Technical note: an improved method to quantify nonesterified fatty acids in bovine plasma

Abstract

Nonesterified fatty acids (NEFA) are measured routinely in the study of nutrition and growth physiology. To improve the efficiency with which this variable is measured, a two-reaction, enzymatic-based assay was adapted and validated to quantify NEFA in bovine blood plasma using 96-well microtiter plates. The effects of incubation time, temperature, and of sample volume were examined in addition to possible interfering substances, recovery, reagent stability, and range of linearity. Incubation for 30 min at 21 degrees C for each of the two reactions resulted in maximal slope and r2 values (1.94 and .999, respectively). Percentage of recovery was 107% when using 5 microL and 100% when using 10 microL of heparinized bovine plasma in the assay. Uniform linear standard curves (r2 > .99) were obtained using reagents stored at 4 degrees C for 9 d. An equal volume of 1 mM acetate, propionate, valerate, and butyrate added to plasma did not affect results. The assay was linear from 125 to > 1,000 microEq/L. Intra- and interassay CV for the 5-microL sample volume were 3.6 and 3.7%, respectively. This modified assay provides results comparable to the standard assay yet reduces reagent and labor requirements and increases sample capacity.

Consumo, digestibilidade de nutrientes e parâmetros sanguíneos de cabras mestiças moxotó suplementadas com óleos de licuri ou mamona

“…Após a coleta, o material foi centrifugado a 3.500rpm, por 15 minutos, e o soro resultante foi congelado a -20ºC, para análises posteriores. As determinações das concentrações de ureia e glicose foram feitas com kits disponíveis no comércio (Labtest diagnóstica SA), empregando-se o método enzimático-colorimétrico, já a concentração de Ácidos Graxos não Esterificados (AGNE) foi determinada pelo método de JOHNSON & PETERS (1993), utilizando-se o kit comercial teste NEFA C wako. Todas essas determinações foram analisadas pelo aparelho espectofotômetro.…”

Detection of Nonesterified (Free) Fatty Acids in Bovine Serum: Comparative Evaluation of Two Methods

“…However, a modified procedure using an automated microtechnique with the kit offers several advantages as has been reported elsewhere. 8,18,19 Two published reports were found in which the NEFA C kit was modified for the use of a 96-well microtiter plate. One study used a modified enzymatic procedure based on an nicotinamide adenine dinucleotide reduced-luciferase system applied to serum (2 l) and milk (5 l) samples with the use of a luminometer.…”

“…8,18,19 However, to authors' knowledge, published reports that deal with method comparison for detection and quantitation of NEFA have used least-square regression analysis and comparison of extinction coefficients to assess agreement between the methods. 5,7,8,10,18,19,30,39 This approach, though widely used in analytical data evaluation, is inappropriate for use in method comparison studies.…”

“…8,18,19 However, to authors' knowledge, published reports that deal with method comparison for detection and quantitation of NEFA have used least-square regression analysis and comparison of extinction coefficients to assess agreement between the methods. 5,7,8,10,18,19,30,39 This approach, though widely used in analytical data evaluation, is inappropriate for use in method comparison studies. 15,25,27,32,33 It has been shown that 2 methods can have a high correlation, but may never agree, suggesting that correlation is only a measure of association between the methods and not a measure of agreement.…”

“…As previously reported, an adaptation of the ''NEFA C'' kit to an automated microtechnique offers several advantages over the manual colorimetric UVS method, including higher sample throughput, the ability to simultaneously analyze samples and standards, need for smaller sample sizes (10 vs. 50 l) and decreased cost per assay. 8,18,19 Cost savings are due to the ability to run more tests per kit (250 vs. 50) and need for less technician time for sample analysis.…”