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Journal of Lipid Research, Vol. 8, 589-597, November 1967
Copyright © 1967 by Lipid Research, Inc.

Rapid colorimetric micromethod for free fatty acids

Robert D. Mackenzie , Thomas R. Blohm , Edward M. Auxier , and Andrew C. Luther

Department of Biochemistry, The Wm. S. Merrell Company, Division of Richardson-Merrell Inc., Cincinnati, Ohio 45215

Free fatty acids (FFA), the uranyl ion, and the basic dye Rhodamine B form colored complexes, which are extractable into toluene or benzene. Fatty acids of different chain lengths above C₁₀ and different degrees of unsaturation gave constant molar yield. Complexes in toluene alone are unstable, especially in the light, but a small amount of aqueous uranyl acetate stabilizes them sufficiently for determination.

At constant uranyl and Rhodamine B concentrations, a plot of optical density vs. FFA concentration yields two straight lines of different slope, i.e., a biphasic standard curve. Phospholipids interfere, and must be removed with zeolite during FFA extraction. Recovery of FFA added to rat plasma was very similar to that with titration. Assay of rat and dog plasma samples under fasting and fed conditions gave good agreement with the titration method. Values of human plasma samples tended to be higher by the colorimetric procedure; a few samples gave significant disagreement.

The method compares well with previous methods in sensitivity and accuracy, and offers advantages in speed, simplicity, and possibly specificity.

Supplementary key words free fatty acids • colorimetric assay • Rhodamine B • uranyl ion

Submitted on December 29, 1966
Accepted on July 7, 1967

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