- Email: [email protected]
The occurrence of urocanic acid in human sweat
VOL. 12 (1953)
SI~ORT COMMUNICATIONS,PRELIMINARYNOTES
479
higher values found in physiological contraction, the extensibility in rigor tends t o w a r d s the values associated w i t h contracted muscle. F u r t h e r m o r e , the relation b e t w e e n s h o r t e n i n g and extensibility, indicating a diminished cross-bond f o r m a t i o n w i t h increased shortening, suggests t h a t individual b o n d s become available d u r i n g the onset of rigor for either shortening or an increase in resistance to extension. On this view the difference b e t w e e n s h o r t e n i n g and decreased extensibility would be largely one of b o n d direction; b o n d f o r m a t i o n b e t w e e n side-by-side chains or filanlents would give the cross-linked s t r n c t u r e suggested b y BATE-SMITH 2, while b o n d formation either intranlolecularly or between longitudinally a d j a c e n t filaments would result in shortening. This work is published b y pernlission of the Director, D o m i n i o n L a b o r a t o r y , \Vellington, New Zealand. REFERENCES 1 E. C. BATE-SMITH, ,]. Physiol., 96 (1939) 1762 E. C. ~3ATE-SMITH, Advances in Food Research, i (1948) ~. 3 E. C. ]~ATE-SMITH AND J. R. BENDALL, J. Physiol., i i o (I949) 47. 4 j. R. BENDALL, J. Physiol., 114 (1951 ) 71 . 5 M. G. M. PRYOR, Progress in Biophysics, 1 (195 o) 216. Received A u g u s t 5th, 1953
T H E OCCURRENCE OF UROCANIC ACID IN HUMAN SWEAT by A. ~ E N ~ E K
AND J. A. KRA.L
Institute o[ 34edicine as Applied to Physical Education and Sport, Charles University, Prague (Czechoslovakia)
Since the d e m o n s t r a t i o n of urocanic (imidazole-acrylic) acid in animal material 1, the i m p o r t a n c e of this substance as a m a j o r i n t e r m e d i a r y in histidine metabolism has been stressed b y several a u t h o r s 2, b u t the physiological significance of this p a t h w a y has been questioned b y others 3. The formation of this substance from histidine b y surviving liver tissue seems to have been obscured by the simultaneous presence of a s y s t e m t h a t splits urocanic acid (via formylisoglutamine). In skin, on the o t h e r hand, the occurrence of an enzymic deamination p r o d u c t of histidine which contains intact imidazole ring could readily be d e m o n s t r a t e d b y BORGHI AND TARANTINO4. The a b s t r a c t does n o t r e p o r t the identification of this substance or a discussion of this finding, in relation to the p r o d u c t i o n of sweat. I n view of the d e m o n s t r a t i o n of urocanic acid in h u m a n s w e a t b y H n l s and the p r e s e n t a u t h o r s ~, the physiological significance of the oxidative histidine deaminase should be w o r t h investigation. I t is interesting to note t h a t a n o t h e r enzymic feature of skin s h o w n b y the same I t a l i a n authors6 - - the presence of an " a r g i n a s e " leading to the liberation of a m m o n i a - - i s parallelled b y the occurrence of citrulline in sweat, as d e m o n s t r a t e d b y KRAL, HAIS AND ~ENf~EKT, 8 and in dry skin water-wipings, as found b y ROTHMAN AND SULLIVAN9. On every p a p e r c h r o m a t o g r a m of h u m a n sweat detected w i t h the P a u l y reagent, KRXL, HAIS AND ~ENfgEKT, 8 found an unidentified (crimson) spot. I t s position in the butanol-formic acid m i x t u r e was near t h a t of tyrosine b u t it could be readily distinguished from it on c o - c h r o m a t o g r a p h y , owing to a different shade of the colour reaction and different shape of the spot. I t s R F differed from histamine and dihydroxyphenylalanine. A l t h o u g h the concentration of this substance was considerable in sweat, it was n o t found in the urine of the same person. The presence of occasional faint traces of a similar substance in the blood s e r u m could not be ruled out. Only s w e a t of h e a l t h y males w a s studied. All samples were electrolytically desalted before application 1°,Ix. The substance was resistant to acid hydrolysis (6 N HC1; 24 hours). I n the course of later work, the s u b s t a n c e responsible for this spot was partially purified and concentrated b y p r e p a r a t i v e p a p e r c h r o m a t o g r a p h y and ionophoresis. The presence of the p r i m a r y aliphatic a m i n o group seemed improbable, as it was found t h a t the s u b s t a n c e does not show the colour reaction w i t h ninhydrin. No colour was formed by t r e a t m e n t w i t h n i t r o u s fumes and s p r a y i n g w i t h the Bratton-Marshall reagent, t h u s excluding diazotizable p r i m a r y aromatic amino group, Electrical mobility of the substance was t o w a r d s the cathode in the acidic and n e u t r a l reaction; it was similar to t h a t of histidine in o.5 % acetic acid. A p p r o a c h i n g
SHORT COMMUNICATIONS, PRELISfINARY NOTES
4~qO
VOL. 12
(i953'
p H 9, ulobility took tile opposite sign. An a m p h o t e r i c n a t u r e was t h u s established, ruling o u t a n y n i t r o g e n - f r e e phenolic s u b s t a n c e s w h i c h couht also react w i t h diazotized s u l p h a n i l a m i d e . Adrenaline, w h o s e R F w a s similar in t h e b u t a n o l - f o r m i c acid m i x t u r e , was ruled o u t b y t h e failure of t h e s u b s t a n c e to ltuoresce in alkaline m e d i u m a n d to affect a p p r e c i a b l y t h e blood pressure of st)inal cat. O u r a t t e n t i o n w a s t h u s directed to imidazole s u b s t i t u t e d carboxylic acids, for exanlple urocanic acid, w h i c h is k n o w n as a n a t u r a l l y occurring s u b s t a n c e 1. However, according to ERSPAMER12, urocalliC acid does n o t give a red spot w i t h t h e l~auly-reagent. It was found t h a t a n a u t h e n t i c s a m p l e of imidazole propionic acid e x a c t l y c o r r e s p o n d s to t h e u n k n o w n s u b s t a n c e , b o t h in colour reaction (crimson red) a n d position of t h e s p o t s in p a p e r c h r o m a t o g r a p h i c (Table 1, Spot x) a n d p a p e r ionophoretic e x p e r i m e n t s . Acetic acid or disodium h y d r o g e n p h o s p h a t e was used as electrolyte for t h e latter. TA B L E I RF-value~
n-butanol formic acid (85 : zS, water saturated)
n-butanol-N hydrochloric acid (~ : i)
n-butanol;acetic acid;water (4 : :~ : 5)
Pauly colour
U r o c a n i c acid Spot y (undesalted) Inlidazole propionic acid Spot x (desalted) Tyrosine
o.4o 0.40 0.3(, 0.34 o.34
o.28 o.28 0.20 0.33 o.33
o.65 o.65 o.5o o.5o o.45
b r o w n orange b r o w n orange c r i m s o n red c r i m s o n red b r o w n orange
H o w e v e r , w h e n a n u n d e s a l t e d s a m p l e of s w e a t w a s used, this s u b s t a n c e could n o t be d e t e c t e d . T h e m a i n P a u l y positive s p o t s corresponded to t h e positions of histidine, t y r o s i n e a n d u r o c a n i c acid (Table I, s p o t y). T h e n c e it w a s c o n c l u d e d t h a t imidazole-propionic acid, whose presence in a n i m a l m a t e r i a l h a s n o t y e t been reported, arises from h y d r o g e n a t i o n in t h e course of t h e electrodialytic d e s a l t i n g procedure. T h i s conversion of urocanic acid into imidazole-propionic could be verified b y c o m p a r i s o n of a n u n d e s a l t e d a n d a desalted s p e c i m e n from t h e s a m e p e r s o n ; it a g a i n e m p h a s i z e s t h e desirability of suitable control a n d c a u t i o u s i n t e r p r e t a t i o n w h e n electrolytic d e s a l t i n g is used. T h e i d e n t i t y of u r o c a n i c acid was f u r t h e r s u b s t a n t i a t e d b y t h e characteristic shift of t h e a b s o r p t i o n n l a x i m u m from 264 m/~ (pH 4.3) to 280 in/t (pH I 1). T h e c o n c e n t r a t i o n of urocanic acid in h u m a n s w e a t is n o t negligible; in a few s a m p l e s w h i c h h a v e been i n v e s t i g a t e d b y q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h y it a v e r a g e d o.i m g / m l . I t s concent r a t i o n in s w e a t a f t e r p h y s i c a l exercise a p p e a r s h i g h e r t h a n in t h e r m a l sweat. O u r t h a n k s are due to Dr. I. M. }{AIS for close collaboration a n d fruitful discussions, to Dr. K. SLAViK for t h e U.V. s p e c t r u m a n d to Prof. Dr. V. ERSPAMER (Bari) for t h e g e n e r o u s gift of imidazole-acetic, imidazole-propionic a n d urocanic acid. REFERENCES 1 M. JAFF£, Bet., 7 (1874) 1669; M. SIEGFRIED, Z. physiol. Chem., 24 (I898) 399. 2 y . KOTAKE AND M. KONISHI, Z. physiol. Chem., 122 (I922) 23o; M. KIVOI
SI~ORT COMMUNICATIONS,PRELIMINARYNOTES
479
higher values found in physiological contraction, the extensibility in rigor tends t o w a r d s the values associated w i t h contracted muscle. F u r t h e r m o r e , the relation b e t w e e n s h o r t e n i n g and extensibility, indicating a diminished cross-bond f o r m a t i o n w i t h increased shortening, suggests t h a t individual b o n d s become available d u r i n g the onset of rigor for either shortening or an increase in resistance to extension. On this view the difference b e t w e e n s h o r t e n i n g and decreased extensibility would be largely one of b o n d direction; b o n d f o r m a t i o n b e t w e e n side-by-side chains or filanlents would give the cross-linked s t r n c t u r e suggested b y BATE-SMITH 2, while b o n d formation either intranlolecularly or between longitudinally a d j a c e n t filaments would result in shortening. This work is published b y pernlission of the Director, D o m i n i o n L a b o r a t o r y , \Vellington, New Zealand. REFERENCES 1 E. C. BATE-SMITH, ,]. Physiol., 96 (1939) 1762 E. C. ~3ATE-SMITH, Advances in Food Research, i (1948) ~. 3 E. C. ]~ATE-SMITH AND J. R. BENDALL, J. Physiol., i i o (I949) 47. 4 j. R. BENDALL, J. Physiol., 114 (1951 ) 71 . 5 M. G. M. PRYOR, Progress in Biophysics, 1 (195 o) 216. Received A u g u s t 5th, 1953
T H E OCCURRENCE OF UROCANIC ACID IN HUMAN SWEAT by A. ~ E N ~ E K
AND J. A. KRA.L
Institute o[ 34edicine as Applied to Physical Education and Sport, Charles University, Prague (Czechoslovakia)
Since the d e m o n s t r a t i o n of urocanic (imidazole-acrylic) acid in animal material 1, the i m p o r t a n c e of this substance as a m a j o r i n t e r m e d i a r y in histidine metabolism has been stressed b y several a u t h o r s 2, b u t the physiological significance of this p a t h w a y has been questioned b y others 3. The formation of this substance from histidine b y surviving liver tissue seems to have been obscured by the simultaneous presence of a s y s t e m t h a t splits urocanic acid (via formylisoglutamine). In skin, on the o t h e r hand, the occurrence of an enzymic deamination p r o d u c t of histidine which contains intact imidazole ring could readily be d e m o n s t r a t e d b y BORGHI AND TARANTINO4. The a b s t r a c t does n o t r e p o r t the identification of this substance or a discussion of this finding, in relation to the p r o d u c t i o n of sweat. I n view of the d e m o n s t r a t i o n of urocanic acid in h u m a n s w e a t b y H n l s and the p r e s e n t a u t h o r s ~, the physiological significance of the oxidative histidine deaminase should be w o r t h investigation. I t is interesting to note t h a t a n o t h e r enzymic feature of skin s h o w n b y the same I t a l i a n authors6 - - the presence of an " a r g i n a s e " leading to the liberation of a m m o n i a - - i s parallelled b y the occurrence of citrulline in sweat, as d e m o n s t r a t e d b y KRAL, HAIS AND ~ENf~EKT, 8 and in dry skin water-wipings, as found b y ROTHMAN AND SULLIVAN9. On every p a p e r c h r o m a t o g r a m of h u m a n sweat detected w i t h the P a u l y reagent, KRXL, HAIS AND ~ENfgEKT, 8 found an unidentified (crimson) spot. I t s position in the butanol-formic acid m i x t u r e was near t h a t of tyrosine b u t it could be readily distinguished from it on c o - c h r o m a t o g r a p h y , owing to a different shade of the colour reaction and different shape of the spot. I t s R F differed from histamine and dihydroxyphenylalanine. A l t h o u g h the concentration of this substance was considerable in sweat, it was n o t found in the urine of the same person. The presence of occasional faint traces of a similar substance in the blood s e r u m could not be ruled out. Only s w e a t of h e a l t h y males w a s studied. All samples were electrolytically desalted before application 1°,Ix. The substance was resistant to acid hydrolysis (6 N HC1; 24 hours). I n the course of later work, the s u b s t a n c e responsible for this spot was partially purified and concentrated b y p r e p a r a t i v e p a p e r c h r o m a t o g r a p h y and ionophoresis. The presence of the p r i m a r y aliphatic a m i n o group seemed improbable, as it was found t h a t the s u b s t a n c e does not show the colour reaction w i t h ninhydrin. No colour was formed by t r e a t m e n t w i t h n i t r o u s fumes and s p r a y i n g w i t h the Bratton-Marshall reagent, t h u s excluding diazotizable p r i m a r y aromatic amino group, Electrical mobility of the substance was t o w a r d s the cathode in the acidic and n e u t r a l reaction; it was similar to t h a t of histidine in o.5 % acetic acid. A p p r o a c h i n g
SHORT COMMUNICATIONS, PRELISfINARY NOTES
4~qO
VOL. 12
(i953'
p H 9, ulobility took tile opposite sign. An a m p h o t e r i c n a t u r e was t h u s established, ruling o u t a n y n i t r o g e n - f r e e phenolic s u b s t a n c e s w h i c h couht also react w i t h diazotized s u l p h a n i l a m i d e . Adrenaline, w h o s e R F w a s similar in t h e b u t a n o l - f o r m i c acid m i x t u r e , was ruled o u t b y t h e failure of t h e s u b s t a n c e to ltuoresce in alkaline m e d i u m a n d to affect a p p r e c i a b l y t h e blood pressure of st)inal cat. O u r a t t e n t i o n w a s t h u s directed to imidazole s u b s t i t u t e d carboxylic acids, for exanlple urocanic acid, w h i c h is k n o w n as a n a t u r a l l y occurring s u b s t a n c e 1. However, according to ERSPAMER12, urocalliC acid does n o t give a red spot w i t h t h e l~auly-reagent. It was found t h a t a n a u t h e n t i c s a m p l e of imidazole propionic acid e x a c t l y c o r r e s p o n d s to t h e u n k n o w n s u b s t a n c e , b o t h in colour reaction (crimson red) a n d position of t h e s p o t s in p a p e r c h r o m a t o g r a p h i c (Table 1, Spot x) a n d p a p e r ionophoretic e x p e r i m e n t s . Acetic acid or disodium h y d r o g e n p h o s p h a t e was used as electrolyte for t h e latter. TA B L E I RF-value~
n-butanol formic acid (85 : zS, water saturated)
n-butanol-N hydrochloric acid (~ : i)
n-butanol;acetic acid;water (4 : :~ : 5)
Pauly colour
U r o c a n i c acid Spot y (undesalted) Inlidazole propionic acid Spot x (desalted) Tyrosine
o.4o 0.40 0.3(, 0.34 o.34
o.28 o.28 0.20 0.33 o.33
o.65 o.65 o.5o o.5o o.45
b r o w n orange b r o w n orange c r i m s o n red c r i m s o n red b r o w n orange
H o w e v e r , w h e n a n u n d e s a l t e d s a m p l e of s w e a t w a s used, this s u b s t a n c e could n o t be d e t e c t e d . T h e m a i n P a u l y positive s p o t s corresponded to t h e positions of histidine, t y r o s i n e a n d u r o c a n i c acid (Table I, s p o t y). T h e n c e it w a s c o n c l u d e d t h a t imidazole-propionic acid, whose presence in a n i m a l m a t e r i a l h a s n o t y e t been reported, arises from h y d r o g e n a t i o n in t h e course of t h e electrodialytic d e s a l t i n g procedure. T h i s conversion of urocanic acid into imidazole-propionic could be verified b y c o m p a r i s o n of a n u n d e s a l t e d a n d a desalted s p e c i m e n from t h e s a m e p e r s o n ; it a g a i n e m p h a s i z e s t h e desirability of suitable control a n d c a u t i o u s i n t e r p r e t a t i o n w h e n electrolytic d e s a l t i n g is used. T h e i d e n t i t y of u r o c a n i c acid was f u r t h e r s u b s t a n t i a t e d b y t h e characteristic shift of t h e a b s o r p t i o n n l a x i m u m from 264 m/~ (pH 4.3) to 280 in/t (pH I 1). T h e c o n c e n t r a t i o n of urocanic acid in h u m a n s w e a t is n o t negligible; in a few s a m p l e s w h i c h h a v e been i n v e s t i g a t e d b y q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h y it a v e r a g e d o.i m g / m l . I t s concent r a t i o n in s w e a t a f t e r p h y s i c a l exercise a p p e a r s h i g h e r t h a n in t h e r m a l sweat. O u r t h a n k s are due to Dr. I. M. }{AIS for close collaboration a n d fruitful discussions, to Dr. K. SLAViK for t h e U.V. s p e c t r u m a n d to Prof. Dr. V. ERSPAMER (Bari) for t h e g e n e r o u s gift of imidazole-acetic, imidazole-propionic a n d urocanic acid. REFERENCES 1 M. JAFF£, Bet., 7 (1874) 1669; M. SIEGFRIED, Z. physiol. Chem., 24 (I898) 399. 2 y . KOTAKE AND M. KONISHI, Z. physiol. Chem., 122 (I922) 23o; M. KIVOI