B I O L O G I C A L T R A C E E L E M E N T R E S E A R C H 2, 247-254
Trypsin Activity in the Small Intestine of Rats after Application of Copper and Zinc RENATE WIENINGER-RUSTEMEYER, MANFRED
KIRCHGESSNER,*
AND
HANS STEINHART Institute of Nutrition Physiology, Technical University of Munich, D-8050 Freising- Weihenstephan, West Germany Received February 6, 1980; Accepted May 9, 1980
Abstract In vivo experiments with Spra/~ue-Dawley rats were conducted in order to 9 2+ 2+ 9 9 explore the influence of Cu , Zn as well as of the combinations of both on the activity of trypsin9 The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by using N~-benzoylL-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu 2+ ions are applied, there is a minimum activity at 10-5 mol Cu2§ and a maximum at 10-4 mol Cu2§ When giving Zn 2§ ions, a minimum of trypsin activity is found at 10-5 mol Zn2§ and a maximum at 5 • l 0 -6 mol Zn2§ On the whole, the trypsin activity is lower when the Cu2§ 2§ combinations are applied compared to the addition of the single trace elements9 On principle, a good conformity of the in vivo results was found with in vitro results. Index Entries: Activity of trypsin in the small intestine; rat trypsin activity, and copper and zinc; L-BAPA, and Cu and Zn effects on trypsin activity; addition of copper, zinc, and combinations to rat intestine; comparison of in vivo and in vitro results of Cu and Zn on trypsin activity9
Introduction A striking influence of the trace elements Cu, Zn, Fe, Ni, and Co on the activity of trypsin using N"-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate was found when in vitro experiments were conducted in order 9 1980 by The H u m a n a Press Inc. All rights o f any nature whatsoever reserved. 0163 4 9 8 4 / 8 0 / 1200 4)247502.00
247
248
WIENINGER-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
to determine enzyme activity. Depending on the trace elements used and on their concentrations, activating and inhibitory effects could be demonstrated (1, 2). Additional in vitro experiments were done in order to explore the interactions of copper with other trace elements. A predominant influence of copper was found (3). It was the aim of this work to research whether the effects found in vitro can also be shown in the animal. The experiments in this trial were restricted to the trace elements copper and zinc. For this purpose, solutions of trace elements were applied per os into the gastrointestinal tract with a stomach tube, and, after killing the rats, the trypsin activity was determined in the juice of the small intestine.
Methods Female SPF Sprague-Dawley rats were used for the experiments. At the beginning of the experiments, the animals were six weeks old and they weighed about 130 g. Their weight at the end of the experiments was about 150 g. Four animals were used for each applied trace element concentration. Drinking water, which was renewed every day (aqua dest.; brought up to normal osmolarity with 0.014% NaCI), was available ad libitum. The animals were fed a standard feed that contained 6.8% fish meal (65% crude protein); 6.3% meat meal (65% crude protein); 2.7% dried yeast; 1.2% bone meal, 48% barley; 18% oat: 12% corn; 4% alfalfa meal; 1% CaHPO4"2H20. During the night, the feed was removed in order to train the animals to consume 5 g feed within 1 h at the morning meal. With this procedure, the presupposition was created for an equal feed intake by each animal. At the same time, the animals got used to the stomach tube with which 0.3 or 0.6 mL of distilled water were applied per os every day, and the respective trace element solutions were given in the same way on the day of the experiment. The applied trace element concentrations are shown in Table 1. The animals were decapitated under ether narocsis exactly 1 h after the beginning of the feeding and 0.5 h after having applied the respective trace element solution. The abdominal cavity was opened and the first 20-cm segment of the small intestine was removed. The contents of this part of the small intestine were put into a 10-mL flask and filled with distilled water. The trypsin activity in the juice of the small intestine was determined with the method of Erlanger et al. (4) modified by Wieninger-Rustemeyer et al. (5) in order to make it applicable for these experiments. Pappas (6) recommended this method for in vivo experiments because the trypsin activity is determined specifically by using L-BAPA as a substrate. The reaction time was 10 min. p-Nitroaniline which is liberated from L-BAPA, was measured spectrophotometrically at 410 nm and the extinction was used as a criterion of the relative trypsin activity.
249
TRYPSIN ACTIVITY IN RATS
TABLE 1 Ion Concentration (tool Me~+/L) and Quantity (mL) of the Applied Trace Element Solutions Me 2+
[Me ~§
Cu 2+ as CuSO4.5H20
Zn 2+ as ZnSO4 ' 7H20
m
5 • 10-6 10-5 5 • 10-5 10-4 5 X 10 -4
_
-0.3 0.3 0.3 0.3
Cu2+(10-4mol/L) + Zn 2§
_
-
0.3 0.3 0.3 0.3 --
-
Each Each Each Each
0.3 0.3 0.3 0.3
--
The + values given in the tables are standard deviations. The significance was calculated with the Newman-Keuls test. Groups that do not share one or more c o m m o n letters are different with the probability given in the respective tables.
Results Trypsin Activation in the Small Intestine Dependent on the Concentration of C u 2+ Ions The results of the experiments with per os application of Cu 2§ ions as well as the standard deviations and the statistical significances are summarized in Table 2. The trypsin activity in the juice of the small intestine decreases first somewhat when 10-5 tool Cu2+/L are applied, and then it increases strongly with increasing copper additions. The highest activity is reached with 10-4 tool Cu2§ Application of even more Cu 2§ ions results again in a decrease of the trypsin activity. When comparing the trypsin activities in the small TABLE 2 Trypsin Activity in the Juice of Rat Small Intestine After per os Application of Different Amounts of Cu 2§ ions (P < 5%) 0 0.48
10-5 _
0 . 1 3 a'b
0.37 4- 0.14 a
5 X 10-5 0.56
- - 0 . 1 1 b'c
10-4
5 • 10-4 mol Cu2+/L
0.68 4- 0.20 c
0.56 4- 0.13 b'c
25O
WIENINGER-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
O.d.
0.7
0.6
0.5
\ \
\ \
0.4
0.3
0.2
0.1
I/
i
5.10-5
[
10-4
I
5.10-4
9.10 -8 4.5.10-7 9.10-7
i
10-3
mol Cu++/I
i
510-3 invivo
L.510 -69.10 -6
/..510 -59,10 -5 in vitro
FIG. 1. Influence of Cu2+ ions on the tryptic hydrolysis of L-BAPA in vitro (x--x) and in vivo (o--o). intestine with results from in vitro experiments (Fig. 1), one can recognize a good conformity. The initial activities of the trypsin are similar. The increase, and after reaching a maximum, the decrease are as pronounced in vitro as in vivo. But the Cu ~-§ concentrations needed in vivo are about 5 X 10z times higher than those needed in vitro in order to get similar results.
Trypsin Activities in the Small Intestine Dependent on the Concentrations of Zn 2§ Ions The results of the experiments with per os application of Zn 2§ ions are summarized in Table 3. Although the mean trypsin activity is essentially higher at the Zn 2§ concentration of 5 • 10 -6 mol Zn2*/L compared to the control experiment, this difference cannot be secured by statistical methods because of the large variations of the individual values. A considerable decrease of the trypsin activity can be seen when 10-5 mol Zn2+/L are
251
TRYPSIN ACTIVITY IN RATS
TABLE 3 Trypsin Activity in the Juice of Rat Small Intestine After per os Application of Different Amounts of Zn 2§ Ions (P < 5%) 0
5 X 10-6
0.48 + 0.13 a
10-5
5 X 10-5
10-4molZn2§
0.60 -t- 0.13 a 0.27 + 0.05 b 0.52 + 0.12 a
0.49 9
0.08
a
applied. When the Zn 2+ concentration is increased further, the activities of the control experiment are reached again. In Fig. 2, the in vivo results are compared to in vitro results. It can be seen that a certain parallelism exists between both trials. The Zn 2§ concentrations used in the in vivo experiments are, however, about 10 times higher than those in the in vitro experiments in order to get comparable effects. It is, however, a fact that the variations of the trypsin activity are higher in the experiments with the juice of the small intestine than in the in vitro trials. o.d.i
0.6 7 f~
f/O,~
--% O.L
0.3
\
\ \
0.2
\ \ \ \
0.1 mot Zn§
I 10 -6
~/
I 9.10-8
I 5.10 -6
I
I 10-5
I
L.5.10-7 9.10-7
I 5.10 -5
t
I 10-L
1-
/,.5.10-6 9.10-6
I 5.10 -3 in vlvo
I
~__
L.5.10 -5 in vitro
FIG. 2. Influence of Zn 2§ions on the tryptic hydrolysi~ Of L-BAPA without Cu 2§ addition in vivo (o--o) and in vitro (x--x) as well as with addition of Cu 2§ in vivo ( ~ - e ) and in vitro (u--tu).
252
WIEN1NGER-RUSTEMEYER,
KIRCHGESSNER,
AND STEINHART
Influence of the Interactions Between Zn 2§ and Cu2+ Ions on the Trypsin Activity In order to investigate the influence of the interactions between Zn 2§ and Cu 2§ application on the trypsin activity, a Cu 2§ solution in equal quantity and concentration was applied per os. When 0.3 mL of a 10-4 molar Cu 2§ solution were given alone, the increase in the trypsin activity was highest. 9 9 ' Together with Cu 2+ , Zn 2+ solutions were added m the same concentratmns as in the experiments with isolated Zn 2§ additions. The results of these experiments are summarized in Table 4. The trypsin activity decreases markedly when Cu 2§ and Zn 2§ ions are added together compared to the control experiment. The reason may be that the concentration of the trace elements is twice as high as in the experiments in which each trace element was applied alone. This decrease is, however, not linear. The trypsin activity reaches a minimum at 10-5 mol Zn2§ then increases to a maximum at 5 • 10~ mol Zn2§ and then decreases again. The trypsin activity curves of the experiments with the combined addition of Cu 2§ and Zn 2§ ions of the in vivo and of the corresponding in vitro trials are shown in Fig. 2. It can be seen from this figure that the curves tend to be similar. The effects, however, are more distinct in the in vivo than in the in vitro experiments.
Discussion It is known from in vitro experiments that trace elements influence the activity of pepsin (7, 8) and trypsin (1, 2). The change of their activity depends on the element and its concentration; it may be either positive or negative. There are also interactions between trace elements in regard to their effect on the activity of pepsin (9) and trypsin (3) in vitro. Therefore, it was reasonable to conduct experiments in order to find out whether these effects are also present in vivo. But in vivo, the parameters that may influence the activity of proteolytic enzymes (for instance, enzyme-substrate ratio, pH value, or trace element concentration) are not as constant as in in vitro experiments. The in vivo experiments were conducted with Zn and Cu TABLE 4 Trypsin Activity in the Juice of Rat Small Intestine after per os Application of 10-4mol CuZ+/L together with Increasing Amounts of Zn 2+ Ions (P < 5%) 0
0.48 + 0.13 a
5 X 10-6
10-s
5 X 10-5
0.26 + 0.05 b 0.13 + 0.04 c 0.31 + 0.13 b
10-4toolZn:+/L + 10-4tool Cu:+/L 0.23 + 0.03 b
TRYPSIN ACTIVITY IN RATS
253
because these elements influenced the strongest trypsin activity in vitro. These trace elements were given per os by stomach tube in order to avoid additional influences that might be encountered if applied with the feed. It was not possible to apply the same quantity of liquid in the in vivo as in the in vitro experiments (1 mL) because rats are not able to consume such a quantity all at once. Therefore the quantity of the applied solution was reduced to 0.3 mL. The in vivo experiments were conducted with higher concentrations of trace elements than the in vitro experiments because Zn 2+ and Cu :+ ions are bound in the digestive tract by different ligands. The method of Pappas (6) was used in order to determine the trypsin activity in the juice of the small intestine. Taking L-BAPA as trypsin substrate seemed to be best, because L-BAPA is a relatively specific substrate of trypsin. L-BAPA is only split by trypsin, papain, and thrombin (10, 11). Because the papain does not occur in animal material, and thrombin only in the blood, it may be assumed that only the tryptic activity was determined in the juice of the small intestine of the rats. Therefore it was possible to use the nonpurified juice as the enzyme source. Pappas (6) found it to be important to use only the juice of the first or second part of the small intestine to determine trypsin activity because the activity is already strongly reduced in the juice of the third part. This result is in agreement with observations of Bird (12), who found the highest trypsin activity in the last quarter of the duodenum, and in the first part of the jejunum. The results of these experiments show that the trypsin activity in the juice of the small intestine is influenced by Cu 2+ions as well as by Z n 2+ ions. The effect is dependent on the concentration of the applied trace elements. When the Cu 2+ ions are applied per os, there is a similar change in the trypsin activity as can be observed in the in vitro experiments. The concentration of Cu 2+ ions is, however, about 5 • 102 mol higher in the in vivo experiments than in the in vitro experiments. One reason for these differences between in vivo and in vitro experiments is probably the fact that Cu 2+ions are partly complexed in the heterogeneous environment of the gastrointestinal tract, and therefore, it is obvious that not the whole amount of the applied Cu 2+ ions may interact with the trypsin. Another reason is that Cu ~+ ions are partly absorbed during the time between application and killing of the rats. There is also a good agreement between the in vivo and the in vitro results when Zn is considered. To get similar effects, the Zn :+ concentration was in vivo 10 times higher than in the in vitro experiments. The differences to the even higher Cu 2+ concentrations in vivo compared to in vitro experiments may be explained by the fact that Cu 2+ ions are complexed stronger in the gastrointestinal tract than Zn 2+ ions do. Metabolic effects of the applied trace elements on trypsin synthesis in the pancreas may be excluded because the period of time of 30 min between application of the trace elements and killlng of the animals is too short. When a constant Cu 2+quantity (10-4 tool Cu 2 /L) is applied together with increasing amounts of Zn 2+ ions, trypsin
254
WlENINGEI~-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
activity is changed in the same way in the in vitro as well as in the in vivo experiments. At any rate, the activity is decreased to about the a m o u n t as that which is caused by the Cu 2§ ions. With these experiments it was possible to demonstrate that effects found in an isolated system are transferable, up to a certain degree, to living systems.
References 1. M. Kirchgessner, R. Wieninger-Rustemeyer, and H. Steinhart, Z. Ticrphysiol., Tierern~hrg. Futtermittelkde 43, 130 (1980). 2. R, Wieninger-Rustemeyer, M. Kirchgessner, and H. Steinhart, Nutr. Metab, in press. 3. M. Kirchgessner, H. Steinhart, and R. Wieninger-Rustemeyer, Internar Z. Vir Ern, Forsch., in press. 4. B. F. Erlanger, F. Edel, and A. G. Cooper, Arch. Biochem. Biophys. 115, 206 (1966). 5. R. Wieninger-Rustemeyer, H, Steinhart, and M. Kirchgessner, Landwtrtsch. Forsch., in press. 6. P. W. Pappas, J. Parasitol. 64, 562 (1978). 7. M. Kirchgessner, M. G. Beyer, and H. Steinhart, Brit. J. Nutr. 36, 15 (1976). 8. M.G. Beyer, M. Kirchgessner, and H. Steinhart, Landwirtsch, Forsch. 29, 53 (1976). 9. H. Steinhart, M. Kirchgessner, and M. G. Beyer, Arch. Tierernhhrung26, 629 (1976). 10. W. Nagel, F. Willig, W. Peschke, and F. H. Schmidt, Hoppe-Seylers Z. Physiol. Chem. 340, 1 (1965). 11. H. Schiessler, H. Fritz, M. Arnold, E. Fink, and H, Tschesche, Hoppe-Seylers Z. Physiol. Chem. 353, 1638 (1972). 12. F. H. Bird, Brit. Poultry Sci. 12, 373 (1971).
Trypsin Activity in the Small Intestine of Rats after Application of Copper and Zinc RENATE WIENINGER-RUSTEMEYER, MANFRED
KIRCHGESSNER,*
AND
HANS STEINHART Institute of Nutrition Physiology, Technical University of Munich, D-8050 Freising- Weihenstephan, West Germany Received February 6, 1980; Accepted May 9, 1980
Abstract In vivo experiments with Spra/~ue-Dawley rats were conducted in order to 9 2+ 2+ 9 9 explore the influence of Cu , Zn as well as of the combinations of both on the activity of trypsin9 The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by using N~-benzoylL-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu 2+ ions are applied, there is a minimum activity at 10-5 mol Cu2§ and a maximum at 10-4 mol Cu2§ When giving Zn 2§ ions, a minimum of trypsin activity is found at 10-5 mol Zn2§ and a maximum at 5 • l 0 -6 mol Zn2§ On the whole, the trypsin activity is lower when the Cu2§ 2§ combinations are applied compared to the addition of the single trace elements9 On principle, a good conformity of the in vivo results was found with in vitro results. Index Entries: Activity of trypsin in the small intestine; rat trypsin activity, and copper and zinc; L-BAPA, and Cu and Zn effects on trypsin activity; addition of copper, zinc, and combinations to rat intestine; comparison of in vivo and in vitro results of Cu and Zn on trypsin activity9
Introduction A striking influence of the trace elements Cu, Zn, Fe, Ni, and Co on the activity of trypsin using N"-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate was found when in vitro experiments were conducted in order 9 1980 by The H u m a n a Press Inc. All rights o f any nature whatsoever reserved. 0163 4 9 8 4 / 8 0 / 1200 4)247502.00
247
248
WIENINGER-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
to determine enzyme activity. Depending on the trace elements used and on their concentrations, activating and inhibitory effects could be demonstrated (1, 2). Additional in vitro experiments were done in order to explore the interactions of copper with other trace elements. A predominant influence of copper was found (3). It was the aim of this work to research whether the effects found in vitro can also be shown in the animal. The experiments in this trial were restricted to the trace elements copper and zinc. For this purpose, solutions of trace elements were applied per os into the gastrointestinal tract with a stomach tube, and, after killing the rats, the trypsin activity was determined in the juice of the small intestine.
Methods Female SPF Sprague-Dawley rats were used for the experiments. At the beginning of the experiments, the animals were six weeks old and they weighed about 130 g. Their weight at the end of the experiments was about 150 g. Four animals were used for each applied trace element concentration. Drinking water, which was renewed every day (aqua dest.; brought up to normal osmolarity with 0.014% NaCI), was available ad libitum. The animals were fed a standard feed that contained 6.8% fish meal (65% crude protein); 6.3% meat meal (65% crude protein); 2.7% dried yeast; 1.2% bone meal, 48% barley; 18% oat: 12% corn; 4% alfalfa meal; 1% CaHPO4"2H20. During the night, the feed was removed in order to train the animals to consume 5 g feed within 1 h at the morning meal. With this procedure, the presupposition was created for an equal feed intake by each animal. At the same time, the animals got used to the stomach tube with which 0.3 or 0.6 mL of distilled water were applied per os every day, and the respective trace element solutions were given in the same way on the day of the experiment. The applied trace element concentrations are shown in Table 1. The animals were decapitated under ether narocsis exactly 1 h after the beginning of the feeding and 0.5 h after having applied the respective trace element solution. The abdominal cavity was opened and the first 20-cm segment of the small intestine was removed. The contents of this part of the small intestine were put into a 10-mL flask and filled with distilled water. The trypsin activity in the juice of the small intestine was determined with the method of Erlanger et al. (4) modified by Wieninger-Rustemeyer et al. (5) in order to make it applicable for these experiments. Pappas (6) recommended this method for in vivo experiments because the trypsin activity is determined specifically by using L-BAPA as a substrate. The reaction time was 10 min. p-Nitroaniline which is liberated from L-BAPA, was measured spectrophotometrically at 410 nm and the extinction was used as a criterion of the relative trypsin activity.
249
TRYPSIN ACTIVITY IN RATS
TABLE 1 Ion Concentration (tool Me~+/L) and Quantity (mL) of the Applied Trace Element Solutions Me 2+
[Me ~§
Cu 2+ as CuSO4.5H20
Zn 2+ as ZnSO4 ' 7H20
m
5 • 10-6 10-5 5 • 10-5 10-4 5 X 10 -4
_
-0.3 0.3 0.3 0.3
Cu2+(10-4mol/L) + Zn 2§
_
-
0.3 0.3 0.3 0.3 --
-
Each Each Each Each
0.3 0.3 0.3 0.3
--
The + values given in the tables are standard deviations. The significance was calculated with the Newman-Keuls test. Groups that do not share one or more c o m m o n letters are different with the probability given in the respective tables.
Results Trypsin Activation in the Small Intestine Dependent on the Concentration of C u 2+ Ions The results of the experiments with per os application of Cu 2§ ions as well as the standard deviations and the statistical significances are summarized in Table 2. The trypsin activity in the juice of the small intestine decreases first somewhat when 10-5 tool Cu2+/L are applied, and then it increases strongly with increasing copper additions. The highest activity is reached with 10-4 tool Cu2§ Application of even more Cu 2§ ions results again in a decrease of the trypsin activity. When comparing the trypsin activities in the small TABLE 2 Trypsin Activity in the Juice of Rat Small Intestine After per os Application of Different Amounts of Cu 2§ ions (P < 5%) 0 0.48
10-5 _
0 . 1 3 a'b
0.37 4- 0.14 a
5 X 10-5 0.56
- - 0 . 1 1 b'c
10-4
5 • 10-4 mol Cu2+/L
0.68 4- 0.20 c
0.56 4- 0.13 b'c
25O
WIENINGER-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
O.d.
0.7
0.6
0.5
\ \
\ \
0.4
0.3
0.2
0.1
I/
i
5.10-5
[
10-4
I
5.10-4
9.10 -8 4.5.10-7 9.10-7
i
10-3
mol Cu++/I
i
510-3 invivo
L.510 -69.10 -6
/..510 -59,10 -5 in vitro
FIG. 1. Influence of Cu2+ ions on the tryptic hydrolysis of L-BAPA in vitro (x--x) and in vivo (o--o). intestine with results from in vitro experiments (Fig. 1), one can recognize a good conformity. The initial activities of the trypsin are similar. The increase, and after reaching a maximum, the decrease are as pronounced in vitro as in vivo. But the Cu ~-§ concentrations needed in vivo are about 5 X 10z times higher than those needed in vitro in order to get similar results.
Trypsin Activities in the Small Intestine Dependent on the Concentrations of Zn 2§ Ions The results of the experiments with per os application of Zn 2§ ions are summarized in Table 3. Although the mean trypsin activity is essentially higher at the Zn 2§ concentration of 5 • 10 -6 mol Zn2*/L compared to the control experiment, this difference cannot be secured by statistical methods because of the large variations of the individual values. A considerable decrease of the trypsin activity can be seen when 10-5 mol Zn2+/L are
251
TRYPSIN ACTIVITY IN RATS
TABLE 3 Trypsin Activity in the Juice of Rat Small Intestine After per os Application of Different Amounts of Zn 2§ Ions (P < 5%) 0
5 X 10-6
0.48 + 0.13 a
10-5
5 X 10-5
10-4molZn2§
0.60 -t- 0.13 a 0.27 + 0.05 b 0.52 + 0.12 a
0.49 9
0.08
a
applied. When the Zn 2+ concentration is increased further, the activities of the control experiment are reached again. In Fig. 2, the in vivo results are compared to in vitro results. It can be seen that a certain parallelism exists between both trials. The Zn 2§ concentrations used in the in vivo experiments are, however, about 10 times higher than those in the in vitro experiments in order to get comparable effects. It is, however, a fact that the variations of the trypsin activity are higher in the experiments with the juice of the small intestine than in the in vitro trials. o.d.i
0.6 7 f~
f/O,~
--% O.L
0.3
\
\ \
0.2
\ \ \ \
0.1 mot Zn§
I 10 -6
~/
I 9.10-8
I 5.10 -6
I
I 10-5
I
L.5.10-7 9.10-7
I 5.10 -5
t
I 10-L
1-
/,.5.10-6 9.10-6
I 5.10 -3 in vlvo
I
~__
L.5.10 -5 in vitro
FIG. 2. Influence of Zn 2§ions on the tryptic hydrolysi~ Of L-BAPA without Cu 2§ addition in vivo (o--o) and in vitro (x--x) as well as with addition of Cu 2§ in vivo ( ~ - e ) and in vitro (u--tu).
252
WIEN1NGER-RUSTEMEYER,
KIRCHGESSNER,
AND STEINHART
Influence of the Interactions Between Zn 2§ and Cu2+ Ions on the Trypsin Activity In order to investigate the influence of the interactions between Zn 2§ and Cu 2§ application on the trypsin activity, a Cu 2§ solution in equal quantity and concentration was applied per os. When 0.3 mL of a 10-4 molar Cu 2§ solution were given alone, the increase in the trypsin activity was highest. 9 9 ' Together with Cu 2+ , Zn 2+ solutions were added m the same concentratmns as in the experiments with isolated Zn 2§ additions. The results of these experiments are summarized in Table 4. The trypsin activity decreases markedly when Cu 2§ and Zn 2§ ions are added together compared to the control experiment. The reason may be that the concentration of the trace elements is twice as high as in the experiments in which each trace element was applied alone. This decrease is, however, not linear. The trypsin activity reaches a minimum at 10-5 mol Zn2§ then increases to a maximum at 5 • 10~ mol Zn2§ and then decreases again. The trypsin activity curves of the experiments with the combined addition of Cu 2§ and Zn 2§ ions of the in vivo and of the corresponding in vitro trials are shown in Fig. 2. It can be seen from this figure that the curves tend to be similar. The effects, however, are more distinct in the in vivo than in the in vitro experiments.
Discussion It is known from in vitro experiments that trace elements influence the activity of pepsin (7, 8) and trypsin (1, 2). The change of their activity depends on the element and its concentration; it may be either positive or negative. There are also interactions between trace elements in regard to their effect on the activity of pepsin (9) and trypsin (3) in vitro. Therefore, it was reasonable to conduct experiments in order to find out whether these effects are also present in vivo. But in vivo, the parameters that may influence the activity of proteolytic enzymes (for instance, enzyme-substrate ratio, pH value, or trace element concentration) are not as constant as in in vitro experiments. The in vivo experiments were conducted with Zn and Cu TABLE 4 Trypsin Activity in the Juice of Rat Small Intestine after per os Application of 10-4mol CuZ+/L together with Increasing Amounts of Zn 2+ Ions (P < 5%) 0
0.48 + 0.13 a
5 X 10-6
10-s
5 X 10-5
0.26 + 0.05 b 0.13 + 0.04 c 0.31 + 0.13 b
10-4toolZn:+/L + 10-4tool Cu:+/L 0.23 + 0.03 b
TRYPSIN ACTIVITY IN RATS
253
because these elements influenced the strongest trypsin activity in vitro. These trace elements were given per os by stomach tube in order to avoid additional influences that might be encountered if applied with the feed. It was not possible to apply the same quantity of liquid in the in vivo as in the in vitro experiments (1 mL) because rats are not able to consume such a quantity all at once. Therefore the quantity of the applied solution was reduced to 0.3 mL. The in vivo experiments were conducted with higher concentrations of trace elements than the in vitro experiments because Zn 2+ and Cu :+ ions are bound in the digestive tract by different ligands. The method of Pappas (6) was used in order to determine the trypsin activity in the juice of the small intestine. Taking L-BAPA as trypsin substrate seemed to be best, because L-BAPA is a relatively specific substrate of trypsin. L-BAPA is only split by trypsin, papain, and thrombin (10, 11). Because the papain does not occur in animal material, and thrombin only in the blood, it may be assumed that only the tryptic activity was determined in the juice of the small intestine of the rats. Therefore it was possible to use the nonpurified juice as the enzyme source. Pappas (6) found it to be important to use only the juice of the first or second part of the small intestine to determine trypsin activity because the activity is already strongly reduced in the juice of the third part. This result is in agreement with observations of Bird (12), who found the highest trypsin activity in the last quarter of the duodenum, and in the first part of the jejunum. The results of these experiments show that the trypsin activity in the juice of the small intestine is influenced by Cu 2+ions as well as by Z n 2+ ions. The effect is dependent on the concentration of the applied trace elements. When the Cu 2+ ions are applied per os, there is a similar change in the trypsin activity as can be observed in the in vitro experiments. The concentration of Cu 2+ ions is, however, about 5 • 102 mol higher in the in vivo experiments than in the in vitro experiments. One reason for these differences between in vivo and in vitro experiments is probably the fact that Cu 2+ions are partly complexed in the heterogeneous environment of the gastrointestinal tract, and therefore, it is obvious that not the whole amount of the applied Cu 2+ ions may interact with the trypsin. Another reason is that Cu ~+ ions are partly absorbed during the time between application and killing of the rats. There is also a good agreement between the in vivo and the in vitro results when Zn is considered. To get similar effects, the Zn :+ concentration was in vivo 10 times higher than in the in vitro experiments. The differences to the even higher Cu 2+ concentrations in vivo compared to in vitro experiments may be explained by the fact that Cu 2+ ions are complexed stronger in the gastrointestinal tract than Zn 2+ ions do. Metabolic effects of the applied trace elements on trypsin synthesis in the pancreas may be excluded because the period of time of 30 min between application of the trace elements and killlng of the animals is too short. When a constant Cu 2+quantity (10-4 tool Cu 2 /L) is applied together with increasing amounts of Zn 2+ ions, trypsin
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WlENINGEI~-RUSTEMEYER, KIRCHGESSNER, AND STEINHART
activity is changed in the same way in the in vitro as well as in the in vivo experiments. At any rate, the activity is decreased to about the a m o u n t as that which is caused by the Cu 2§ ions. With these experiments it was possible to demonstrate that effects found in an isolated system are transferable, up to a certain degree, to living systems.
References 1. M. Kirchgessner, R. Wieninger-Rustemeyer, and H. Steinhart, Z. Ticrphysiol., Tierern~hrg. Futtermittelkde 43, 130 (1980). 2. R, Wieninger-Rustemeyer, M. Kirchgessner, and H. Steinhart, Nutr. Metab, in press. 3. M. Kirchgessner, H. Steinhart, and R. Wieninger-Rustemeyer, Internar Z. Vir Ern, Forsch., in press. 4. B. F. Erlanger, F. Edel, and A. G. Cooper, Arch. Biochem. Biophys. 115, 206 (1966). 5. R. Wieninger-Rustemeyer, H, Steinhart, and M. Kirchgessner, Landwtrtsch. Forsch., in press. 6. P. W. Pappas, J. Parasitol. 64, 562 (1978). 7. M. Kirchgessner, M. G. Beyer, and H. Steinhart, Brit. J. Nutr. 36, 15 (1976). 8. M.G. Beyer, M. Kirchgessner, and H. Steinhart, Landwirtsch, Forsch. 29, 53 (1976). 9. H. Steinhart, M. Kirchgessner, and M. G. Beyer, Arch. Tierernhhrung26, 629 (1976). 10. W. Nagel, F. Willig, W. Peschke, and F. H. Schmidt, Hoppe-Seylers Z. Physiol. Chem. 340, 1 (1965). 11. H. Schiessler, H. Fritz, M. Arnold, E. Fink, and H, Tschesche, Hoppe-Seylers Z. Physiol. Chem. 353, 1638 (1972). 12. F. H. Bird, Brit. Poultry Sci. 12, 373 (1971).
