Free fatty acid assay kit

Lipids were extracted and dried down with nitrogen gas from equal amounts of purified WT and mutant forms of LBP-8 using the Bligh and Dyer method as described above^{47 (link)}. The dried lipid extracts were resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH = 8.0). The total amount of fatty acid for each sample was determined using the Free Fatty Acid Assay Kit (Colorimetric), Cell Biolabs, San Diego, CA, USA. Data was analyzed in GraphPad Prism 7. All data represents the average of three replicates.

Triglycerides (TAG) are the digestive end product of breaking down dietary fats, and serve as an energy source and play a key role in metabolism. Secreted enzyme lipases hydrolyze the triglyceride ester bond, yielding glycerol and free fatty acids (FFA) in a process called lipolysis. Measurement of free fatty acids is important in metabolic diseases and cancer. We quantitated FFAs by using Free Fatty Acid Assay Kit (Cell Biolabs, San Diego, CA) that measures non-esterified fatty acids (NEFA) in serum and plasma by a coupled enzymatic reaction system (ACS-ACO Method). First, Acyl CoA Synthetase (ACS) catalyzes fatty acid acylation of coenzyme A. Next, the acyl-CoA product is oxidized by Acyl CoA Oxidase (ACO); producing hydrogen peroxide, which reacts with the colorimetric probe and gives absorbance at 570 nm. Palmitic acid was used as standard.

The hepatic metabolite levels for glycogen, triglyceride, non-esterified free fatty acid (NEFA) and total cholesterol were determined using commercially available kits from Cell Biolabs (CA, USA). Ultrasonic disruption in PBS containing 0.5% Triton X-100 was used to homogenize 200 mg of liver tissue, which was centrifuged at 5000 xg for 10 minutes at 4°C. The supernatant was collected, stored on ice, and used for glycogen and triglyceride quantification using the Glycogen Assay Kit (Cell Biolabs), and Serum Triglyceride Quantification Kit (Cell Biolabs), respectively. For cholesterol and free fatty acid quantification [26 (link)], 10 mg of liver tissue was extracted with 200 μL of a chloroform:isopropanol:NP-40 (7:11:0.1) mixture in a micro-homogenizer. The extract was centrifuged at 15,000 xg for 10 minutes, and the organic phase was transferred to a new tube and air dried at 50ºC to remove the chloroform. The dried lipids were dissolved and homogenized in 200 μL of 1X Assay Diluent via vortexing. Cholesterol and NEFA were quantified using Total Cholesterol Assay Kit (Cell Biolabs) and Free Fatty Acid Assay Kit (Cell Biolabs), respectively.