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The isolation of a new compound from urine of humans with porphyria cutanea tarda
433 Biochimica et Biophysica Acta, 500 (1977) 433--435
© Elsevier/North-Holland Biomedical Press
B B A Report BBA 21461
THE ISOLATION OF A NEW COMPOUND FROM URINE OF HUMANS WITH PORPHYRIA CUTANEA T A R D A
MOISES GRINSTEIN, ANA MARIA FERRAMOLA DE SANCOVICH and HORACIO A. SANCOVICH Laboratorio de Porfirinas, Departamento de Qufmica Bioldgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina)
(Received May 24th, 1977)
Summary A m e t h o d is reported for the isolation of phyriaviolin, a new c o m p o u n a from h u m a n porphyria cutanea tarda urine. The substance has been crystallized and some of its properties are described.
The use of anion exchange resin De Acidite FFiP [1] for isolation of porphyrins in urine of humans with porphyria cutanea tarda, revealed the presence of an u n k n o w n substance in relatively high quantities. The present report deals with the isolation in crystalline form of this new c o m p o u n d which we named 'phyriaviolin', and with some of its properties. A volume of urine with pH > 3, is mixed with De Acidite FFiP, chloride form, mesh 14--52 (Permutit Co., U.K.) at a rate of 20 g per 100 ml urine. It was shaken for approx. 30 min, to ensure a complete combination of the porphyrins and phyriavi61in with the resin. After standing a few minutes, the supernatant was decanted and the resin washed three times with distilled water and three more times with methanol. The washed resin was suspended in the esterification mixture, methanol/sulfuric acid (10:1, v/v). If the mixture is examined with a spectroscope after about 30 min, the appearance of a broad absorption band at 500--600 nm, can be observed, coinciding with the development of a peculiar red violet colour. The same phenomena was observed using methanol/HC1 as esterification mixture. Preliminary semiquantitative studies showed t h a t approx. 75% of the urine phyriaviolin content was liberated from the resin into the esterification mixture during the first 24 h. The remaining 25% could be recovered from the resin by repeated washing with methanol. The esterification solution was mixed with chloroform and an excess of distilled water; phyriaviolin was extracted into the organic layer together
434
with the porphyrin esters. The chloroform solution was separated, washed, and concentrated under vacuum. When the concentrated solution, containing 1--2 ~g of porphyrins, was applied as a narrow strip into a sheet of Whatmann No. 1 paper and the chromatogram developed according to the procedure of Falk and Benson [2], phyriaviolin separated from the porphyrins as a single violet band located above the coproporphyrin one. Phyriaviolin can be isolated in a relatively pure form, by the application of the calcium carbonate column chromatography as described previously [3]. The adsorbed pigments are separated by eluting with benzene] chloroform (1:1, v/v). Phyriaviolin separates as a violet band, usually at the upper part of the column. By repeating the chromatography it is possible to obtain a single phyriaviolin band, free of porphyrins and other pigments present, which is cut out and eluted with chloroform. When the chloroform solution is concentrated, phyriaviolin crystallizes in red brown needles. A complete crystallization is achieved by adding to the chloroform solution one or two vols. petroleum ether 60--80 ° C. By repeated crystallization from chloroform it is possible to get uniform type crystals, consisting of red brown needles often occurring in rosettes {Fig. 1).
J Fig. I . Crystals of phyriaviolin.
The pure crystalline material is insoluble in water, dilute acid, alkaline solutions and petroleum ether. It is soluble in sulfuric acid, glacial acetic acid, pyridine and dimethyl sulfoxide and slightly less soluble in chloroform, ethyl acetate, ethylether, benzene, acetone, methanol, ethanol, toluol and xylol. Upon silica gel thin layer chromatography it behaves as a single substance, as could be seen by using development mixtures of different polarities, e.g. benzene/acetone {9:1, v/v), Rf 0.80; benzene/chloroform {9:1, v/v), Rf 0.40; dioxane/kerosene (1:25, v/v), Rf 0.15. Upon paper chromatography, following the m e t h o d of Falk and Benson [2] it behaves as a single c o m p o u n d with a Rf: 0.68--0.70.
435
The chloroform solution exhibits an spectra absorbance with maxima at 532, 360, and 292 nm (A29~ > A53~ > A360 ); with an extinction coefficient at 5 3 2 n m E 1% =298, and at 292 nm E 1% =691 ClTI ClTI We have not Seen able to observe a typic~ melting point. Using a Leitz-Wetzlar micro hot stage apparatus for melting point determinations it is seen that at 230°C the crystals start to decrease in size and at 250-260°C most of them disappear. Elementary analysis gave: C, 57.9; H, 4.0; N, 7.11; O, 30.99. Microanalysis were carried out in the Department of Chemistry, University College, Cardiff, U.K. through the courtesy of Professor A.H. Jackson. Further studies are in progress in Professor Jackson's laboratories {Cardiff) with the aim to determine its chemical structure. We have been able to perform semiquantitative determinations of the phyriaviolin content in urine of 10 different patients with porphyria cutanea tarda. Results so far obtained indicate a daily excretion ranging from 1 to 5 mg. We have been able to detect phyriaviolin also in the urine of two normal humans, but in far smaller quantities. Our thanks to Dr. Carlos Naranjo for performing the photograph of the phyriaviolin crystals. Part of this work was supported by a research grant from Consejo Nacional de Investi~aciones Cientfficas y Tgcnicas, Argentina.
References 1 2 3
Grinstein, M, (1977) Anal. Biochem. 7 7 , 5 2 2 Falk, J.E. and Benson, A, (1953) Biochem. J. 55, 101 Grinstein, M., Schwartz, S. and Watson, C.J. (1945) J. Biol. Chem. 157, 323
© Elsevier/North-Holland Biomedical Press
B B A Report BBA 21461
THE ISOLATION OF A NEW COMPOUND FROM URINE OF HUMANS WITH PORPHYRIA CUTANEA T A R D A
MOISES GRINSTEIN, ANA MARIA FERRAMOLA DE SANCOVICH and HORACIO A. SANCOVICH Laboratorio de Porfirinas, Departamento de Qufmica Bioldgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina)
(Received May 24th, 1977)
Summary A m e t h o d is reported for the isolation of phyriaviolin, a new c o m p o u n a from h u m a n porphyria cutanea tarda urine. The substance has been crystallized and some of its properties are described.
The use of anion exchange resin De Acidite FFiP [1] for isolation of porphyrins in urine of humans with porphyria cutanea tarda, revealed the presence of an u n k n o w n substance in relatively high quantities. The present report deals with the isolation in crystalline form of this new c o m p o u n d which we named 'phyriaviolin', and with some of its properties. A volume of urine with pH > 3, is mixed with De Acidite FFiP, chloride form, mesh 14--52 (Permutit Co., U.K.) at a rate of 20 g per 100 ml urine. It was shaken for approx. 30 min, to ensure a complete combination of the porphyrins and phyriavi61in with the resin. After standing a few minutes, the supernatant was decanted and the resin washed three times with distilled water and three more times with methanol. The washed resin was suspended in the esterification mixture, methanol/sulfuric acid (10:1, v/v). If the mixture is examined with a spectroscope after about 30 min, the appearance of a broad absorption band at 500--600 nm, can be observed, coinciding with the development of a peculiar red violet colour. The same phenomena was observed using methanol/HC1 as esterification mixture. Preliminary semiquantitative studies showed t h a t approx. 75% of the urine phyriaviolin content was liberated from the resin into the esterification mixture during the first 24 h. The remaining 25% could be recovered from the resin by repeated washing with methanol. The esterification solution was mixed with chloroform and an excess of distilled water; phyriaviolin was extracted into the organic layer together
434
with the porphyrin esters. The chloroform solution was separated, washed, and concentrated under vacuum. When the concentrated solution, containing 1--2 ~g of porphyrins, was applied as a narrow strip into a sheet of Whatmann No. 1 paper and the chromatogram developed according to the procedure of Falk and Benson [2], phyriaviolin separated from the porphyrins as a single violet band located above the coproporphyrin one. Phyriaviolin can be isolated in a relatively pure form, by the application of the calcium carbonate column chromatography as described previously [3]. The adsorbed pigments are separated by eluting with benzene] chloroform (1:1, v/v). Phyriaviolin separates as a violet band, usually at the upper part of the column. By repeating the chromatography it is possible to obtain a single phyriaviolin band, free of porphyrins and other pigments present, which is cut out and eluted with chloroform. When the chloroform solution is concentrated, phyriaviolin crystallizes in red brown needles. A complete crystallization is achieved by adding to the chloroform solution one or two vols. petroleum ether 60--80 ° C. By repeated crystallization from chloroform it is possible to get uniform type crystals, consisting of red brown needles often occurring in rosettes {Fig. 1).
J Fig. I . Crystals of phyriaviolin.
The pure crystalline material is insoluble in water, dilute acid, alkaline solutions and petroleum ether. It is soluble in sulfuric acid, glacial acetic acid, pyridine and dimethyl sulfoxide and slightly less soluble in chloroform, ethyl acetate, ethylether, benzene, acetone, methanol, ethanol, toluol and xylol. Upon silica gel thin layer chromatography it behaves as a single substance, as could be seen by using development mixtures of different polarities, e.g. benzene/acetone {9:1, v/v), Rf 0.80; benzene/chloroform {9:1, v/v), Rf 0.40; dioxane/kerosene (1:25, v/v), Rf 0.15. Upon paper chromatography, following the m e t h o d of Falk and Benson [2] it behaves as a single c o m p o u n d with a Rf: 0.68--0.70.
435
The chloroform solution exhibits an spectra absorbance with maxima at 532, 360, and 292 nm (A29~ > A53~ > A360 ); with an extinction coefficient at 5 3 2 n m E 1% =298, and at 292 nm E 1% =691 ClTI ClTI We have not Seen able to observe a typic~ melting point. Using a Leitz-Wetzlar micro hot stage apparatus for melting point determinations it is seen that at 230°C the crystals start to decrease in size and at 250-260°C most of them disappear. Elementary analysis gave: C, 57.9; H, 4.0; N, 7.11; O, 30.99. Microanalysis were carried out in the Department of Chemistry, University College, Cardiff, U.K. through the courtesy of Professor A.H. Jackson. Further studies are in progress in Professor Jackson's laboratories {Cardiff) with the aim to determine its chemical structure. We have been able to perform semiquantitative determinations of the phyriaviolin content in urine of 10 different patients with porphyria cutanea tarda. Results so far obtained indicate a daily excretion ranging from 1 to 5 mg. We have been able to detect phyriaviolin also in the urine of two normal humans, but in far smaller quantities. Our thanks to Dr. Carlos Naranjo for performing the photograph of the phyriaviolin crystals. Part of this work was supported by a research grant from Consejo Nacional de Investi~aciones Cientfficas y Tgcnicas, Argentina.
References 1 2 3
Grinstein, M, (1977) Anal. Biochem. 7 7 , 5 2 2 Falk, J.E. and Benson, A, (1953) Biochem. J. 55, 101 Grinstein, M., Schwartz, S. and Watson, C.J. (1945) J. Biol. Chem. 157, 323