Isolation of Glycocyamine from Urine

Abstract

The isolation of glycocyamine (guanido acetic acid) from urine is of interest because of its possible relationship to the origin of creatine. Hunter has critically reviewed the literature on this subject in his monograph on “Creatine and Creatinine”.

My interest in this subject began with the finding that an extract of urine prepared with Lloyd's reagent gave a positive Sakaguchi reaction. I also found that the Lloyd's extract of urine from a case of pseudohypertrophic muscular dystrophy gave a stronger Sakaguchi reaction than did urine from normal individuals. Urine from this patient (8-year-old boy) was therefore used in attempting to isolate the substance responsible for the positive reaction. The patient received 15 gm. of glycine daily during the period of urine collection. The urine was collected in 2- or 3-day periods using toluene as a preservative.

The procedure used is briefly as follows: The urine after filtering was made acid to congo red with hydrochloric acid and extracted with 100 gm. Lloyd's reagent. The extractives were removed from the Lloyd's with barium hydroxide. The barium was removed from the filtrate with sulphuric acid. The filtrate containing creatine, creatinine and substances giving a positive Sakaguchi reaction was concentrated in vacuo to a volume of approximately 300 ml. This concentrated extract was kept in the refrigerator until there had accumulated a series of extracts from a total of 14.8 liters of urine representing a collection period of 18 days. These concentrates were then combined and evaporated to a volume of 1400 ml. and treated in the usual manner with phosphotungstic acid. Sixty per cent of the substances responsible for the positive Sakaguchi reaction remain in the phosphotungstic acid filtrate. The filtrate is freed of phosphotungstic acid with barium hydroxide and the excess barium removed with sulfuric acid. The filtrate is evaporated almost to dryness and extracted with absolute alcohol.