55
Clinica Chimica Acta, 79 (1977) 55-61 @ ElsevierlNorth-Holland Biomedical Press
CCA 8659
DETERMINATION OF ENTEROPEPTIDASE DUODENAL ASPIRATES
R.J. BARNS
ACTIVITY
IN HUMAN
* and R.G. ELMSLIE
Department of Surgery, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011 (Australia) (Received
February
llth,
1977)
Summary A sensitive procedure is described for the determination in duodenal aspirates of enteropeptidase activity based on the activation of trypsinogen and the estimation of trypsin formed with benzoyl-arginine-p-nitroanilide. Using the recovery approach where a known amount of purified human enteropeptidase is diluted in duodenal fluid and the recoverable activity determined, this method was shown to give a sensitive and reliable estimate of the enteropeptidase activity in duodenal fluid although it was shown that the enzyme was subject to a 10% activation by components in the duodenal fluid. The reported 5-fold stimulation of enteropeptidase activity by bile salts could not be demonstrated.
Introduction Enteropeptidase (formerly known as enterokinase, EC 3.4.21.9) is the duodenal proteolytic enzyme which liberates trypsin from the inactive precursor, trypsinogen. Trypsin subsequently activates the other proenzymes of the pancreatic secretion. This action of enteropeptidase has now been shown to be the only significant means of in vivo activation of trypsinogen [ 1,2]. Where enteropeptidase activity is absent or limiting, the free tryptic activity in duodenal aspirates is negligible and marked disruptions to protein digestion result. In 1965, a condition referred to as trypsinogen deficiency disease was described by Townes [3] and subsequently further reports of this condition have appeared [4,5]. It was characterised by the absence of tryptic activity in duodenal aspirates which resulted in the secondary failure of activation of the other pancreatic proenzymes. These same observations characterised the * To whom correspondence
should be addressed.
56
enteropeptidase deficiency described a few years later [1,2]. However where enteropeptidase was deficient addition of exogenous enteropeptidase but not trypsin to the duodenal aspirates showed trypsinogen to be present but unable to be activated. Consequently a distinction between enteropeptidase and trypsinogen deficiencies requires the demonstration that enteropeptidase activity and not trypsinogen is absent in the duodenal aspirates since the net result in both cases is the absence of tryptic activity. Current procedures for the determination of enteropeptidase are all based on the two-step technique originally described by Kunitz [6] and involve the initial activation of trypsinogen by enteropeptidase and the subsequent determination of the amount of trypsin liberated in the initial incubation. However most of the published procedures for the determination of enteropeptidase activity can be criticised on one or more grounds. All use a very low trypsinogen concentration [ 7 ] well below the K, for this substrate in an attempt to prevent the autoactivation of the proenzyme with the result that these assays are markedly dependent on the purity and concentration of the trypsinogen. Incubation times are usually excessively long (30-120 min) and some have no added of this metal ion has now been recognised [7Ca*+, although the importance 10]. Also the conditions used to stop the initial incubation have been found insufficient to inactivate all traces of enteropeptidase prior to the trypsin determination with the result that in the trypsin determination these traces of enteropeptidase continue to liberate more trypsin from the trypsinogen carried through from the initial incubation. In this laboratory a procedure for the determination of enteropeptidase activity has been developed which takes into account all of the above factors [ 7,111. In this report the use of this assay procedure for the determination of enteropeptidase activity in human duodenal aspirates is examined. Materials and method N-(Y-Benzoyl-DL-arginine p-nitroanilide and bovine trypsinogen were products of Sigma Chemical Co. All other reagents were of reagent grade where possible .
A trypsinogen solution (25 mg/ml, 5 mM HCl) was dialysed against 1 mM HCl at 4°C and then centrifuged to remove insoluble material. The protein concentration was adjusted to 10 mg/ml (A: r$ml = 1.5 at 280 nm [12]) and the solution stored frozen. Human enteropeptidase was obtained from pooled duodenal aspirates. The enzyme was partially purified by DEAE-cellulose, pH 6.0, and Sephadex G200, pH 6.0, chromatography as previously described for the pig enzyme [ll]. Porcine enteropeptidase was isolated from fresh pig duodenal fluid [ 111. Duodenal fluid was obtained from fasting patients undergoing pancreatic function tests using secretin. The fluid was frozen until required and prior to use insoluble material was removed by centrifuging at 3000 rpm for 5 min. Recovery experiments where known amounts of enteropeptidase were added to both bile-stained and bile-free duodenal fluid and the residual enteropeptidase activity determined were performed. A series of tubes, in ice, were set up containing 0.3 ml assay buffer, 0.1 ml bile-stained or bile-free duodenal
57
fluid and various aliquots of partially purified human enteropeptidase made to 0.1 ml with water. ,This 5-fold dilution of the duodenal fluid corresponds to the standard dilution used in this laboratory in enteropeptidase determinations. A control series of tubes was set up containing water instead of duodenal fluid. The recoverable enterobeptidase activity was then determined on aliquots of these dilutions by conip$ing the control activity with that in duodenal fluid.
Determination of en teropep tidase activity The procedure used was aspreviously described [ 7,111. The activation of trypsinogen was followed using assay mixtures (total volume 0.2 ml) containing (in pmol): 3,3’dimethyl glutarate (sodium salt) (pH 5.8), 10: calcium chloride, 0.6; trypsinogen, 0.5 mg. The reaction was initiated with 50 1.11of enteropeptidase and stopped after 5 min by the addition of 50 ~1 0.55 M HCl. The incubation was at 30°C. The assay mixtures were then incubated at 50°C for 10 min to ensure the complete inactivation of the enteropeptidase [ 111. No trypsin was inactivated during this incubation. The trypsin liberated during this incubation was estimated spectrophotometrically on a 20-1.11aliquot with benzoyl-arginine-p-nitroanilide as substrate as previously described in detail [ 131. One unit of trypsin catalyses the hydrolysis of 1 pmol of benzoyl-arginine-p-nitroanilide per min under the conditions of the assay; one unit of enteropeptidase will liberate one unit of trypsin from trypsinogen per min under the specified conditions. Results Various dilutions of fresh duodenal fluid were made and the time course of trypsin release was determined at each dilution (Fig. 1). Trypsin release was
1.6. 5 .
5
Time (mln) Fig. 1. Time course of trypsin release using different dilutions of duodenal fluid as indicated. Bulk assay solutions (1.6 ml) were incubated at 30°C and the reaction was started by the addition of 0.4 ml of duodenal fluid diluted as indicated with water. Aliquota (0.2 ml) were removed at 0, 2, 4. 6. 9. 12 and 15 min and diluted into 50 ~1 0.55 M HCl. The reaction mixtures were incubated for 10 min at 50°C and the trypsin released was determined on 20-~1 aliquots using benzoyl-arginine-p-nitroanilide.
58
essentially linear with time at least up to 10 min at all dilutions greater than two-fold indicating that despite the high concentration of trypsinogen in this assay autoactivation was not kinetically significant. This is similar to that shown previously with the pig enzyme [ 71 and confirms that the requirement for the low trypsinogen concentrations to avoid autoactivation has no experimental basis. However when the trypsin released at a particular time was corrected for dilution, the activity after a two-fold dilution was lower than expected especially with heavily bile-stained samples presumably because of coprecipitation (and losses) of trypsin with bile salts and other material following acidification. These losses can only partly be reversed by neutralisation of assay mixtures prior to the trypsin determination. A routine five-fold dilution of the duodenal aspirate was subsequently chosen for enteropeptidase determinations with an incubation time of up to 10 min for the more dilute samples. Standard amounts of enteropeptidase were diluted in the presence of Ca” and duodenal fluid and the residual or recoverable activity was determined using the standard assay procedure. Both bile-stained and bile-free duodenal samples were used to determine whether bile influenced the recovery of enteropeptidase activity as has previously been reported [14--161. These results are presented as plots of the enteropeptidase activities determined following the dilution of the enzyme in the duodenal fluid against the values obtained following the same dilution of enteropeptidase in buffer (Fig. 2). In both cases there was a linear relationship between the recovered activity and the control activity over a wide range of enteropeptidase activities and the degree of correlation was highly significant in both cases (Fig. 2). The recovery of enteropeptidase in the bile-free duodenal fluid was 113.2 2 9.8% while in the bile-stained duodenal fluid the recovery was 109.5 t 16.1% (n = 43 in both cases). shows a similar response to pH as The activity of human enteropeptidase
A i;
Fig.
2.
peptidasc
Plots
of
recowrrd
in bilvstained
entrropcptidase (A)
and
bilr-free
actlvitv (B)
duodenal
against fluid.
control
activity
following
dilution
of
entero-
59
does the pig enzyme [ 71. Similarly it is also dependent on ionic strength with the result that in many published assays where the buffer concentration (and consequently the buffering capacity) is kept low (
Clinica Chimica Acta, 79 (1977) 55-61 @ ElsevierlNorth-Holland Biomedical Press
CCA 8659
DETERMINATION OF ENTEROPEPTIDASE DUODENAL ASPIRATES
R.J. BARNS
ACTIVITY
IN HUMAN
* and R.G. ELMSLIE
Department of Surgery, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011 (Australia) (Received
February
llth,
1977)
Summary A sensitive procedure is described for the determination in duodenal aspirates of enteropeptidase activity based on the activation of trypsinogen and the estimation of trypsin formed with benzoyl-arginine-p-nitroanilide. Using the recovery approach where a known amount of purified human enteropeptidase is diluted in duodenal fluid and the recoverable activity determined, this method was shown to give a sensitive and reliable estimate of the enteropeptidase activity in duodenal fluid although it was shown that the enzyme was subject to a 10% activation by components in the duodenal fluid. The reported 5-fold stimulation of enteropeptidase activity by bile salts could not be demonstrated.
Introduction Enteropeptidase (formerly known as enterokinase, EC 3.4.21.9) is the duodenal proteolytic enzyme which liberates trypsin from the inactive precursor, trypsinogen. Trypsin subsequently activates the other proenzymes of the pancreatic secretion. This action of enteropeptidase has now been shown to be the only significant means of in vivo activation of trypsinogen [ 1,2]. Where enteropeptidase activity is absent or limiting, the free tryptic activity in duodenal aspirates is negligible and marked disruptions to protein digestion result. In 1965, a condition referred to as trypsinogen deficiency disease was described by Townes [3] and subsequently further reports of this condition have appeared [4,5]. It was characterised by the absence of tryptic activity in duodenal aspirates which resulted in the secondary failure of activation of the other pancreatic proenzymes. These same observations characterised the * To whom correspondence
should be addressed.
56
enteropeptidase deficiency described a few years later [1,2]. However where enteropeptidase was deficient addition of exogenous enteropeptidase but not trypsin to the duodenal aspirates showed trypsinogen to be present but unable to be activated. Consequently a distinction between enteropeptidase and trypsinogen deficiencies requires the demonstration that enteropeptidase activity and not trypsinogen is absent in the duodenal aspirates since the net result in both cases is the absence of tryptic activity. Current procedures for the determination of enteropeptidase are all based on the two-step technique originally described by Kunitz [6] and involve the initial activation of trypsinogen by enteropeptidase and the subsequent determination of the amount of trypsin liberated in the initial incubation. However most of the published procedures for the determination of enteropeptidase activity can be criticised on one or more grounds. All use a very low trypsinogen concentration [ 7 ] well below the K, for this substrate in an attempt to prevent the autoactivation of the proenzyme with the result that these assays are markedly dependent on the purity and concentration of the trypsinogen. Incubation times are usually excessively long (30-120 min) and some have no added of this metal ion has now been recognised [7Ca*+, although the importance 10]. Also the conditions used to stop the initial incubation have been found insufficient to inactivate all traces of enteropeptidase prior to the trypsin determination with the result that in the trypsin determination these traces of enteropeptidase continue to liberate more trypsin from the trypsinogen carried through from the initial incubation. In this laboratory a procedure for the determination of enteropeptidase activity has been developed which takes into account all of the above factors [ 7,111. In this report the use of this assay procedure for the determination of enteropeptidase activity in human duodenal aspirates is examined. Materials and method N-(Y-Benzoyl-DL-arginine p-nitroanilide and bovine trypsinogen were products of Sigma Chemical Co. All other reagents were of reagent grade where possible .
A trypsinogen solution (25 mg/ml, 5 mM HCl) was dialysed against 1 mM HCl at 4°C and then centrifuged to remove insoluble material. The protein concentration was adjusted to 10 mg/ml (A: r$ml = 1.5 at 280 nm [12]) and the solution stored frozen. Human enteropeptidase was obtained from pooled duodenal aspirates. The enzyme was partially purified by DEAE-cellulose, pH 6.0, and Sephadex G200, pH 6.0, chromatography as previously described for the pig enzyme [ll]. Porcine enteropeptidase was isolated from fresh pig duodenal fluid [ 111. Duodenal fluid was obtained from fasting patients undergoing pancreatic function tests using secretin. The fluid was frozen until required and prior to use insoluble material was removed by centrifuging at 3000 rpm for 5 min. Recovery experiments where known amounts of enteropeptidase were added to both bile-stained and bile-free duodenal fluid and the residual enteropeptidase activity determined were performed. A series of tubes, in ice, were set up containing 0.3 ml assay buffer, 0.1 ml bile-stained or bile-free duodenal
57
fluid and various aliquots of partially purified human enteropeptidase made to 0.1 ml with water. ,This 5-fold dilution of the duodenal fluid corresponds to the standard dilution used in this laboratory in enteropeptidase determinations. A control series of tubes was set up containing water instead of duodenal fluid. The recoverable enterobeptidase activity was then determined on aliquots of these dilutions by conip$ing the control activity with that in duodenal fluid.
Determination of en teropep tidase activity The procedure used was aspreviously described [ 7,111. The activation of trypsinogen was followed using assay mixtures (total volume 0.2 ml) containing (in pmol): 3,3’dimethyl glutarate (sodium salt) (pH 5.8), 10: calcium chloride, 0.6; trypsinogen, 0.5 mg. The reaction was initiated with 50 1.11of enteropeptidase and stopped after 5 min by the addition of 50 ~1 0.55 M HCl. The incubation was at 30°C. The assay mixtures were then incubated at 50°C for 10 min to ensure the complete inactivation of the enteropeptidase [ 111. No trypsin was inactivated during this incubation. The trypsin liberated during this incubation was estimated spectrophotometrically on a 20-1.11aliquot with benzoyl-arginine-p-nitroanilide as substrate as previously described in detail [ 131. One unit of trypsin catalyses the hydrolysis of 1 pmol of benzoyl-arginine-p-nitroanilide per min under the conditions of the assay; one unit of enteropeptidase will liberate one unit of trypsin from trypsinogen per min under the specified conditions. Results Various dilutions of fresh duodenal fluid were made and the time course of trypsin release was determined at each dilution (Fig. 1). Trypsin release was
1.6. 5 .
5
Time (mln) Fig. 1. Time course of trypsin release using different dilutions of duodenal fluid as indicated. Bulk assay solutions (1.6 ml) were incubated at 30°C and the reaction was started by the addition of 0.4 ml of duodenal fluid diluted as indicated with water. Aliquota (0.2 ml) were removed at 0, 2, 4. 6. 9. 12 and 15 min and diluted into 50 ~1 0.55 M HCl. The reaction mixtures were incubated for 10 min at 50°C and the trypsin released was determined on 20-~1 aliquots using benzoyl-arginine-p-nitroanilide.
58
essentially linear with time at least up to 10 min at all dilutions greater than two-fold indicating that despite the high concentration of trypsinogen in this assay autoactivation was not kinetically significant. This is similar to that shown previously with the pig enzyme [ 71 and confirms that the requirement for the low trypsinogen concentrations to avoid autoactivation has no experimental basis. However when the trypsin released at a particular time was corrected for dilution, the activity after a two-fold dilution was lower than expected especially with heavily bile-stained samples presumably because of coprecipitation (and losses) of trypsin with bile salts and other material following acidification. These losses can only partly be reversed by neutralisation of assay mixtures prior to the trypsin determination. A routine five-fold dilution of the duodenal aspirate was subsequently chosen for enteropeptidase determinations with an incubation time of up to 10 min for the more dilute samples. Standard amounts of enteropeptidase were diluted in the presence of Ca” and duodenal fluid and the residual or recoverable activity was determined using the standard assay procedure. Both bile-stained and bile-free duodenal samples were used to determine whether bile influenced the recovery of enteropeptidase activity as has previously been reported [14--161. These results are presented as plots of the enteropeptidase activities determined following the dilution of the enzyme in the duodenal fluid against the values obtained following the same dilution of enteropeptidase in buffer (Fig. 2). In both cases there was a linear relationship between the recovered activity and the control activity over a wide range of enteropeptidase activities and the degree of correlation was highly significant in both cases (Fig. 2). The recovery of enteropeptidase in the bile-free duodenal fluid was 113.2 2 9.8% while in the bile-stained duodenal fluid the recovery was 109.5 t 16.1% (n = 43 in both cases). shows a similar response to pH as The activity of human enteropeptidase
A i;
Fig.
2.
peptidasc
Plots
of
recowrrd
in bilvstained
entrropcptidase (A)
and
bilr-free
actlvitv (B)
duodenal
against fluid.
control
activity
following
dilution
of
entero-
59
does the pig enzyme [ 71. Similarly it is also dependent on ionic strength with the result that in many published assays where the buffer concentration (and consequently the buffering capacity) is kept low (
