Free fatty acids, also known as non-esterified fatty acids, are derived from dietary or the metabolism of adipose tissue. In adipose tissue, hormone-sensitive lipase (HLS) decomposes triglycerides to produce glycerol and fatty acids. Circulating in the body with free fatty acids combined with plasma albumin, used as an energy source easily absorbed by muscles, brains, and other tissues and organs.

1.   The samples should be fresh collected and detect within 24 hours.

2.   The supernatant after centrifugation must be clarified for the pretreatment of tissue and cell samples. Otherwise take the turbid supernatant to another centrifuge tube and centrifuge again.

3.   The reagent has a pungent smell. Please operate in the draught cupboard.

                                  [Note]: A-H, standard wells；S1-S40, samples wells；S1'-S40', control wells.

1)    Standard tube: Add 0.5 mL of standard with different concentrations and add 0.25 mL of Reagent 4.

Control tube: Take 0.5 mL of the supernatant of sample and add 0.25 mL of Reagent 3. 

Sample tube: Take 0.5 mL of the supernatant of sample and add 0.25 mL of Reagent 4. 

2)    Oscillate for 3 min and stand at room temperature for 3 min.

3)    Take 0.3 mL of the upper layer liquid to micro-plate and measure the OD value at 715 nm with Microplate reader.

Dilute 10 mmol/L standard solution with reagent 1 to a serial concentration. The recommended dilution gradient is as follows: 1.5, 1.2, 1.0, 0.9, 0.6, 0.4, 0.3, 0 mmol/L. Then carry the assay according to the operation table.

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

Example analysis

For Rat liver tissue, take 0.1 g of rat liver tissue, add 1.2 mL reagent 1, oscillate at 4℃ for 2 hours to extract the NEFA, centrifuge at 10000×g for 10 min, dilute the supernatant with reagent 1 for 3 times, then carry the assay according to the operation table.

The results are as follows:

standard curve: y = 0.09174 x – 0.0026, the average OD value of the sample tube is 0.108, the average OD value of the control tube is 0.049, and the calculation result is:

NEFA content(μmol/g)=(0.108-0.049+0.0026)÷0.09174×1.2÷0.1×3=24.17

Detect rat heart tissue (m=0.1 g, V3=1.2 mL), rat liver tissue (m=0.1 g, V3=1.2 mL), rat kidney tissue (m=0.1 g, V3=1.2 mL) and HepG2 cells (5×106 cells, V4=1.2 mL) according to the protocol, the result is as follows: