Lipolysis is the breakdown of lipids, whereby triglycerides are hydrolysed to produce free fatty acids (FFA) (also known as non-esterified fatty acids).  The non-esterified fatty acids (NEFA) are then degraded further by a process known as beta oxidation which produces ketones.  Therefore measurement of NEFA can provide information about metabolic states and energy balance.  Serum NEFA can be increased due to starvation, cold, fear or smoking and can be decreased by physical exercise.  Changes in NEFA are also observed in individuals with diabetes, hepatic or endocrine diseases.  

NEFA measurement may be of interest to those investigating, diabetes mellitus, insulin resistance, obesity, metabolic syndrome, nutrition, sports metabolism and dairy herd health.

The Wako NEFA-HR(2) reagents can be used to measure NEFA in serum and are suitable for both manual and automated methods.  (A range of applications for automated chemistry analysers are available – please enquire. A manual microplate protocol is also available on request). NEFA-HR(2) replaces the popular NEFA-C Kit which was withdrawn in 2010 and utilises the same method principles.

The Wako NEFA assay is a enzymatic, colorimetric method (ACS-ACOD-MEHA).  NEFA present in the test sample is first converted to Acyl-CoA, AMP and pyrophosphoric acid (PPi) by the action of Acyl-CoA synthetase (ACS) in the presence of coenzyme A (CoA) and adenosine 5-triphosphate disodium salt (ATP).  The resulting Acy-CoA is then oxidised by the action of Acyl-CoA oxidase (ACOD) to yield 2,3-trans-Enoyl-CoA and hydrogen peroxide.  In the presence of peroxidase (POD) the hydrogen peroxide yields a blue-purple pigment by quantitative oxidation condensation with 3-Methyl-N-Ethyl-N-(beta-Hydroxyethyl)-Aniline (MEHA) and 4-aminoantipyrine (4-AA).  The concentration of

NEFA in the sample is subsequently determined by measuring the absorbance of the blue-purple pigment at 546nm. 

NEFA-HR(2) Reagent 2 (436-91995) is one component of the Wako NEFA Kit and should be used with Reagent 1 (434-91795) and the NEFA Standard (270-77000).   The Wako NEFA assay has a measurable range of 0.01-4.00 mEq/L and is not significantly effected by haemolysis or ascorbic acid. Once reconstituted: reagent 1 is stable for 10 days when stored at 2-10C and reagent 2 is stable for up to 3 weeks at 2-10C.