Reagents of the kit comprise the first reagent composed of isooctane, the second reagent formed by dissolving sodium acetate in distilled water, then adding pyridine and mixing the constituents uniformly and the third reagent formed by dissolving copper acetate in distilled water, then adding pyridine and mixing the constituents uniformly. The method comprises the steps that free fatty acid in a sample is extracted through the isooctane, then the free fatty acid reacts with copper salt to generate copper soap under the weak acidic condition, and the content of free fatty acid in the reaction sample at the light absorption value of 715 nm is measured.
Free fatty acid (FFA) is both a product of fat hydrolysis and a substrate of fat synthesis. The concentration of free fatty acids is related to lipid metabolism, glucose metabolism and endocrine-related functions, it also reflects the quality changes of food in storage.
Free Fatty Acid Assay Kit
Reagent 1: 60mL × 1 bottle, 4°C
Reagent 2: 25mL × 1 bottle, 4°C
Reagent 3: 25mL × 1 bottle, 4°C
Materials Required but Not Supplied:
Mortar, desktop centrifuge, vibration meter, visible spectrophotometer, 1mL glass cuvette.
Free fatty acids extract in the sample:
1) Blood: Place blood sample 1 hour at room temperature, centrifuge 15 min at 3500 rpm, 4°C, take 0.1mL supernatant, add 1.2mL reagent 1, shock 3h and centrifuge 10 minutes at 8000rpm, 4°C, the supernatant to be tested.
2) Tissue: Rinse the tissue with distilled water, absorb surface moisture with absorbent paper, take about 0.1g mashed tissue to a centrifuge tube. Add reagent 1 according to the ratio of organization's quality (g) and reagent volume (mL) is 1:12. Shock 3h and centrifuge 10 minutes at 8000rpm, 4°C, the supernatant to be tested.
3) Bacteria, fungi: Add reagent 1 according to the ratio of cells amount (10^4) and reagent volume (mL) is (500~1000): 1.2 (Recommend add 1.2mL reagent 1 for 5 million cells), ultrasionic cell-break on ice (Power 300w, ultrasonic 2 seconds, interval 3 seconds, the total time is 3 minutes). Shock 3h and centrifuge 10 minutes at 8000rpm, 4°C, the supernatant to be tested.
1. Preheat the spectrophotometer 30 min, adjust wavelength to 715 nm.
2. Control tube: take 1mL supernatant, add 0.5mL reagent 2, fully shock 5min, place at room temperature for 5min, take 0.8mL supernatant to 1mL glass cuvette, adjust to zero.
3. Test tube: take 1mL supernatant, add 0.5mL reagent 3, fully shock 5min, place at room temperature for 5min, take 0.8mL supernatant to 1mL glass cuvette, measured the absorbance as A.
Note: The control tube is required for each test sample.
Standard curve: y = 0.0075 x + 0.0055, R2 = 0.994
1. free fatty acid content in the blood is calculated as:
FFA (nmol/mL) = (A - 0.0055)/(0.0075 * Vr)/ Vs = 1333*(A - 0.0055)
2. free fatty acid content in the tissue is calculated as:
1) calculated by sample protein concentration:
FFA (nmol/mg prot) = (A-0.0055)/0.0075 * Vr/ (Vs * Cpr)= 133*(A - 0.0055)/Cpr
2) calculated by the sample quality:
FFA (nmol/g fresh weight) = (A-0.0055)/0.0075 * Vr/(Vs/Vst * W) = 160*(A-0.0055)/W
3. free fatty acid content in the bacteria, fungi is calculated as:
FFA (nmol/10^4 cell) = (A-0.0055)/0.0075 * Vr/(Vs/Vst * cell number) = 160*(A-0.0055)/cell number
Vst = 1.2 mL, represents the total volume of the supernatant;
Vr = 1mL, represents the total reaction volume;
Vs = 1mL, represents the sample volume;
W (g) represents fresh weight of sample;
Cpr (mg/mL) represents protein concentration of the sample.