Photometric Adaptation of Dole's Microdetermination of Free Fatty Acids.

The method is based on the measurement of color changes in phenol red barbital buffer in heptane-ethanol. It has been applied to human serum and to incubation media containing fatty acids liberated from rat epididymal adipose tissue. The values obtained were found to agree with those using the titrimetric method, within the limit of experimental error. A PROCEDURE FOR the determination of long-chain fatty acids based on measurement of the optical density of copper or cobalt soaps extracted into chloroform has been devised by Ayers (1). Even if the sensitivity of this procedure is increased by means of triethanolamine buffer (2) and particularly by the use of diethyldithiocarbamate for copper detection (3-5), some circumstances limit the use of such methods for rapid serial analyses of biological material. For instance, there is appreciable interference by phospholipids, a decrease of selectivity with increasing concentration of triglyceride (4), the necessity for prolonged and vigorous extraction on a mechanical shaker if albumin is present,' the necessity of a somewhat cumbersome separation of the chloroform phase from the water-copper nitrate-triethanolamine mixture, and incomplete recovery of fatty acids added to human serum.2, In the method to be described, fatty acids are extracted first into heptane in a biphasic system as recommended by Dole (6) and then determined photometrically using a simple principle described by Croxatto et al. (7) and suitable modifications for work in a nonaqueous medium. The fatty acids are added to a solution of sodium barbital and phenol red in heptane-ethanol and the optical density is measured. Liberated free diethylbarbituric acid converts the alkaline phenol red into its yellow acid form in almost proportionate amounts since the dissociation constants of phenol red and barbital are within 0.1 unit of each other. The stock buffer indicator consisted of 1% phenol red (Lachema, Brno, Czechoslovakia) in 0.12 M sodium barbital (0.25 g per 10 ml). This solution was diluted before use: 99 ml of absolute ethanol and then 200 ml of heptane were added to 1 ml of the buffer and placed in the automatic burette. Similarly the heptane, distilled water, and extraction mixture (isopropyl alcohol-heptane-N sulphuric acid 40 : 10 : 1) were stored in and added from an automatic burette. The changes during storing of these solutions was negligible and without influence on the final analytical values. Procedure. One milliliter of fatty acids dissolved in heptane, and heptane alone (blank), were placed in clean, dry, matched cuvettes. In this study tubes of 1 cm diameter were used. Heptane was measured out from a 1 ml pipette operated by an injection syringe connected to the pipette by means of plastic tubing. In some cases Reagents.