Print

VETSPEC™ REAGENTS - Nonesterified Fatty Acids (NEFA)

Intended Use

For IN VITRO diagnostic use in the manual or automated, quantitative determination of Nonesterified Fatty Acids (NEFA) in serum or plasma.

Method Principle

Most previously used procedures for Nonesterified Fatty Acids were based on organic solvent extractions, titration and gas liquid chromatography. These procedures are complicated, time consuming and restricted to manual assays. The Catachem NEFA procedure is based on the enzymatic synthesis of thiols esters of CoA, known as Acyl-CoA by the activity of Acyl-CoA Synthatase (ACS) in the presence of ATP and CoA. The Acyl-CoA thus formed is then oxidized in a second reaction by Acyl-CoA Oxidase (ACOX) to produce 2,3-trans-trans-enoyl-CoA and Hydrogen Peroxide. The Hydrogen Peroxide is then quantitated by the oxidative condensation of N-Ethyl-(3-sulfopropyl) aniline (ALPS) with 4-aminoantipyrine to produce a Quinoneindamine dye with maximum absorption at 550 nm. The increase in absorbance is directly proportional to the concentration of NEFA  in the original serum sample. The following reaction scheme illustrates the reactions that take place in this NEFA procedure.

ACS
      NEFA + ATP + CoA —————> Acyl-CoA + AMP + PPi

ACOX
                      Acyl-CoA + O2 ——————> 2,3-trans-Enoyl-CoA + H2O2

Peroxidase
                 H2O2 + 4AA + ALPS ——————> Quinoneindamine Dye + 4H2O

 

Reagent Storage And Stability

Store the Catachem NEFA reagents at 2-8°C. When stored as directed, these reagents are stable until expiration date stated on the label.

Working Reagent Preparation

Reconstitute the required number of vials of Catachem NEFA Sample Diluent (R1) and Enzyme Color Reagent (R2) with the volume of Reagent Diluent indicated on the label of each reagent vial. Mix reconstituted reagents by inversion, gently to prevent foaming. Store the Working Reagents at 2-8°C. When stored and prepared as directed, the Catachem NEFA Working Reagents are stable for 30 days.

Method Performance Characteristics

Sensitivity: Using a path length of 1 cm, a D-absorbance of 0.1-0.20 per mmol/L should be obtained.
Linearity: This procedure is linear over the range of 0-2.5 mmol/L.
Precision: Precision data was obtained using five levels of protein based controls and following the NCCLS EP5-T2 procedure (5). The following results were observed:

Nonesterified Fatty Acids (NEFA)

Within-Run Precision

Total Precision

MEAN

SD

CV

SD

CV

mmol/L

mmol/L

%

mmol/L

%

0.169

0.0060

3.356

 

 

0.443

0.0087

1.953

 

 

1.020

0.0149

1.461

 

 

1.515

0.0169

1.078

 

 

Accuracy:

Using an automated analyzer, correlation studies were carried out between this Catachem NEFA procedure (Y) and a commercially available NEFA test kit as reference (X). Serum samples were assayed and the results compared by the least squares regression. The following statistics were observed:

Correlation Data

Parameter

Data Observed

N

44

Range (mmol/L)

0.1-2.3

Mean of Y

0.5295

Mean of X

0.5841

Regression

Y = 0.956x + 0.28

Correlation

r = 0.9949

Sy,x

0.0054