Use of acetyl chloride/methanol for assumed selective methylation of plasma nonesterified fatty acids results in significant methylation of esterified fatty acids

The albumin-bound nonesterified fatty acid pool in plasma, which represents a very small percentage of total plasma fatty acids, has previously been quantitated by a variety of methods. In the present study we determined that the nonesterified fatty acid concentrations in the plasma, quantitated by a popular method using acetyl chloride and methanol which is reported to be specific for methylation of nonesterified fatty acids in the presence of esterified fatty acids (i.e., without prior isolation of the plasma nonesterified fatty acids), were significantly overestimated due to cleavage and methylation of esterified fatty acids. Quantitation of the contaminating fatty acid from the esterified pool demonstrated that the amount of fatty acid cleaved from the esterified pool was enough to exceed the entire mass of nonesterified fatty acids. As an established method for comparison, we isolated nonesterified fatty acids from the plasma by thin-layer chromatography prior to methylation, using a number of simple precautions to limit oxidation. By performing all thin-layer chromatography steps in an atmosphere of nitrogen and by including fatty acid standards in the plasma with 0, 1, 2 or 4 double bonds, we were able to accurately and reproducibly determine the concentration of nonesterified fatty acids in the plasma, including arachidonate. We demonstrated that no oxidation occurred in the thin-layer chromatographic isolation of nonesterified fatty acids and that the coefficients of variation for repeat measurements of the same sample were < 11% using our reference method. Our data indicate that the use of acetyl chloride and methanol for assumed selective methylation of plasma nonesterified fatty acids results in significant methylation of esterified fatty acids.