DETERMINATION OF CITRULLINE AND ALLANTOIN AND DEMONSTRATION OF CITRULLINE IN BLOOD PLASMA

Abstract

Fearon (1) introduced the use of diacetylmonoxime, CHs+ C:NOH. C0.CH3, in strongly acid solution (trichloroacetic) as a reagent for the calorimetric determination of citrulline. The color produced is a reddish yellow peach shade. Fearon recognized that allantoin and urea, when present, vitiated the results by producing colors which are somewhat more yellowish. Gornall and Hunter (2) used Fearon’s reaction to measure citrulline aft,er removal of urea with urease. They realized that the urease contributed a rather appreciable blank. We have found that allantoin or allantoic acid and canavanine present in commercial preparations of urease are responsible for most, if not all, of this blank and have removed these materials by dialysis (3). When dialyzed urease is used to destroy urea, no urease blank need be run. Interference by allantoin is prevented by adsorbing the citrulline with Amberlite at pH 6 to 7. Allantoin is not adsorbed. The difference in chromogenic material before and after adsorption is a measure of the citrulline. Interference by other plasma extractives besides allantoin is also eliminated by this procedure. It is desirable to keep at a minimum the color formed by allantoin in the mixture analyzed before adsorption of the citrulline. This object has been attained by forming the color in a solution acidified with a 1: 3 mixt,ure of sulfuric and phosphoric acids, and heating for 10 minutes. Under these conditions color development from citrulline is almost maximal, while the amount of color formed by allantoin is only about one-fiftieth as much as that from equimolar amounts of citrulline. The nature of the carbamido-diacetyl reaction and the structure of the products formed remain unknown. Formation of the colored product is apparently favored by the presence of an oxidizing agent and to some extent by light. The product, however, is at the same time photolabile and is destroyed by excess oxidant. Color formation takes place slowly at room temperature. The following observations on the nature of the alternative reagents and the subsequent discussion of the specificity of the reaction relate to