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Biochemical Assay Kits - LabAssay™ Series -

Non-Esterified Fatty Acid (NEFA) Assay Kits

Free fatty acids, among the fatty acids that make up lipids (fats), refer to those in a free state (not esterified with glycerol) that are used as an energy source in the body. Free fatty acids in serum make up about 4-5% of total fatty acids and serve as an important energy source for peripheral tissues.

LabAssay™ NEFA is a kit for the determination of non-esterified fatty acids (NEFA) in blood (serum/plasma) or cell culture supernatants using the ACS-ACOD method. With the use of a microplate, this kit provides a quick and convenient method for measuring free fatty acids in samples.

What is Non-Esterified Fatty Acid (NEFA)?

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.

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Figure 1. Breakdown of neutral fats (triglycerides) and formation of fatty acids

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).

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Figure 2. Fats and energy production2)

Methods of NEFA Measurement

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 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.

Principle of the NEFA assay using the ACS-ACOD method

Figure 3. Principle of the NEFA assay using the ACS-ACOD method

LabAssay™ NEFA

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LabAssay™ NEFA is a kit used for the determination of free fatty acids (NEFA) in blood (serum/plasma) or cell culture supernatants using the ACS-ACOD method. With the use of a microplate, this kit provides a quick and convenient method for measuring free fatty acids in samples.

[Note] LabAssay™ series are reagents for research purposes. They cannot be used for diagnostic purposes.

Kit Performance

Animal Human, Mouse, Rat, Dog, Cat
Sample Serum, Plasma, Culture supernatants
Calibration curve range 0.4-1.97 mEq/L
*Oleic acid 1 mEq=1 mmol
Sample volume 4 μL
Measurement duration Approx. 20 min
Wavelength 550 nm

References

  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)

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LabAssay™ Series

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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