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The quantitative determination of pyrrole-2-carboxylic acid in urine
SHORT COMMUNICATIONS
185
tions on one blood sample to which 70 mg/roo ml xylose was added; a mean of 70.8 mg/roo ml with a standard deviation of 1.7 was obtained. This investigation was supported in part by a training grant from the National Institutes of Health, 2 A-5321. W. H. KLASSEN*
University of Utah, College of Mqiicine, Salt Lake City, Utah (U.S.A.)
PHILIP LANZKOWSKY * *
1 J. A. BENSON, JR., P. J. CULVER, S. RAGLAND, C. M. JONES, G. D. DRUMMEY AND E. BO~GAS. New Engl. J. Med., 256 (1957) 335. * F. S. BRIEN, D. A. TURNER, E. M. WATSON AND J. H. GBDDES, Gastroentevology,zo (1952) 287. * I. CHANARIN AND M. C. BENNETT, &it. Med. J., I (1962) 985. 4 J. M. FINLAY AND K. J. R. WIGHTMAN, Ann. Internal Med., 4g (1958) 1332. 6 F. H. GARDNER AND E. PEREZ-SANTIAGO,A.M.A. Arch. Internal Med., g8 (1956) 467. * J. G. ROBERTS, I. T. BECK, J. KALLOS AND D. S. HAHN, Can. Med. Assoc. J., 83 (1960) 112. 7 J. H. ROE AND E. W. RICE, J. Biol. Chem., 173 (1948) 507. * E. H. THAYSEN AND S. M~LLERTZ, Acta Med. Sand., 171 (1962) 521. * G. E. YOUNGBURG, J. Biol. Chem., 73 (1927) 599.
Received September gth, 1963. * Research Associate, Department of Pediatrics. * * Cecil John Adams Memorial Traveling Fellow and Research Fellow in Pediatric Hematology. Clin.
The quantitative
Chim.
Acta,
g (1964)
183-185
determination of pyrrole-2-carboxylic acid in urine*
Studies on the metabolism of collagen have made it desirable to determine quantitatively the amount of pyrrole-2-carboxylic acid (PCA), a metabolic conversion product of hydroxyprolinel, excreted in the urine of man and rat. This communication describes a method for the determination of urinary PCA based on the hydrogen-ion-catalyzed decarboxylation of PCA to pyrrole. The pyrrole is removed from the reaction mixture by distillation and estimated using Ehrlich’s reagent. Reagents Hydrochloric acid: 6.84 N HCl, aqueous. Ehrlich’s reagent (ER): 2.5% dimethyl aminobenzaldehyde in 95% ethanol. Pyrrole (Matheson, Coleman and Bell Co.) : freshly distilled before use. Xanthydrol (Eastman Kodak Co.) : 0.5% solution in methanol. Acetic acid : glacial. * This investigation was supported in whole by U.S. Public Health Research Grant CA 06698 from the National Institute of Cancer. Clin. Chim. Acta, g (1964) 185-187
186
SHORT COMMUNICATIONS
PROCEDURE
Fifteen ml of urine are made 0.5 N with respect to hydrochloric acid and placed in a 50-ml round-bottom flask fitted with a short stillhead and a Liebig condenser. In the case of rat urine it may be necessary to add 3-4 drops of octyl alcohol to avoid
TABLE EXCRETION
Subject J.H. K.O. P.B. P.L. P.L.
OF
PCA
I IN
HUMAN
URINE
pmoles of PCA excreted in 24 h I.39 2.46 I
.07
2.18
3.52
frothing. The distilling flask is heated with a Bunsen burner until IO ml of distillate have been collected. Under these conditions the first 8.5-9.0 ml of the distillate will contain all of the pyrrole. If the specific activity of [X] pyrrole is to be determined (vide ircfra) smaller collections may be more convenient. To a a.3-ml aliquot of the distillate is added 0.65 ml of ER and 0.35 ml of 6.84 N HCl. Exactly 5 min after the addition of HCl the optical density at 560 ma is determined using a Beckman Spectrophotometer or a similar instrument. Precision in the timing of this measurement is important as the color of the solution changes appreciably on standing. A standard curve must be prepared for each set of determinations, using a blank of distilled water and several aqueous standard samples containing from I.49 . IO--~ to I.49 - 10-l micromoles of pyrrole per 2.3 ml. The size of the aliquot of the distillate may be reduced to 1.0 ml with good results. The amounts of ER and HCl must be reduced proportionately. This is advantageous in situations where as much of the distillate as possible must be kept for the determination of radioactivity. Results of an application of this method to human urine are shown in Table I. Determi?zation of the WI-activity of pywole present in the distillate The procedure for obtaining pyrrole from PCA can also be applied when data on the specific %-activity of PCA are desired. It must, of course, be kept in mind that one carbon atom of PCA will be lost as a result of the decarboxylation. In a typical experiment in which urine from rats injected with U-[ “C] proline had been collected, 6 ml of urinary distillate was obtained. One ml of this distillate was used for the quantitative determination of pyrrole. To the remainder of the distillate were added 2.0 ml of glacial acetic acid and 0.1 ml of a 0.5% methanolic solution of xanthydroll. The resulting solution is then evaporated to dryness on a rippled, stainless steel planchet and the X-activity of the residue determined. From the data Clilz. Chim. A&,
g (1964) 185-187
SHORTCOMMUNICATIONS
187
obtained the specific activity is calculated. The quantity of material present on the planchet under these conditions is small enough to constitute an infinitely thin layerGEORGEB. GER~R KNUT R. T~LNES DAVID WOOD* KURT I. ALTMAN
Division of Ex@rimental Radiology and the Departments of Radiation Biology and Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. (U.S.A.) 1 A. N. RADHAKRISHNANAND A. M&ESTER,J. BioZ. Gem.,
226 (1957) 55~.
Received September 3rd, 1963 * Trainee on the Biochemistry Training Grant to the University of Rochester School of Medicine and Dentistry (NIH Grant 2G-137X5)_ Cl& Chins. Ada, g (x964) 185-187 Chelation in human sweat The clinical observation that nickel sensitization might be preceded by a transport of nickel ions through the sweat glands lead us to investigate the interaction of nickel chloride with human sweat. Sweat, obtained by thermal stimulus, was filtered before use. Five ml of the clear liquid and 5 ml 0.1 it4 nickel chloride were diluted to 50 ml and the difference in transmission was measured against 5 ml of the same sweat, diluted to 50 ml, with a Zeiss spectrophotometer PMQ II in the region 220-350 mp. A shift was obtained, having a form independent of the origin of the sweat (Fig. I, x-3). The experiment was repeated with a solution proposed by SPIERAND PASCHER’ as a sweat imitation. This solution contains lactic, citric, uric, formic, pyroglutamic, urocanic, aspartic, and glutamic acids, glycine, serine, threonine, alanine, tyrosine, citrulline, valine, leucine, phenylalanine, proline, tryptophan, histidine, omithine, lysine, arginine, urea, glucose, ribose, glycogen, glucosamine, c.+ketoglutaric acid, creatinine and inorganic substances. In this case, the solutions to be measured had to be diluted I : 2 to avoid excessive absorption. The same result was obtained as with natural sweat (Fig. I, 4}_ When urocanic acid was left out of this mixture, a different shift was obtained (Fig. I, 5). Nickel chloride (concentration in the measured solution 10~~44) with combinations of urocanic acid (IO-*M) and all other constituents of this mixture in comparable concentrations did not give results differing in shape from that of nickel chloride with urocanic acid only (Fig. I, 6), except with serine (5 - IO-*M, Fig- I, 7), citrulline fz - IO-PM, Fig. I, S}, and histidine (IO-*M, Fig. I, 9). Without urocanic acid and histidine, no effect could be observed with the artificial sweat. A comparison was made with zinc because of its similarity to nickel*, and with calcium and magnesium because of their occurrence in sweat in relatively large amounts. Zinc sulfate (IO-‘M) with urocanic acid (IO-~&.?)and with urocanic acid and histidine (IO-*M) showed a similar behaviour as nickel chloride (Fig. 2, A, B). Ma~esium chloride (IO-SM) reacted with urocanic acid (IO-*M) in the same manner as nickel chloride (Fig. 2, C). Combinations of uronacic acid with all constituents of the artificial sweat and magnesium chloride gave different results only for lactic acid (5 * x0-44, Fig. 2, D), pyroglutamic acid (4 - IO-4M, Fig. 2, E), and proline (IO-*M, Fig. z, F). Sweat with magnesium chloride gives non-comparable results, probably due to interaction with unknown metabolic products (Fig. 2, G-I). GE&&. Chim. A&, g (1964) x87-rgo
185
tions on one blood sample to which 70 mg/roo ml xylose was added; a mean of 70.8 mg/roo ml with a standard deviation of 1.7 was obtained. This investigation was supported in part by a training grant from the National Institutes of Health, 2 A-5321. W. H. KLASSEN*
University of Utah, College of Mqiicine, Salt Lake City, Utah (U.S.A.)
PHILIP LANZKOWSKY * *
1 J. A. BENSON, JR., P. J. CULVER, S. RAGLAND, C. M. JONES, G. D. DRUMMEY AND E. BO~GAS. New Engl. J. Med., 256 (1957) 335. * F. S. BRIEN, D. A. TURNER, E. M. WATSON AND J. H. GBDDES, Gastroentevology,zo (1952) 287. * I. CHANARIN AND M. C. BENNETT, &it. Med. J., I (1962) 985. 4 J. M. FINLAY AND K. J. R. WIGHTMAN, Ann. Internal Med., 4g (1958) 1332. 6 F. H. GARDNER AND E. PEREZ-SANTIAGO,A.M.A. Arch. Internal Med., g8 (1956) 467. * J. G. ROBERTS, I. T. BECK, J. KALLOS AND D. S. HAHN, Can. Med. Assoc. J., 83 (1960) 112. 7 J. H. ROE AND E. W. RICE, J. Biol. Chem., 173 (1948) 507. * E. H. THAYSEN AND S. M~LLERTZ, Acta Med. Sand., 171 (1962) 521. * G. E. YOUNGBURG, J. Biol. Chem., 73 (1927) 599.
Received September gth, 1963. * Research Associate, Department of Pediatrics. * * Cecil John Adams Memorial Traveling Fellow and Research Fellow in Pediatric Hematology. Clin.
The quantitative
Chim.
Acta,
g (1964)
183-185
determination of pyrrole-2-carboxylic acid in urine*
Studies on the metabolism of collagen have made it desirable to determine quantitatively the amount of pyrrole-2-carboxylic acid (PCA), a metabolic conversion product of hydroxyprolinel, excreted in the urine of man and rat. This communication describes a method for the determination of urinary PCA based on the hydrogen-ion-catalyzed decarboxylation of PCA to pyrrole. The pyrrole is removed from the reaction mixture by distillation and estimated using Ehrlich’s reagent. Reagents Hydrochloric acid: 6.84 N HCl, aqueous. Ehrlich’s reagent (ER): 2.5% dimethyl aminobenzaldehyde in 95% ethanol. Pyrrole (Matheson, Coleman and Bell Co.) : freshly distilled before use. Xanthydrol (Eastman Kodak Co.) : 0.5% solution in methanol. Acetic acid : glacial. * This investigation was supported in whole by U.S. Public Health Research Grant CA 06698 from the National Institute of Cancer. Clin. Chim. Acta, g (1964) 185-187
186
SHORT COMMUNICATIONS
PROCEDURE
Fifteen ml of urine are made 0.5 N with respect to hydrochloric acid and placed in a 50-ml round-bottom flask fitted with a short stillhead and a Liebig condenser. In the case of rat urine it may be necessary to add 3-4 drops of octyl alcohol to avoid
TABLE EXCRETION
Subject J.H. K.O. P.B. P.L. P.L.
OF
PCA
I IN
HUMAN
URINE
pmoles of PCA excreted in 24 h I.39 2.46 I
.07
2.18
3.52
frothing. The distilling flask is heated with a Bunsen burner until IO ml of distillate have been collected. Under these conditions the first 8.5-9.0 ml of the distillate will contain all of the pyrrole. If the specific activity of [X] pyrrole is to be determined (vide ircfra) smaller collections may be more convenient. To a a.3-ml aliquot of the distillate is added 0.65 ml of ER and 0.35 ml of 6.84 N HCl. Exactly 5 min after the addition of HCl the optical density at 560 ma is determined using a Beckman Spectrophotometer or a similar instrument. Precision in the timing of this measurement is important as the color of the solution changes appreciably on standing. A standard curve must be prepared for each set of determinations, using a blank of distilled water and several aqueous standard samples containing from I.49 . IO--~ to I.49 - 10-l micromoles of pyrrole per 2.3 ml. The size of the aliquot of the distillate may be reduced to 1.0 ml with good results. The amounts of ER and HCl must be reduced proportionately. This is advantageous in situations where as much of the distillate as possible must be kept for the determination of radioactivity. Results of an application of this method to human urine are shown in Table I. Determi?zation of the WI-activity of pywole present in the distillate The procedure for obtaining pyrrole from PCA can also be applied when data on the specific %-activity of PCA are desired. It must, of course, be kept in mind that one carbon atom of PCA will be lost as a result of the decarboxylation. In a typical experiment in which urine from rats injected with U-[ “C] proline had been collected, 6 ml of urinary distillate was obtained. One ml of this distillate was used for the quantitative determination of pyrrole. To the remainder of the distillate were added 2.0 ml of glacial acetic acid and 0.1 ml of a 0.5% methanolic solution of xanthydroll. The resulting solution is then evaporated to dryness on a rippled, stainless steel planchet and the X-activity of the residue determined. From the data Clilz. Chim. A&,
g (1964) 185-187
SHORTCOMMUNICATIONS
187
obtained the specific activity is calculated. The quantity of material present on the planchet under these conditions is small enough to constitute an infinitely thin layerGEORGEB. GER~R KNUT R. T~LNES DAVID WOOD* KURT I. ALTMAN
Division of Ex@rimental Radiology and the Departments of Radiation Biology and Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. (U.S.A.) 1 A. N. RADHAKRISHNANAND A. M&ESTER,J. BioZ. Gem.,
226 (1957) 55~.
Received September 3rd, 1963 * Trainee on the Biochemistry Training Grant to the University of Rochester School of Medicine and Dentistry (NIH Grant 2G-137X5)_ Cl& Chins. Ada, g (x964) 185-187 Chelation in human sweat The clinical observation that nickel sensitization might be preceded by a transport of nickel ions through the sweat glands lead us to investigate the interaction of nickel chloride with human sweat. Sweat, obtained by thermal stimulus, was filtered before use. Five ml of the clear liquid and 5 ml 0.1 it4 nickel chloride were diluted to 50 ml and the difference in transmission was measured against 5 ml of the same sweat, diluted to 50 ml, with a Zeiss spectrophotometer PMQ II in the region 220-350 mp. A shift was obtained, having a form independent of the origin of the sweat (Fig. I, x-3). The experiment was repeated with a solution proposed by SPIERAND PASCHER’ as a sweat imitation. This solution contains lactic, citric, uric, formic, pyroglutamic, urocanic, aspartic, and glutamic acids, glycine, serine, threonine, alanine, tyrosine, citrulline, valine, leucine, phenylalanine, proline, tryptophan, histidine, omithine, lysine, arginine, urea, glucose, ribose, glycogen, glucosamine, c.+ketoglutaric acid, creatinine and inorganic substances. In this case, the solutions to be measured had to be diluted I : 2 to avoid excessive absorption. The same result was obtained as with natural sweat (Fig. I, 4}_ When urocanic acid was left out of this mixture, a different shift was obtained (Fig. I, 5). Nickel chloride (concentration in the measured solution 10~~44) with combinations of urocanic acid (IO-*M) and all other constituents of this mixture in comparable concentrations did not give results differing in shape from that of nickel chloride with urocanic acid only (Fig. I, 6), except with serine (5 - IO-*M, Fig- I, 7), citrulline fz - IO-PM, Fig. I, S}, and histidine (IO-*M, Fig. I, 9). Without urocanic acid and histidine, no effect could be observed with the artificial sweat. A comparison was made with zinc because of its similarity to nickel*, and with calcium and magnesium because of their occurrence in sweat in relatively large amounts. Zinc sulfate (IO-‘M) with urocanic acid (IO-~&.?)and with urocanic acid and histidine (IO-*M) showed a similar behaviour as nickel chloride (Fig. 2, A, B). Ma~esium chloride (IO-SM) reacted with urocanic acid (IO-*M) in the same manner as nickel chloride (Fig. 2, C). Combinations of uronacic acid with all constituents of the artificial sweat and magnesium chloride gave different results only for lactic acid (5 * x0-44, Fig. 2, D), pyroglutamic acid (4 - IO-4M, Fig. 2, E), and proline (IO-*M, Fig. z, F). Sweat with magnesium chloride gives non-comparable results, probably due to interaction with unknown metabolic products (Fig. 2, G-I). GE&&. Chim. A&, g (1964) x87-rgo