- Email: [email protected]
Chelation in human sweat
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
188
SHORT COMMUNICATIONS
Calcium chloride failed to show any reaction with sweat or artificial sweat in this experiment. In all experiments the PH was kept at about 6. Potentiometric titrations showed that a series of compounds exists between urocanic acid, histidine and nickel, if the PH is kept close to 7. In competition with dimethylglyoxime, increased histidine concentration reduced the extinction at 445 m,uconsiderably (dimethylglyoxime,nickel chloride, urocanic acid 2 * IO-@M, histidine o-4 * IO-EM). A slight competition was observed between EDTA and urocanic acid in the ratio I : I (0.008 M). Addition of histidine or more urocanic acid increased the extinction at 600 m,u obtained for the same concentration of nickel chloride with excess EDTA; the combination of nickel with histidine and urocanic acid, however, absorbs in the same region. Zinc-urocanic AT
(%)-
1
i : mixed
sweat
of seven1
60
2:perSon x 3: ,, Y 4 : artificial sweat 5: .* I 6: umcunic acid
70
a:
7:
80 J
240
240
9: I 300
280 Effect
of
persons
without ur-ic
”
n
+ serine
.. ,*
,, ”
+ citrulline + histidine
/ 320
I
340
acic
I-
nickel
Fig. I. Clin.Chim.
Ada,
g (1964)
187-190
SHORT COMMUNICATIONS
189
acid complexes have been described by WEITZEL AND FRETZDORFF~. Contrary to the opinion of these authors it may be concluded that the complex formation is due to a shift in the conjugated system of urocanic acid and histidine. The result with magnesium suggests that histidine is of secondary importance. To produce the same effect as in normal sweat, with nickel or zinc and urocanic acid, histidine must be present in excess. From the difference found between calcium and magnesium follows that the steric factor must be considered. In healthy persons, urocanic acid and histidine occur in sweat in considerable amounts 4s&.A possible function of these substances might be the removing of unwanted metal ions from the body. In skin diseases, sweat combined with nickel chloride mostly gives a shift differing from normal sweat.
AT
(%)
20. 30. + histicline
F: ., G: mixed
,, + proline sweat
of
severd
persons.
H:person x 1: ,, Y
80 1 220
240
240
&
&
3BO
3;0
IA X&Z)
Effect
of zinc
and magnesium Fig. 2. Clin.Chim.
A&,
g (1964) 187-190
SHORTCOMMUNICATIONS
190
This provides us with a tool to observe at least a qualitative deviation in an important part of histidine metabolism. Most of the experiments reported have been carried out by Miss N. DE GROOT. Department of Dermatology, Roman Catholic University, Nijmegen (The Netherlands).
J. W. H. MALI D. SPRUIT E. SEUTTER
1 H. W. SPIER AND F. PASCHER, in R. SCHUPPLI (Ed.), Aktuelle Pvobleme der Dermatologie, Vol.I,Karger,Basel,1959,p. 17. a H. FREISER, Q. FERNANDO AND G. E. CHENEY, J. Phys. Ckem., 63 (1959) 250. a G. WEITZEL AND A. M. FRBTZDORFF, Z. Pkysiol. Ckem., 305 (1956) I. 4 J. KRAL, M. KUTOVA, A. &N~SEK AND I. M. HAIS, Czech. Dermatol., 33 (1958) 289. 6 J. KRAL, I. M. HAIS, E. KREJEI AND A. &Nf&zK, Casopis L&karrS Ceskfck, g8 (1959) 1268.
Received October z3rd, 1963 Clin. Chim. Acta, 9 (1964) I87-rgo
Hydrolysis of aromatic esters by human duodenal contents In the majority of studies concerning the enzymatic activity of humanintestinal contents, only the chief enzymes taking part in digestion, i.e. proteases, amylase and lipase, have been investigated, mostly in connection with various pathological conditions of the digestive tract, pancreas and liver. The presence of esterase distinct from lipase was observed in duodenal contents by RICK l,but the properties of this enzyme TABLE
I
HYDROLYSIS OF ACETATES AND BUTYRATES OF VARIOUSPHENOLS BY HUMAN DUODENAL CONTENTS Activity
units are expressed in pmoles of hydrolysed ester per min at 37O. The numbers brackets represent the lowest and the highest activity values.
Substrate p-Hydroxybenzoic acid acetate Indoxyl acetate p-Nitrophenyl acetate Phenyl acetate Guaiacol acetate p-Hydroxybenzoic acid butyrate p-Nitrophenyl butyrate Guaiacol butyrate
No. of determinations 7 7
22 8 8 7 16 8
in
Activity Relative
Units/ml 0.02 ( oo0.16) 2.0 ( 0.7- 3.4 ) 5,o ( 1.2- 9.3 ) 10.7 ( 2.2-20.5 ) 14.2 ( 4.4-24.6 ) 1.2 ( 0.3- I.7 ) 24.7 44.4
(11.5-49.0 (15.2-86.9
) 1
0.04 4.5 11.3 24.1 32.0 2.7 55.8 roo.00
have not been thoroughly studied. In the present study esterolytic activity of human duodenal contents has been tested against a series of aromatic esters differing with respect to the length of fatty acyl chain as well as the type of phenol. Duodenal contents were obtained from II healthy adults by means of the Einhom duodenal tube. All subjects were previously fasted for 12 h and any trace of gastric juice was sucked out before the tube was introduced into the duodenum. Samples of duodenal contents were collected in test tubes immersed in crushed ice. Esterolytic activity was determined by incubation of suitably diluted duodenal fluid with substrate solution and 0.66 M phosphate buffer, pH = 7, at 37’. The rate of substrate decomposition was measured by means of the HESTRIN hydroxamic method2 or, in the case of fi-nitrophenyl esters, the HUGGINS AND LAPIDES method’. Clin. Chim. Ada, g (1964) Igo-192
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
188
SHORT COMMUNICATIONS
Calcium chloride failed to show any reaction with sweat or artificial sweat in this experiment. In all experiments the PH was kept at about 6. Potentiometric titrations showed that a series of compounds exists between urocanic acid, histidine and nickel, if the PH is kept close to 7. In competition with dimethylglyoxime, increased histidine concentration reduced the extinction at 445 m,uconsiderably (dimethylglyoxime,nickel chloride, urocanic acid 2 * IO-@M, histidine o-4 * IO-EM). A slight competition was observed between EDTA and urocanic acid in the ratio I : I (0.008 M). Addition of histidine or more urocanic acid increased the extinction at 600 m,u obtained for the same concentration of nickel chloride with excess EDTA; the combination of nickel with histidine and urocanic acid, however, absorbs in the same region. Zinc-urocanic AT
(%)-
1
i : mixed
sweat
of seven1
60
2:perSon x 3: ,, Y 4 : artificial sweat 5: .* I 6: umcunic acid
70
a:
7:
80 J
240
240
9: I 300
280 Effect
of
persons
without ur-ic
”
n
+ serine
.. ,*
,, ”
+ citrulline + histidine
/ 320
I
340
acic
I-
nickel
Fig. I. Clin.Chim.
Ada,
g (1964)
187-190
SHORT COMMUNICATIONS
189
acid complexes have been described by WEITZEL AND FRETZDORFF~. Contrary to the opinion of these authors it may be concluded that the complex formation is due to a shift in the conjugated system of urocanic acid and histidine. The result with magnesium suggests that histidine is of secondary importance. To produce the same effect as in normal sweat, with nickel or zinc and urocanic acid, histidine must be present in excess. From the difference found between calcium and magnesium follows that the steric factor must be considered. In healthy persons, urocanic acid and histidine occur in sweat in considerable amounts 4s&.A possible function of these substances might be the removing of unwanted metal ions from the body. In skin diseases, sweat combined with nickel chloride mostly gives a shift differing from normal sweat.
AT
(%)
20. 30. + histicline
F: ., G: mixed
,, + proline sweat
of
severd
persons.
H:person x 1: ,, Y
80 1 220
240
240
&
&
3BO
3;0
IA X&Z)
Effect
of zinc
and magnesium Fig. 2. Clin.Chim.
A&,
g (1964) 187-190
SHORTCOMMUNICATIONS
190
This provides us with a tool to observe at least a qualitative deviation in an important part of histidine metabolism. Most of the experiments reported have been carried out by Miss N. DE GROOT. Department of Dermatology, Roman Catholic University, Nijmegen (The Netherlands).
J. W. H. MALI D. SPRUIT E. SEUTTER
1 H. W. SPIER AND F. PASCHER, in R. SCHUPPLI (Ed.), Aktuelle Pvobleme der Dermatologie, Vol.I,Karger,Basel,1959,p. 17. a H. FREISER, Q. FERNANDO AND G. E. CHENEY, J. Phys. Ckem., 63 (1959) 250. a G. WEITZEL AND A. M. FRBTZDORFF, Z. Pkysiol. Ckem., 305 (1956) I. 4 J. KRAL, M. KUTOVA, A. &N~SEK AND I. M. HAIS, Czech. Dermatol., 33 (1958) 289. 6 J. KRAL, I. M. HAIS, E. KREJEI AND A. &Nf&zK, Casopis L&karrS Ceskfck, g8 (1959) 1268.
Received October z3rd, 1963 Clin. Chim. Acta, 9 (1964) I87-rgo
Hydrolysis of aromatic esters by human duodenal contents In the majority of studies concerning the enzymatic activity of humanintestinal contents, only the chief enzymes taking part in digestion, i.e. proteases, amylase and lipase, have been investigated, mostly in connection with various pathological conditions of the digestive tract, pancreas and liver. The presence of esterase distinct from lipase was observed in duodenal contents by RICK l,but the properties of this enzyme TABLE
I
HYDROLYSIS OF ACETATES AND BUTYRATES OF VARIOUSPHENOLS BY HUMAN DUODENAL CONTENTS Activity
units are expressed in pmoles of hydrolysed ester per min at 37O. The numbers brackets represent the lowest and the highest activity values.
Substrate p-Hydroxybenzoic acid acetate Indoxyl acetate p-Nitrophenyl acetate Phenyl acetate Guaiacol acetate p-Hydroxybenzoic acid butyrate p-Nitrophenyl butyrate Guaiacol butyrate
No. of determinations 7 7
22 8 8 7 16 8
in
Activity Relative
Units/ml 0.02 ( oo0.16) 2.0 ( 0.7- 3.4 ) 5,o ( 1.2- 9.3 ) 10.7 ( 2.2-20.5 ) 14.2 ( 4.4-24.6 ) 1.2 ( 0.3- I.7 ) 24.7 44.4
(11.5-49.0 (15.2-86.9
) 1
0.04 4.5 11.3 24.1 32.0 2.7 55.8 roo.00
have not been thoroughly studied. In the present study esterolytic activity of human duodenal contents has been tested against a series of aromatic esters differing with respect to the length of fatty acyl chain as well as the type of phenol. Duodenal contents were obtained from II healthy adults by means of the Einhom duodenal tube. All subjects were previously fasted for 12 h and any trace of gastric juice was sucked out before the tube was introduced into the duodenum. Samples of duodenal contents were collected in test tubes immersed in crushed ice. Esterolytic activity was determined by incubation of suitably diluted duodenal fluid with substrate solution and 0.66 M phosphate buffer, pH = 7, at 37’. The rate of substrate decomposition was measured by means of the HESTRIN hydroxamic method2 or, in the case of fi-nitrophenyl esters, the HUGGINS AND LAPIDES method’. Clin. Chim. Ada, g (1964) Igo-192