The most frequently used method for estimating free fatty acids (FFA) in biological tissues is the titrimetric procedure of Dole (1, 2). This technique requires appreciable experience and observer bias can easily affect, the titration values (3).
A sensitive calorimetric method was proposed by Duncombe (4). It is based on the transfer of copper soaps into chloroform (5) and detection of copper by diethyldithiocarbamate.
Itaya and Ui (6) applied Duncombe's procedure for determining FFA in biological fluids and determined the conditions for the transfer of FFA from an aqueous phase into chloroform. Attempts to use their technique for in vitro assay of lipoprotein lipase activity (measurement of FFA liberated from a triglyceride emulsion) were unsuccessful. The high albumin concentration required in the incubation system always resulted in the production of an emulsion when an aliquot of the assay system was extracted with the chloroform phosphate mixture, as described by Itaya and Ui (6). Substitution of this extraction system by Dole's extraction mixture (1 N H,SO, 0.1 vol, heptane 1 vol, isopropyl alcohol 4 vol) followed by direct addition of chloroform to the heptane phase was also unsatisfactory. Very high blank values were obtained, probably due to the presence of isopropanol retaining copper in the organic phase.
The procedure described here combines the best features of several previously described methods: (a) it uses the sensitive calorimetric reaction; (b) Dole's extraction method (1) does not cause emulsification even when the aqueous phase contains a high concentration of albumin; (c) washing the heptane phase with dilute H,SO, as described by Trout et al. (7) reduces the blank to a low value; (d) use of a dense aqueous