A Practical Method for Preparation and Isolation of l -beta-Hydroxybutyric Acid. ∗

Abstract

The increasing number of studies on the metabolism of ketone bodies has made a method for the preparation of pure l-beta-hydroxybutyric acid desirable. The easiest way to accomplish this is to synthesize it in the animal body. Although it may be obtained from the urine of fasting human subjects, it may also be readily produced in large amounts from the urine of fasted rats fed sodium butyrate according to the earlier procedures from this laboratory (0.237 g of butyric acid daily per 100 g rat in solution by stomach tube in divided doses morning and evening). When 85 female rats weighing 125-150 g were used, 58 g of l-beta-hydroxybutyric acid were excreted in a 4-day period of which 34 g were recovered as the pure Ca-Zn double salt. Although a similar procedure might tie employed with a smaller number of clogs, our experience has been that rats are particularly well adapted because they can not regurgitate. The following method was employed in separating the acid in pure form.

To the pooled urines is added sufficient solid copper sulfate to obtain a concentration of 20%. To the resulting solution is added 0.5 g calcium hydroxide (in 10% aqueous suspension) for each gram of copper sulfate present. This amount of the base usually suffices to bring the pH slightly alkaline to litmus. If the pH is not alkaline to litmus after standing 1/2 hour more calcium hydroxide should be added. The mixture is then filtered by suction, working the precipitate with a spatula to a dry, hard cake, to express as much of the liquid as possible.

The filtrate is evaporated under reduced pressure to a syrup. At this point the precipitated salts may be filtered off.