The colorimetric estimation of alpha-amino nitrogen in tissue fluids.

Various authors have reported methods for the colorimetric estimation of alpha-amino nitrogen with ninhydrin (Moore and Stein, 1948, 1954; Smith and Agiza, 1951 ; Troll and Cannan, 1953; Yemm and Cocking, 1955; Kalant, 1956; Rosen, 1957). Only Kalant has applied this method to the direct measurement of alpha-amino nitrogen in biological fluids; he reported on the determination of total alpha-amino nitrogen in dog plasma. This paper describes a colorimetric ninhydrin method for amino-acid determination, based on that of Troll and Cannan. It differs in using disodium ethylenediamine tetra-acetic acid (E.D.T.A.) instead of pyridine to enhance colour development. This improves reproducibility by eliminating trace metal interferences, and yields lower blanks than the pyridine reagent. In addition, we use a separate buffer to maintain the pH optimal at 5.1. For the first time a technique has been evolved for applying the colorimetric ninhydrin procedure to biological fluids generally, and, in particular, it has been successfully applied to urine. The optimum conditions for pH and concentration of cyanide, ninhydrin, and other reagents have been studied, and the optimal time for heating has been established. With the exception of the E.D.T.A. concentration, all the factors are moderately critical.