Non-esterified free fatty acids NEFA enzymatic colorimetric assay serum 0.01-4.00 mEq/L enzymatic colorimetric method FUJIFILM Wako Reagent 2 Set

Non-esterified free fatty acids NEFA HR-2 Assay Reagent 2 Set manufactured by FUJIFILM Wako Pure Chemicals for measurement of NEFA in serum.

NEFA (non-esterified fatty acids) are free fatty acids (FFA) as opposed to triglycerides (esterified fatty acids) and measuring serum NEFA provides information about energy balance and fat mobilisation. NEFA in serum binding albumin, is used as an important energy source of peripheral tissues. The amount of NEFA in serum depends on a balance between intake in liver and peripheral tissues, and the release from adipose tissues. Amount of NEFA decreases by physical exercise, increases by starvation, cold, fear or smoking. And then increase or decrease of NEFA is observed in diabetes, hepatic diseases or endocrine diseases.

NEFA had been assayed by organic solvent extraction method, which was complicated to operate. Enzymatic method using acyl-CoA-oxidase (ACOD) has become widespread due to excellent specificity and concise procedure. NEFA-HR(2) is the reagent kit for NEFA assay based on enzymatic method using 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline (MEHA) as a violet colour agent.

Firstly the NEFA present in the test sample is 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 Acyl-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). NEFA concentration is obtained by measuring absorbance of the blue-purple colour.

NEFA-HR(2) Reagent 2 (990-91901) is one component of the Wako NEFA Kit and should be used with Reagent 1 (994-91801) and the NEFA Standard (991-91791).
The Wako NEFA assay has a measurable range of 0.01-4.00 mEq/L and is not significantly affected by haemolysis, bilirubin. Intralipid, or ascorbic acid.

Once reconstituted both reagents are stable for up to 1 month when stored at 2-10C.