Automated Determination of Free Amino Groups in Serum and Plasma Using 2,4,6-

A simple automated method is describedfor determining the level of total free amino acids in the blood. The method utilizes the AutoAnalyzer, and is basedon the forma-tion of colored complexes by uniting free amino groups with 2,4,6-trinitrobenzene sulfonate (TNBS). Proteins do not interfere because the free amino acids are first separated by dialysis. Characteristics of the reaction and potential clinical appli-cations of the procedure are discussed. DESPITE THE FACT that the free amino acids of the blood are an im-portant metal)ohc pool, levels of free amino acids are not routinely determined. This report describes an AutoAnalyzer procedure that simplifies the analysis so that measurement of serum amino acid levels may attract wider clinical interest and application in the future. The method utilizes 2,4,6-trinitrobenzene sulfonate (TNBS), a rea-gent intro(luced in 1960 by Satake (2). in contrast to ninhydriui, the most commonly used color reagent for amino acid analysis, TXBS is water-soluble, miafm’eeted by air,and unreactive with both ammonia and urea. As shown ill the equation, TNBS reacts under relatively mild alkaline coll(hitiolls to produce an unstable Meisenheimer complex. Sub-sequent acidification to pH 1 rapidly converts the orange unstable prod-uct to a yellow stable trinitrophenol (TNP) derivative