ChemkjaIBasis of the CarbamidodiacetylMicromethodfor Estima’tionof Urea,Citrulline,andCarbamylDerivatives

The chemical pathway of the carbamidodiacetyl colorimetric assay was investigated. The experimental variables that were studied include reaction temperature, heating time, and the ratio of the mineral acid mixture to the reactants. Evidence is presented establishing the involvement of either 7-or 8-methyl, or 7,8.d imethyltetrahyd roimidazo(4,5-d)imidazole-2,5-d iones as chromogens, all three being equally acceptable. T HE CARBAMIDODIACETYL colorimetric assay has long been the method of choice in many clinical laboratories for the determination of urea, citrul-line, and carbamyl derivatives (1, 2). Convenient reagent kits (3), as well as a mechanized adaptation (4), have been described for reportedly simple and rapid application of the assay to the analysis of biologic fluids. The addition of either semidine (5) or thiosemicarbazide (6) to the reaction mixtur