A Spectrophotometric Method for Estimating Formimino-glutamic and Urocanic acid

Abstract

A Spectrophotometric Method for Estimating Formimino-glutamic and Urocanic AcidFormimino-glutamic acid, an intermediary in the path- way of histidine breakdown (Borek and Waelsch, 1953), may be excreted in the urine (Bakerman, Silverman, and Daft, 1951) when there is interference with the function of the folic-acid coenzymes.Paper electrophoresis is adequate for identifying this product in the urine of patients excreting relatively large amounts (Knowles, Prankerd, and Westall, 1960; Kohn, Mollin, and Rosen- bach, 1961).However, until now there has been no relatively simple sensitive and quantitative method for estimating this material.This communication describes a new method based on that of Silverman, Gardiner, and Condit (1958) for estimating formimino-glutamic acid.Some of the results in patients with folic-acid deficiency are illustrated.MATERIALSPrinciple.-Acrude liver-enzyme preparation (Futterman, 1957) is employed firstly to reduce folic acid to the active form, and, secondly, to transfer the formimino group of formimino-glutamic acid to this active form of folic acid.The end-product of the reaction is con- verted to 5-10-methenyl-tetrahydrofolic acid with HCl and read spectrophotometrically.Water.-All water used was passed through an ion- exchange resin.Suibstrate Solution.-KH2PO4,20 m-mole (2.72 g.); citric acid, 2 m-mole (0.42 g.); MgSO4.7H20, 2 m-mole (0.49 g.); TPN, 12 smole (0.01 g.); folic acid, 55 /mole (0.024 g.).The KH2PO4 and citric acid were brought into solution and the pH was adjusted to 6 with KOH.The folic acid was brought into solution with the minimum amount of KOH.The TPN and MgSO4 were added, the volume was made to 100 ml., and the solution was stored at -200 C.Enzyme Preparation.-Alloperations were carried out at 2-4' C. Frozen chicken liver was sliced into 2-mm.slices with a scalpel, added to 1.3 volumes of 0.1 M