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

Area of Science:

• Animal Science
• Biochemistry
• Analytical Chemistry

Background:

• Nonesterified fatty acids (NEFA) are crucial biomarkers in bovine nutrition and growth physiology.
• Current measurement methods can be labor-intensive and costly, necessitating more efficient alternatives.

Purpose of the Study:

• To adapt and validate a two-reaction, enzymatic assay for quantifying NEFA in bovine blood plasma.
• To optimize the assay for use with 96-well microtiter plates, enhancing throughput and efficiency.

Main Methods:

• A modified two-reaction enzymatic assay was developed and validated using bovine plasma.
• Parameters such as incubation time, temperature, sample volume, and potential interfering substances were systematically evaluated.
• Assay linearity, reagent stability, and recovery rates were determined.

Main Results:

• Optimal incubation conditions were identified as 30 minutes at 21°C for both reactions, yielding high linearity (r² = 0.999).
• High recovery rates (100-107%) were achieved with sample volumes of 5-10 µL of heparinized plasma.
• The assay demonstrated excellent reagent stability (9 days at 4°C), linearity (125-1000 µEq/L), and low intra- and inter-assay coefficients of variation (3.6-3.7%).

Conclusions:

• The modified enzymatic assay provides a reliable and efficient method for measuring NEFA in bovine plasma.
• This optimized assay reduces reagent and labor requirements while increasing sample capacity compared to standard methods.
• The validated assay is suitable for routine use in nutritional and physiological studies of cattle.