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Formimino-L-glutamic acid, an intermediate in the metabolism of histidine (1, 2), accumulates in the urine of folic acid-deficient rats (37). The urinary formiminoglutamic acid has also been reported to be increased in macrocytic anemia of pregnancy and in patients treated with folic acid antagonists (8-10). These findings can be attributed to a lack of tetrahydrofolic acid (THF), since enzymatic studies have shown (11-14) that THF is required for the further metabolism of formiminoglutamic acid. The accumulation of formiminoglutamic acid in the urine can be used as a test for folic acid deficiency both in rats and in patients. The available assay for formiminoglutamic acid, based on the microbiological assay of the glutamic acid formed from formiminoglutamic acid by heating (4, 9), is relatively inconvenient and inaccurate. The assay is complicated by the fact that glutamic acid and glutamine are normally present in urine, as well as by the variable conversion of these compounds to pyrrolidone carboxylic acid during the heating step. In this paper a sensitive spectrophotometric method is presented for the assay of formimino-Lglutamic acid in the urine. This method is based on enzymatic reactions (1) and (2) [Diagram 1] which we have recently (14) demonstrated to be involved in the metabolism of formiminoglutamic acid. The amount of 5,10-methenyl-THF formed in reaction (2) is easily measured spectrophotometrically, since this compound has a high absorption in the ultraviolet with an extinction coefficient of 25,000 at 350 m,. (15-18). During the course of the reaction, however, 5,10-methenyl-

A Method for the Determination of Formiminoglutamic Acid in Urine1