MATERIALS AND METHODS

For this initial investigation, the procedure of Davis (2) for the determination of UFA in blood plasma was employed, with modifications as follows: Blood samples were taken from antecubital veins except where other-wise noted (a tourniquet being applied briefly), treated with solid sodium oxalate 1.5 mg. per ml. to prevent clotting, and immediately chilled in ice water. Centri-fugation was carried out in the cold, and the extract pre-pared within four hours. These precautions were intended to reduce in vitro lipolytic action which might cause artifactual elevation of the UFA concentration. Cold storage of normal or lipemic plasma samples for periods of up to four hours has been shown not to re-sult in significant changes of UFA concentrations. To 2 ml. of plasma were added, in order, 1 ml. phosphate buffer of pH 6.0 and molarity 0.2, 1 ml. 5 per cent sodium dodecyl sulfate, 9 ml. saturated aqueous sodium sulfate, and finally a slight excess of anhydrous sodium sulfate powder. The reagents utilized were of the best com-mercial grade. Five extractions with 2-ml. portions of ethyl ether were carried out as described by Davis. Af-ter gentle evaporation of the ether, the residue was taken up in 5 ml. 95 per cent alcohol, a drop of approximately. 01 normal sulfuric acid was added, and the solution titrated. The titrant was. 02 normal sodium hydroxide (aqueous solution) deliveredfrom a Gilmont ultramicroburette. CO-free air served to stir the solution, and the titration was followed witha Beckman Model G glass electrode pH meter. Titration between the apparent pH limits of 6 and 10 was found to give excellent agree-ment with the known molarities of standard higher fatty acids. After deduction of an appropriate blank, the concentration of UFA in an unknown plasma could be calculated in milliequivalents per liter. The data have not been corrected for the small change in plasma volume due to addition of sodium oxalate. In the course of the work it was noted that the dried fatty acid extracts, when stored in the refrigerator, were quite stable in terms of their acid equivalence. Not more than 24 hours was permitted to elapse, however, between the preparation and titration of the extracts with which this report is concerned.

The human subjects studied in this investigation are from two sources, inpatients at the Clinical Center of the National Institutes of Health (the normals in this group being primarily conscientious objectors), and a number of laboratory employees who had been found healthy in a routine pre-employment medical work-up, and who were free of acute disease on the day of study.