water to a serial concentration. The recommended dilution gradient is as follows: 2, 1, 0.5, 0.2, 0.1, 0.05, 0 mmol/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.