Non-Esterified Fatty Acid (NEFA) Assay Kits

Among the fatty acids that make up lipids (fats), free fatty acids (FFA), or non-esterified fatty acids (NEFA), refer to those in a free state (not esterified with glycerol) that are used as an energy source in the body. Fatty acids are usually bonded to glycerol and stored as neutral fats (triglycerides or triacylglycerols). When energy is needed, the neutral fats are broken down into fatty acids (Figure 1). Because fatty acids are not readily soluble in water, they are present in the blood bound to albumin.

Free fatty acids play an important role as components of cell membranes and in energy metabolism (Figure 2). In tissues such as muscles and the liver, free fatty acids undergo oxidation in the mitochondria to become acetyl-CoA, and energy is produced through the citric acid cycle. Excessive free fatty acids in the blood, however, can lead to peripheral insulin resistance. Additionally, increased uptake of free fatty acids in the liver can cause non-alcoholic fatty liver disease (NAFLD) and raise blood sugar levels¹⁾.

Free fatty acids in serum make up about 4-5% of total fatty acids and serve as an important energy source for peripheral tissues³⁾.
LabAssay™ NEFA (FFA) is based on an enzymatic colorimetric method (ACS-ACOD method) using the Trinder reagent. With the use of enzymes, this kit provides a convenient method for measuring free fatty acids in samples in a microplate format.

Principle of the NEFA assay using the ACS-ACOD method

Free fatty acids in the sample are converted into acyl-CoA, AMP, and pyrophosphate (PPi) through the action of acyl-CoA synthetase (ACS) in the presence of coenzyme A (CoA) and adenosine-5'-triphosphate disodium (ATP). The resulting acyl-CoA is then oxidized by acyl-CoA oxidase (ACOD), simultaneously generating 2,3-trans-enoyl-CoA and hydrogen peroxide. The resulting hydrogen peroxide induces oxidative condensation of 4-aminoantipyrine and MEHA through the action of peroxidase (POD), forming a blue-purple dye. The absorbance of this blue-purple dye is measured to determine the concentration of free fatty acids in the sample.

1. Ishikawa, K. & Yokote K.: Nihon Naika Gakkai Zasshi, 102(10), 2691(2013).
   Dysfunction of Adipose Tissue and Insulin Resistance (Japanese)
2. Okumura, N., Tozuka, M., Honda, T. and Yatomi, Y.: "Manual of Clinical Laboratory Medicine 35th ed.", Kanehara shuppan, (2010). (Japanese)
3. University of Hyogo [脂肪の代謝とその調節]: https://www.sci.u-hyogo.ac.jp/life/molbio/KOKAI.pdf (Apr. 25th, 2023). (Japanese)