Improvements in the method of separation of guanidino organic acids by column chromatography: Isolation and identification of guanidinosuccinic acid from human urine

Abstract

A procedure is described for the separation and isolation of guanidino organic acid derivatives when present in low concentrations. This comprises fractionation on Dowex 50 (strongly acidic) with a series of buffers ranging upward of pH 2.2, followed by refractionation of the desired peak on Dowex 1 (strongly basic) with a sodium acetate buffer, pH 4.1. Most of the sodium ion can be removed on I.R.C. 50 (methacrylate resin carboxylic acid). The solution is then lyophilized and the guanidino organic acid derivative can be recrystallized from small volumes of water. By using this technique, guanidinosuccinic acid was isolated from the urine of a uremic patient. This is the first time that this substance has been shown to exist in biological material. The guanidinosuccinic acid was identified as the monohydrate by elemental analysis, melting point, comparison with synthetic guanidinosuccinic acid, electrophoretic mobility, paper chromatography, and by comparison of its infrared spectrum with a synthetic preparation of guanidinosuccinic acid monohydrate. The urine contained approximately 27mg per liter of guanidinosuccinic acid. This partially identifies an unidentified peak [ Microchem. J. 7, 63 (1963)], which is now shown to comprise at least two guanidino derivatives; the other major component (approximately 10 mg per liter) still are not identified.