LabAssay (TM) NEFA

※For research purposes only with animal serum/plasma, Not for Diagnostic Use.

Introduction

NEFA (Non‐esterified fatty acid) in the blood is transported complexed with an albumin to peripheral tissues. They are important sources of fuel for the peripheral tissues. The concentration of NEFA in the blood is regulated by a release from the adipose tissues, a consumption in the periphral tissues or a take up from the liver.

Assay principle

NEFA (Non‐esterified fatty acid) forms Acyl-CoA in the presence of Acyl-CoA synthetase (ACS). Hydrogen peroxide, which is produced by a reaction between the Acyl-CoA and Acyl-CoA oxidase (ACOD), promotes oxidative condensation of 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline (MEHA) with 4-aminoantipyrine. LabAssay™ NEFA can be used for the quantitative determination of NEFA in samples by measuring absorbance of a blue purple color which is generated by the oxidative condensation reaction.

• Performance

  Sample	Serum, plasma(Mouse, Rat, dog, cat)
  Standard curve range	0.4～1.97 mEq/L
  Measurement time	approx. 20 min.
  Amount of sample	4 μL
  Measurement wavelength	550 nm

• Standard Curve

  

Assay in a microplate
Perform the assay in the wells according to the following table scheme.

*1: The test sample volume is usually 4 μL, but 8 μL is taken in this case. The NEFA concentration must be corrected accordingly as indicated in the table above.

Spike-and-Recovery Test

Spike and Recovery Test of NEFA, a known amount of standard is spiked into in mouse and rat sample n=2.

• Rat Sample

  	Spike (mEq/L)	Mean (mEq/L)	Recovery (mEq/L)	Recovery (%)
  Serum	-	0.57
  	0.40	1.00	0.43	106.4%
  	0.61	1.21	0.65	106.2%
  	0.78	1.40	0.83	106.4%
  	1.01	1.57	1.01	99.7%
  Plasma (EDTA)	-	0.41
  	0.40	0.86	0.45	111.5%
  	0.61	1.07	0.66	108.5%
  	0.78	1.27	0.86	109.5%
  	1.01	1.44	1.02	101.4%

• Mouse Sample

  	Spike (mEq/L)	Mean (mEq/L)	Recovery (mEq/L)	Recovery (%)
  Serum	-	0.70
  	0.40	1.07	0.37	91.7%
  	0.61	1.31	0.61	100.1%
  	0.78	1.44	0.74	94.5%
  	1.01	1.62	0.92	91.3%
  Plasma (EDTA)	-	0.62
  	0.40	0.99	0.37	92.5%
  	0.61	1.29	0.68	111.5%
  	0.78	1.38	0.77	97.8%
  	1.01	1.62	1.00	99.1%

Linearity-of-Dilution Test

• Rat sample

  

• Mouse sample

  

• D-MEM/10% FBS

  

Examples of measurement

• 20% Diluted Nomal Mouse serum, CD-1(ICR), male
  No.	Mean (mEq/L)	SD	CV(%)
  1	1.118	0.001	0.1
  2	0.788	0.007	0.9
  3	0.715	0.022	3.1
  4	0.794	0.002	0.2
  5	0.829	0.005	0.6

• Nomal Mouse plasma K2EDTA, CD-1(ICR), male
  No.	Mean	SD	CV(%)
  1	*0.309	0.002	0.6
  2	*0.320	0.002	0.6
  3	*0.205	0.004	2.0
  4	*0.179	0.008	4.6
  5	*0.305	0.017	5.6

• 10% Diluted Nomal Rat serum, Sprague Dawley, male
  No.	Mean	SD	CV(%)
  1	*0.300	0	0.0
  2	*0.371	0.004	1.1
  3	*0.289	0.017	5.8
  4	*0.365	0	0.1
  5	*0.382	0.029	7.6

• Nomal Rat plasma K2EDTA, Sprague Dawley, male
  No.	Mean	SD	CV(%)
  1	*0.333	0.004	1.2
  2	*0.356	0.003	0.8
  3	*0.325	0.020	6.0
  4	*0.218	0.005	2.6
  5	0.515	0.010	1.9

• Nomal Canine serum, Beagle, male
  No.	Mean (mEq/L)	SD	CV(%)	Age(years)
  1	0.656	0.0027	0.4	4
  2	0.665	0.0320	4.8	6
  3	0.425	0.0090	2.0	6
  4	*0.38	0.0057	1.5	11
  5	0.882	0.0070	0.8	12
  6	0.599	0.0060	1.1	6
  7	1.006	0.0267	2.7	10
  8	0.506	0.0060	1.2	4

• Nomal Canine plasma EDTA, Beagle, male
  No.	Mean (mEq/L)	SD	CV(%)	Age(years)
  1	0.442	0.0033	0.7	4
  2	0.427	0.0110	2.6	6
  3	*0.308	0.0037	1.2	10
  4	0.478	0.0123	2.6	12
  5	0.473	0.0050	1.0	6
  6	*0.367	0.0047	1.2	11
  7	0.780	0.0133	1.7	10
  8	0.411	0.0387	9.4	12

• Nomal Feline serum, Short hair, male
  No.	Mean (mEq/L)	SD	CV(%)	Age(years)
  1	0.717	0.0117	1.6	3
  2	0.635	0.0100	1.6	3
  3	1.004	0.0053	0.5	6
  4	0.981	0.0093	0.9	4
  5	0.636	0.0170	2.7	6
  6	1.193	0.0267	2.3	6
  7	1.022	0.0893	8.8	1
  8	*0.382	0.0157	4.1	1

• Nomal Feline plasma EDTA, Short hair, male
  No.	Mean (mEq/L)	SD	CV(%)	Age(years)
  1	1.099	0.0103	0.9	0.5
  2	*0.174	0.0033	2.0	1
  3	*0.241	0.0043	1.7	3
  4	*0.320	0.0067	2.2	0.5
  5	*0.359	0.0060	1.7	3
  6	*0.197	0.0120	6.2	3
  7	*0.215	0.0240	11.1	0.5
  8	*0.131	0.0160	12.3	1

References

Kobayashi, Y. et al. : J. Pharmacogn. Nat. Prod., online (2015). Effects of Morus alba L. (Mulberry) Leaf Extract in Hypercholesterolemic Mice on Suppression of Cholesterol Synthesis *Extraction liquid of mouse liver

Gao, F. et al. : Evid. Based Complement. Alternat. Med., 2015, 801291 (2015). Effect of Keishibukuryogan on Genetic and Dietary Obesity Models *Extraction liquid of rat liver

Ogawa, K. et al. : Reprod. Med. Biol., online (2018). Non-esterified fatty acid-associated ability of follicular fluid to support porcine oocyte maturation and development *Pig cumulus-oocyte complexes

Wang, F. et al. : J. Mol. Endocrinol., 52, 133 (2014). LGR4 acts as a link between the peripheral circadian clock and lipid metabolism in liver *Mouse plasma

Uchida, K. et al. : Exp. Anim., 58, 181 (2009). Hyperlipidemia and hyperinsulinemia in the spontaneous osteoarthritis mouse model, STR/Ort *Mouse serum