Abstract
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 C(10) 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.
Highlights
At constant uranyl and Rhodamine B concentrations,a plot of optical density vs. Free fatty acids (FFA) concentration yields two straight lines of different slope, Le., a biphasic standard curve
The proton released from the fatty acid changes Rhodamine B to the cationic form, which complexes with the uranylfatty acid ion
We have found that when the fatty acid has a chain length of four carbons or more, the complex has some solubility in benzene and toluene; if the chain is longer than Clo, the complex has no detectable solubility in water and is completely extracted into the aromatic hydrocarbon phase
Summary
At constant uranyl and Rhodamine B concentrations,a plot of optical density vs. FFA concentration yields two straight lines of different slope, Le., a biphasic standard curve. THERE IS an increasing need for methods that measure microquantities of free fatty acids in biological systems and food products. Dole and Meinertz (1) have described a method for extraction of free fatty acids from plasma and other biological fluids (using a heptane-isopropyl alcoholsulfuric acid system), and their determination by microti-. Baker (2) reported a colorimetric procedure for determining free fatty acids in grain by the formation of copper soaps in benzene. Iwayania ( 3 ) reported the formation of copper soaps in chloroform, and Duncombe (4) increased the sensitivity of this method by using sodium diethyl dithiocarbamate for the detection of copper. Most investigators have used the Dole extraction procedure (1) or its modification as reported by Trout, Estes, and Friedberg (6) for the isolation of free fatty acids. Feigl (10) suggested the use of Rhodamine B for fatty acid detection as a spot test, but did not discuss its suitability for microquantitative analysis
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