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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During clinical trials of a human antihaemophilic globulin concentrate (HAHG) prepared from very fresh plasma by the method of Kekwick and Wolf (1957), it was observed (Maycock, Evans, Vallet, Combridge, Wolf, MacGibbon, French, Wallett, Dacie, Biggs, Handley and Macfarlane, 1963) that the transfusion of this material into haemophiliacs was sometimes followed by reactions similar to those observed by Fox, Goldsmith, Kidd and Lewis (1961) after the intravenous injection of purified bradykinin. A chemically similar fraction (ETF), prepared from aged plasma, has been used for many years for the treatment of hypofibrino‐genaemia without any reported reactions. The reactions described by Maycock et al. (1963) were sufficiently frequent and severe to warrant an investigation of the kininogenic properties of HAHG and, for comparative purposes, of ETF. For reasons not understood, but clearly associated with the adoption of a modification of the original technique (Kekwick and Walton, 1965), reactions occurring during the transfusion of recently prepared batches of HAHG into haemophiliacs are now no greater in number and severity than those occurring during the transfusion of fresh plasma. Human plasma contains prokininogenases, the precursors of enzymes which hydrolyse protein substrates (kininogens, kallidinogens) to produce kinins, polypeptides which cause vasodilation and plain muscle constriction (Lewis, 1960). Activation of two of these proenzymes, kallikrein and plasminogen (profibrinolysin), could lead to the production of kinin in HAHG either during preparation, or during transfusion, and might cause the observed reactions.

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