For investigations concerning the mode of action of the clearing factor, estimations of free fatty acids in mixtures of normal rat plasma and lipaemic dog plasma were performed using a modification of van de Kamer's (1948) method as described for faeces. Because the method developed may be of value for similar or other uses, it is given here. In a 100 ml. flask 2 ml. of the plasma-mixture, containing either heparin or citrate as an anticoagulant, are run into 6 ml. of phosphoric acid solution (metaphosphoric acid, 50 g., NaCl, 250 g. water to 1 1.). After a few minutes 16 ml. of ethanol, containing 0-4 % amyl alcohol, are added, followed by 20 ml. of distilled light petroleum (b.p. 40-60°). The flask is stoppered and the mixture shaken by hand for 1 min. After separation, the upper layer is filtered through dry paper and the funnel covered in order to minimize evaporation. Care should be taken to avoid contamination with traces of the other phase. Ten ml. of the light petroleum extract is evaporated on a boiling-water bath and the residue dried in vacuo. Neutralized ethanol (5 ml.) containing 0-6 mg. of thymol blue per 100 ml. are added and the residue dissolved by boiling gently under reflux for 3 min. Finally, the fatty acids are titrated under a stream of N2 with O-O0N-NaOH.The results are corrected for a blank (usually about 0060 ml. of 001N-NaOH), obtained by running 2 ml. of water through the same procedure.The reproducibility of the method suffices for many purposes. In twenty-four estimations giving results ranging from 021 to 210 0,-equiv. of fatty acids per 2 ml. of plasma tested, the differences between duplicate determinations were found to have a standard deviation of + 0-14 ,u-equiv.

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The estimation of the clotting time of plasma using thromboplastic agents and calcium ions is a valuable test for the assessment of haemorrhagic tendencies, but it is not an indication of frothrombin concentration in a chemical sense. Terms such as "prothrombin time," "accelerated clotting time" and "prothrombin activity'' suggested by various authors all emphasize this point. On the other hand, the so-called two-stage technique elaborated by Warner, Brinkhous ana Smith (1936) allows a quantitative estimation of prothrombin. This method, however, is not sviitable for general use. It was therefore thought that a procedure combining the expediency of the one-stage technique with the e:stimation of prothrombin concentration would answer questions which both techniques have left undecided. There are no known chemical reactions which distinguish prothrombin from proteins of the same group; the most outstanding property of prothrombin however, is its ability to be adsorbed by a variety of compounds. Several substances such as tertiary calcium phosphate (Bordet and Delange, 1914), barium sulphate (Dale and Wa'lpole, 1916), magnesium hydroxide (Fuchs, 1929 andSeegers, 1945) and alumina gel (Quick, 193.5) have been found useful for the removal of prothrombin from oxalated plasma. These preparations were tested for their ability to adsorb prothrombin by using the smallest amount which rendered oxalated plasma noncoagulable, and the adsorbates were analysed for protein content.

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