An approximative colorimetric method for the determination of urea, with an application to the detection and quantitative estimation of arginase

Abstract

The urease method for the determination of urea consists essentially of two distinct operations: first, the enzymatic conversion of the urea into its equivalent of ammonium carbonate, and, secondly, the quantitative determination of the latter. Each of these operations may be accomplished in a variety of ways. In the second, for instance, no less than five different analytical procedures have hitherto been made use of. Either the alkaline ammonium carbonate has been titrated directly with acid (1); or its ammonia has been liberated by a stronger base, conducted into an excess of standard acid, and determined by titration (2); or the ammonia has been estimated colorimetrically by Nesslerization (3); or a measured amount of acid has been added to the alkaline reaction product, and the uncombined excess determined iodometrically (4); or, finally, the ammonium carbonate has been estimated by gasometric determination of its carbon dioxide component (5). One other procedure at least is theoretically possible. The transformation of a molecule of neutral urea into a molecule of ammonium carbonate involves a corresponding reduction in hydrogen ion concentration of the medium in which the change takes place. If, therefore, that medium contains an appropriate indicator, it should be possible to deduce from its change of colour the amount or concentration of the urea hydrolysed. Upon this principle we have devised a method which, although hardly a method of precision, has proved itself in certain special circumstances exceedingly useful. Our procedure is in its essential features identical with one already described by Kay (6), but achieves, through certain refinements, a much closer approach to accuracy than Kay was concerned to attain. The first step in the elaboration of the method was to ascertain what effect upon hydrogen ion concentration is produced by the ammonium carbonate derivable from known quantities of urea. Obviously this must depend upon three factors, of which the concentration of the urea itself is only the first. The others are (i) the original p H of the urea-urease mixture, and (ii) the nature and concentration of the buffer salts present. Only when these two conditions are maintained invariable will there be a fixed correspondence between initial concentration of urea and final concentration of hydrogen ions. It is further evident that if the buffer value of the medium chosen be large, the change of p H effected in a given volume by a given addition of ammonium carbonate will be relatively small; and vice versa . With any selected buffer, therefore, the sensitivity of the reaction is controlled by the concentration in which that buffer is present, and may accordingly be varied to suit the nature of the material, the quantities of urea to be dealt with, and other pertinent circumstances. For the purposes we have had in view the most suitable buffer has been a phosphate mixture of M/20 concentration and of initial p H 6·8. The effect upon this mixture of the enzymatic transformation of known quantities or concentrations of urea was determined in the following way:— 1. A series of about thirty Pyrex test-tubes, 1 x 10 cms. in size and of as nearly as possible equal internal diameter, was graduated at 5 c. c.