Abstract
A modification of the semiautomated assay method of Antonis (1965. J. Lipid Res. 6:307-312) for free fatty acid is presented. Free fatty acids are extracted from serum or plasma into di-n-butyl ether-2-methoxyethanol; the extract is almost free from phospholipids. The acids are analyzed in a portion of extract by a copper soap method using diphenylcarbazide. The extractant, being less dense than water, is easily separated from an aqueous phase both in the extraction of samples and in the assay of copper soaps. The assay is comparable in accuracy with well-tried titrimetric methods and is quicker and easier to operate.
Highlights
We present here a new technique in which free fatty acid (FFA) are extracted manually and can be assayed by a continuous flow method on a Technicon AutoAnalyzer
A novel extraction solvent containing 2-methoxyethanol and di-n-butyl ether which achieves quantitative recovery of FFA without contamination by phospholipids; being less dense than water, it can be conveniently separated from an aqueous phase during the extraction of samples and in the subsequent assay
The extractant consists of 2-methoxyethanol-di-n-butyl ether 1:1 (v/v)
Summary
All reagents are general purpose reagents except where otherwise stated. Bovine serum albumin (BSA; Cohn fraction V) was obtained from Armour Pharmaceuticals, Brighton, U.K., and defatted when so required by the method of Chen (19) using activated charcoal. Radioactive fatty acid, [9,10-3H]palmiticacid and [3H]-n-hexadecanestandard were obtained from the Radiochemical Centre, Amersham, U.K. Lecithin (essential phospholipid, EPL, a polyunsaturated phosphatidylcholine) was obtained from Nattermann, Koln, West Germany. Tripalmitin, oleic acid, cholesterol, and cholesteryl oleate were obtained from Sigma, London, U.K. Extraction of FFA. The extractant consists of 2-methoxyethanol-di-n-butyl ether 1:1 (v/v). The other reagent required for extraction of serum or plasma is a 0.1 M solution of glycine in water, adjusted to pH 2.7 with sulfuric acid
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