Free fatty acid quantitation kit mak044

Serum samples were assessed on an individual mouse basis in duplicate, for enzymatic colorimetric determination of uric acid, total cholesterol, triglycerides, and free fatty acids following the manufacturer’s instructions, using Abcam (Cambridge, MA, USA; Uric Acid Assay Kit, ab65344,), CHRONOLAB SYSTEMS S.L. (Barcelona, Spain; Cholesterol-LQ; Triglycerides), and Sigma-Aldrich (St. Louis, MO, USA, Free Fatty Acid Quantitation Kit, MAK044), respectively. Levels of circulating unbound, free ARA were evaluated on an individual mouse basis, in duplicate or quadruplicate wells, by competitive ELISA using AA (Arachidonic Acid) ELISA Kit (E-EL-0051, Elabscience Biotechnology Co., Ltd., WuHan, China) following the manufacturer’s instructions. Absorbance readings of the uric acid, free fatty acids and ARA standard dilutions were plotted versus concentration values using Excel scatter graph and formula and serum sample concentrations and were expressed as mg/dL, nmole/mL and ng/mL, respectively.

TG, FFA, glycerol, glucose, and insulin levels were measured with various measuring kits according to the manufacturer’s instructions. Triglyceride Quantification Colorimetric/Fluorometric Kit (K622) was from Biovision; Free Fatty Acid Quantitation Kit (MAK044) was from Sigma; Glycerol Quantitation Kit (MAK117) was from Sigma; and Ultra Sensitive Mouse Insulin ELISA Kit (90080) was from Crystalchem. Blood glucose levels were measured using a blood glucose meter (Accu-Chek Aviva Diabetes Blood Glucose Monitoring System, Roche). For the GTT and ITT, mice were fasted for 6 hours, followed by i.p. injection with glucose (2 g/kg) or human insulin (1 unit/kg).

Serums from each rat were centrifuged at 825 g for 20 min and 4 °C to determine lipid ratios. Stored at −80 °C until the analysis. Total cholesterol (Sigma-Aldrich Cholesterol Quantitation Kit MAK043), triglyceride (Sigma Serum Triglyceride Determination Kit TR0100), phospholipid (Sigma-Aldrich Phospholipid Assay Kit MAK122), and free fatty acid (Sigma-Aldrich Free Fatty Acid Quantitation Kit MAK044) analysis were performed in each serum sample.
Liver and kidneys were stored at −80 °C in phosphate buffer (1/10 w/v) to determine total lipid amounts. The liver and kidneys in phosphate buffer (20 mL) were put into an extraction thimble that contained chloroform/methanol (40 mL; 3:2 v/v), separately. The obtained mixture was homogenized at 155 rpm for 15 min. Then, water (8 mL) was added, and the mixture was shaken vigorously to facilitate the transfer of oil into the chloroform and other products into the water–methanol layer. The chloroform layer was then separated via a separation funnel and collected. The extraction steps were repeated two times. The obtained chloroform layers were combined and evaporated using an evaporator (65 °C, for 20–30 min.). Then, the trace amount of chloroform was evaporated in drying oven and weighed remaining oil [59 (link)].