The estimation of acetone bodies.

In a study of the pathogenesis of bovine ketosis it was felt desirable to be able to follow in some detail the changes in. all three ketone bodies (acetone, acetoacetic acid and ,-hydroxybutyric acid) which have been shown by various workers to occur in excessive quantities in blood, milk and urine during the course of that disorder. Hitherto, little or no attempt hasbeenmade at complete differentiationas both acetone and acetoacetic acid are usually determined in the one fraction. Moreover, i8o-propanol which we have found (Robertson, Thin & Stirling, 1950) to be present in this disease and in pregnancy toxaemia of ewes, has been unsuspected hitherto in such conditions and, as it is oxidized to acetone by chromic acid, its presence will undoubtedly have influenced the estimation oftotal ketones by various methods. In this paper the term 'Total Acetone Bodies' is used, therefore, to include acetone, acetoacetic acid, ,B-hydroxybutyric acid and i8opropanol. The accurate determination of free acetone in the presence of acetoacetic acid is difficult. Van Slyke's method, using Denig6s reagent (Van Slyke, 1917, 1929) which has frequently been utilized in ketosis studies, cannot be used to estimate free acetone; in addition, it is not very sensitive as we have found that no precipitate is formed with small amounts of acetone bodies of the order of 5 mg./100 ml. or less. Moreover, using this method, i8opropanol appears to be oxidized to acetone partly in the acetone plus acetoacetic acid fraction, and partly in the P-hydroxybutyric acid fraction, so making the accurate determination ofthe individual acetone bodies impossible. Attempts at using the distillation methods of Shaffer & Marriott (1915) and Behr (1928, 1940) for free acetone by distillation under reduced pressure, or by blowing a fine stream of air through the solution at room temperature, in order to prevent acetoacetic acid decomposing, gave poor results, as with quantities likely to be present in biological fluids, only a small proportion of the acetone could be recovered. The method of Werch (1940, 1941), utilizing diffusion into Nessler's solution in Conway microdiffusion units, though providing a very delicate qualitative test, did not give accurate quantitative results; the time for the appearance of the precipitates varied and they could not be estimated gravimetrically with any degree of success. The following method was therefore devised, combining with some modification the oxidation technique of Greenberg & Lester (1944), the diffusion method of Werch (1940, 1941) and Seifert (1948), and the colorimetric technique of Behr & Benedict (1926). It depends on the development ofan orange to red coloration when an alkaline solution of salicylic aldehyde is left in the presence ofacetone, the depth of colour formed being directly proportional to the amount of acetone present. This can be used to estimate free acetone, the acetone formed by hydrolysis of acetoacetic acid, and that formed by chromic acid oxidation of B-hydroxybutyric acid and of iwopropanol.