[CONTRIBUI ION PROM THE DEPARTMESTS 01: HIOCHEMISTRY ANI) PAlIIOLOGY, LrNIVERSITY O F LVISCONSIN]
Identification of Cyanoacetic Acid as a Urinary Metabolite of p-Aminopropionitrile’ 13Y
s. H. IJIPrON, J. J. LAIIAI(X, J . ’F. (>.\KUUTT
.\N1) It.
A I . STRUNG
KECEIVED JUSE 13, 1958 Urine of rats and rabbits administered p-aminopropionitrile ( B A P S ) contained a metabolite, not present in control urines, which was detected by formation of a red color with diazotized sulfanilic acid. The metabolite was isolated and identified as cyanoacetic acid. A complex of two moles of cyanoacetic acid with one mole of urea was also isolated from the urine and identified with a synthetic sample. \Yhen C14-cyanolabeled BAPN was administered to rats, about SO-SO% of the total administered radioactivity appeared in the urine within 23 hours, and 3 0 4 0 % of this total was extractable from stroiigl~ acidified urine with ethyl acetate. The oiily radioactive substance detected in such extracts was cyanoacetic acid
In studies of the metabolism of P-aminopropionitrile (BAPN) by the rat, a urinary metabolite was observed2 which gave rise to a bright orange-pink zone on paper chromatograms of the urine which were sprayed with diazotized sulfanilic acid.3 This metabolite was isolated from both rat and rabbit urine and was first obtained4 as a crystalline solid, C ~ H I O N (I), ~ O ~melting a t 94-96’. After further fractionation the metabolite was obtained as a crys(11), Z melting a t 87-68’. talline product, C ~ H ~ N O Compound I1 was identical with cyanoacetic acid.5 After I1 was identified, Compound I was determined to consist of a complex of one mole of urea and two moles of cyanoacetic acid. The addition of petroleum ether6 to an ethyl acetate solution of 2 moles of cyanoacetic acid and one mole of urea results in formation of nice crystals, m.p. 94-96’, identical with I. Baum7 previously reported I to result when a mixture of 2 moles of cyanoacetic acid and 1 mole of urea was melted, his product having m.p. 94-95’. Prior to the actual isolation, the properties of the metabolite of RAPN were studied by administering C14-cyanolabeled8 BAPN to white rats. The bulk of the radioactivity was rapidly excreted in the urine in the forin of the metabolite, unchanged BAAPKand possibly other products. The inetabolite was separated from BAPN by extraction from strongly acidified urine by ethyl acetate or ether. Hydrolysis of the urine was unnecessary, as extraction was unaffected by hydrolysis in 1 1V sulfuric acid a t 100’ for 70 minutes. Such extracts were chromatographed on papers developed with three different solvent systemsg and in each case the radioactivity and orange-pink zones coincided. The compound was found to be distillable from the ethyl acetate extract under high vacuum a t temperatures below 80’ and was thus greatly concentrated. (1) Presented in part at the 45Jth meeting of t h e rlmericau Society oi Biological Chemists,4 and the 4Xrd meeting of t h e American Society for Experimental Pathology,z Philadelphia, April, 19S8. Supported in part by grants A538(C8) and h149S from the National Institutes o f 1Ie:rlth. U. S. Pul,lic Health Service. (2) J. J. Lalich, Federation P?oc., 17, ,1 1 4 (1‘338). (3) 11. D. Armstrong. K. N F. Shaw anfl P. E. Wall, J . B i d . Cheni., 218, 293 (1036). (1) J . T. Garbutt, J , J. I,alich, S . 11. Lipton and B.21. Strong, Ped, . ~ ~ l i r i i PVOC., z 1 7 , 220 (19.78). ( 5 ) S. H. 1.ipton. J . J . T.alich a r i d 1’. X I . Strong, l‘ms JOITRNAI., 80, 202‘2 (1Y.58). (13) T h e petroleum ether used was Skellysolve B, b.p. GO-71 ‘. (7) F. Baum, Ber., 41, 5 2 5 (1908). ( 8 ) E. D. Schilling and F. hZ. Strong, J . Oug. C h e ~ i . 23, , 319 (1037). (9) T h e three systems butano1:acetic acid:water ( 8 : 2 : 2 ) , isopropyl xicoho1 :ammonia: water (8: 1: l ) , and benzene: priqiionic arid : water ( 2 : 2 : 1 ) as given in reference 3 were employetl.
The final isolation was carried out on non-radioactive materials. Rabbits were especially suitable for this work, not only because of their larger size but also because they converted more of the administered BAPN into the metabolite. The metabolite from rabbits was chromatographically the same as that excreted by rats. The larger-scale isolation from rabbit urine, which later was repeated on rat urine, consisted in extraction of the acidified urine with ethyl acetate, distillation of the metabolite from the extract under high vacuum, and crystallization of I by addition of petroleum ether to an ethyl acetate solution of the distillate. Compound I displayed a strongly acidic nature,ln PK ca. 2.5, and had a neutral equivalent of about 116. I t showed only end absorption in the ultraviolet and lacked absorption characteristic of phenols.” Since we were unable to assign a structural formula to the compound a t this stage, Compound I was examined on paper chromatograms. An “impurity”12 identified as urea was detected as a trailing green spot by use of a phenol-hypochlorite spray13or as a yellow spot with the $-dimethylamin~benzaldehyde~ spray. The urea contaminant was eliminated by adsorbing I on a stroiig base anion-exchange resin and eluting with 0.1 A4 sodium acetate. Following estraction of the acidified eluate with ethyl acetate and removal of the solvent, the solid residue was sublimed in z.acuo. Compound I1 was thus obtained as a colorless solid which following recrystallization from ethyl acetate-petroleum ether was identical with cyanoacetic acid in chromatographic migration and color reactions. The infrared spectra corresponded closely and the melting point of a mixture of I1 and authentic cyanoacetic acid was undepressed, Simultaneously with thc isolation of 11, it was observedj that only cyanoacetic acid, of a large number of nitriles tested, gave a direct orange-pink color with diazotized sulfanilic acid (i.e., without passage through the animal.) The behavior of I and I1 on paper chromatograms was identical and was duplicated by cyanoacetic acid. I t thus appears that at least in the rat and rabbit RXPN is partly nietnbolized by conversion of the (10) \Ve wish tr, thank Ilr. I
