mdlecine, Universite de Montreal, Montreal, Quebec, Canada H3C 1C5

Transcription

1 J. Phy8iol. (1982), 322, pp With 1 text-figure Printed in Great Britain EXOCRINE PANCREATIC FUNCTION FOLLOWING PROXIMAL SMALL BOWEL RESECTION IN RATS BY MONIQUE D. GELINAS, CLAUDE L. MORIN AND JEAN MORISSET From the Centre de recherche pediatrique and the Detpartement de pediatrie, Service de Gastroenterologie, H6pital Sainte-Justine and Departement de nutrition, Faculte de mdlecine, Universite de Montreal, Montreal, Quebec, Canada H3C 1C5 (Received 8 May 198) SUMMARY 1. In order to assess if proximal enterectomy induces changes in the function of the exocrine pancreas, the exocrine pancreas was studied 1 week, 4 weeks, and 6 months after 5 or 75 % proximal small bowel resection. 2. One week after 5 and 75 % proximal small bowel resections, basal pancreatic bicarbonate outputs, studied by means of an external pancreatic fistula in conscious rats, were increased significantly over control values by 43 and 78 % respectively. Four weeks after a 75 % resection, the bicarbonate output was still significantly higher in resected animals than in sham operated animals. 3. The increase of volume and bicarbonate of the basal pancreatic secretion coincided with a 4-fold increase in plasma secretin concentration 1 week after resection. Both increased pancreatic secretion and plasma secretin concentration were transient. 4. The pancreatic hypersecretion was specifically reversed to control values with an i.p. injection of jejunoileal mucosa homogenate. 5. Serum gastrin and somatostatin values in intestinal mucosa and pancreas were not changed 1 and 4 weeks after enterectomy compared with sham operated animals. 6. The weight of the pancreas and its content of DNA were unaltered by resection. Amylase and chymotrypsinogen per gram pancreatic tissue and per Big DNA were reduced 4 weeks following resections as compared with sham operated rats. After 6 months, chymotrypsinogen appeared further reduced in resected animals. 7. It is concluded that extensive proximal enterectomy in rats produced early, transient and marked increases in basal pancreatic water and bicarbonate secretion and in plasma secretin due to the loss of jejunoileal inhibitorss, and a selective decrease in certain enzymes in pancreatic tissue. INTRODUCTION Proximal intestinal resection induced rapid morphological and functional changes in the gastrointestinal tract (Dowling& Booth, 1967; Nygaard, 1967; Hanson, Osborne & Sharp, 1977; Obertop, Nundy, Malamud & Malt, 1977). Gastric hypersecretion and hyperplasia are well known examples of rapid and transient changes occurring in man, rat and dog following proximal small bowel resection (Osborne, Frederick, Sizer,

2 72 M. D. GELINAS, C. L. MORIN AND J. MORISSET Blair, Cole & Thum, 1966; Caridis, Roberts & Smith, 1969; Seelig, Winborn & Weser, 1977). Gastric hypersecretion could lead to serious clinical problems and could contribute to ulcer formation and diarrhoea sometimes associated with subtotal enterectomy (Osborne et al. 1966; Fielding & Cooke, 197; Buxton, 1974; Weser, 1976). Very few studies have dealt with the influence of small bowel resection on the pancreas. Yet, most of the numerous gut hormones, primarily secretin and cholecystokinin-pancreozymin (CCK-PZ) exert some effects on the exocrine pancreas (Singh & Webster, 1978). It is therefore very likely that enterectomy affects the exocrine pancreas. Baxter (1931) observed a decrease in basal pancreatic secretion of rabbits on the day of 'total' enterectomy. Grossman (1958) noted a marked reduction in basal volume and protein concentration of pancreatic juice in rats two days after 'total' gut resection. Jackson & Linder (1953) reported reduced exocrine pancreatic function correlated with severe atrophy of the pancreas in a young man subjected to extensive small bowel resection 1 year before. However, Lundh & Oldby (1963) noted a normal level of trypsin in the intestinal content of one patient with massive enterectomy and gastric resection. Niessen, Brfigmann, Osswald, Schmidt & Flach (1974) described an increase in volume and bicarbonate of the stimulated pancreatic secretion in two children after subtotal small bowel resection. Similarly, Debas & Yamagishi (1977) reported, in an abstract, that excision of the jejunum and ileum increased the responsiveness of the exocrine pancreas of anaesthetized dogs to exogenous secretin and CCK-PZ, while basal pancreatic secretion was unaltered. However, the same group of researchers (Seal, Debas, Reynolds & Said, 1979) also reported, in another abstract, pancreatic hyposecretion to stimulation by liver extract meal in anaesthetized dogs following small bowel resection. Thus results in the literature span the whole spectrum from pancreatic hyposecretion to hypersecretion. Yet, none of the studies on the pancreas were performed at different time intervals after resection; none were done in rats after proximal enterectomy. The purpose of the present study was to evaluate if proximal small bowel resection done in rats is associated in time with changes of the exocrine pancreas. Changes in pancreatic secretion after intestinal resection are of particular interest because bicarbonate may have a protective role when gastric hypersecretion occurs, and because pancreaticobiliary secretions may be involved in the growth of intestinal mucosa in normal rats (Altmann, 1971) and in enterectomized rats (Weser, Heller & Tawil, 1977; Williamson, Bauer, Ross & Malt, 1978; Hughes, Bates & Dowling, 1978; Gelinas & Morin, 198). The present study suggests that proximal intestinal resection induces transient and marked basal hypersecretion of both plasma secretin and pancreatic bicarbonate outputs due to the loss of jejunoileal inhibitorss. Proximal resection also produces a selective decrease in digestive enzymes of the pancreatic tissue. METHODS Male Sprague-Dawley rats with an initial body weight of 15+-7g (mean+s.e.) were used throughout these experiments. Surgery was performed under pentobarbitone anaesthesia (4-8 mg/1 g body wt i.p.).

3 EXOCRINE PANCREAS AFTER ENTERECTOMY Intestinal surgery At the beginning of each experiment, the rats were subjected either to a sham resection (control groups) or to a proximal intestinal resection (experimental groups). In control rats, division of the jejunum with end-to-end resuture, was carried out at 6 cm distal to the duodenal jejunal junction. In experimental groups, the jejunum and the ileum were measured; removal of either 5 or 75 % of the measured jejunoileum started at 6cm distal to the duodenal jejunal junction with continuity re-established by an end-to-end anastomosis, as described by Lambert (1965). After intestinal surgery, the rats were individually housed and had free access to water and rat chow (Ralston Purina of Canada Ltd.) till the day of pancreatic fistulization or pancreas excision. Pancreatic fistulization In order to collect pure pancreatic juice from conscious rats, short-term external pancreatic fistulae, adapted from Grossman (1958), were installed 1 week, 4 weeks, or 6 months after intestinal surgery. Different groups of rats were used for each period as animals were killed after juice collection, and blood was collected for serum bilirubin analysis. Total serum bilirubin values were found to be within the normal range ( mg/i ml.), with a mean of 57 mg/1 ml. in control and resected animals. Through a mid line abdominal incision, a polyethylene catheter (PE 1 Intramedic, inner diameter -28 mm) was introduced into the uppermost part of the bile duct above a ligature and allowed to drain into the first part of the duodenum to maintain bile flow. A similar tube (25cm long) was pushed for 4mm into the pancreaticobiliary duct immediately above a ligature done at the ductus entry into the duodenum. This catheter was exteriorized through the base of the abdominal incision to collect the pancreatic juice. A silicone tube was introduced into the forestomach to hydrate the rats. The rats were then placed in a Bollman-type cage. A solution of 1% dextrose in -9 % NaCl plus 1 mm-kcl was pumped through the gastrostomy at 1-5 ml./hr till collection time. Measurements of pancreatic secretion Pancreatic juice was collected 2 hr after the fistula was constructed. Basal secretions were measured in microlitres by taking 1 hr collections into a 1 ml. graduated pipette placed 2 in. under the rat and inclined 5 from the horizontal. With a microgasometer (Natelson, 1951), basal bicarbonate concentrations were analysed immediately from the first hour sample collected under light mineral oil. A second lhr basal sample was frozen for protein determination (Lowry, Rosebrough, Farr & Randall, 1951), a convenient measure of enzyme output (De Waele, De Smul, Wissock & Kiekens, 1974; Schneeman & Lyman, 1977). The mean of three 1 hr periods was taken as the basal volume of secretion. Pancreatic secretion was stimulated exqgenously in two subgroups of rats. Forty Crick- Harper-Raper units (CHRU) of secretin and CCK-PZ (Boots Co.) per kg body weight were given within 1 min in two separate i.p. injections. i.p. injections of CCK-PZ have been demonstrated to elicit a response that is more predictable and of a greater magnitude than other routes of administration (Leroy, Morisset & Webster, 1971). Juice was collected for 1 hr immediately following the injections. In another set of experiments, an i.p. injection of jejunoileal mucosa homogenate was given to sham operated and 75 % resected rats. For each injection, mucosa was scraped off 45 cm of the intestine of a normal fasted rat starting 1 cm distal to the duodenal jejunal junction. The mucosa was weighed and homogenized in cold normal saline as described by Ruderman & Nagy (1974); 2-5 ml. of the homogenate (-8 g mucosa) was used for each injection. Pancreatic juice was collected for 2 hr thereafter. The mean of two 1 hr collections was taken to be the basal volume after homogenate injection. A separate control study was done on seven 75 % resected rats with extract of abdominal muscles diluted and injected in the same way as mucosal homogenate. Pre- and post-injection levels of pancreatic flow rate, bicarbonate concentration and output were measured. Pancreas excision Rats with 75% proximal resection and the sham operated rats were killed by stunning and decapitation 4 weeks and 6 months following intestinal surgery. The entire pancreas was excised, trimmed of fat and extraneous tissue, weighed, and homogenized in 4 M-urea as described by Brants & Morisset (1976). Samples of the homogenate (8,ul.) were placed in a SW-5-1 rotor and centrifuged at 43, rev/min for 6 min. Supernatants were immediately collected and diluted in 73

4 74 M. D. GELINAS, C. L. MORIN AND J. MORISSET appropriate buffers for enzyme assays. DNA was measured by the method of Volkin & Cohn (1954) after extraction into 5 N-perchloric acid for 15min at 9TC. Calf thymus DNA was used as standard. Amylase was assayed according to Bernfeld (1955). Lipase activity was measured according to Charbonneau & Morisset (1975) using a ph stat titrimeter (Radiometer). The substrate was prepared as follows: one volume of emulsion (1 volume of 5 % olive oil and 1 volume of 1 % acacia) was mixed with two volumes of NaCl (5 M) and CaCl2 (1 mm); the final ph was set at 9-. Chymotrypsin was determined according to Hummel (1959) and activated by purified bovine trypsin (1 sg/ml.) for 9 min at 4C. All enzyme activities ere expressed in units (1umole product produced per mintue). Protein was determined according to Lowry et al. (1951) with bovine serum albumin as standard. On additional rats, fasted for 4-6 hr, the entire pancreas and total intestinal mucosa were weighed and assayed for somatostatin by the method of Epelbaum, Brazeau, Tsang, Brawer & Martin (1977), 1 and 4 weeks after 5 % small bowel resections, and compared with normal and sham operated rats. Blood was kept for gastrin (Squibb Immunotope New Brunswick, NJ) and secretin determinations by radio-immunoassay. Plasma secretin was assayed according to Tai & Chey (1978). The minimal detectable level was 18pg secretin/ml. plasma, and this was the amount assigned to samples with non-detectable secretin readings. Statistical analysis Results are expressed as means+ standard errors (s.e.). Unless otherwise mentioned, statistical comparisons were made using unpaired Student's t test. When the sample size was relatively small (n < 1), in which case departure from normality easily occurs, a distribution-free method (Mann- Whitney U test) was used in addition to the t test (Colton, 1974). As the t test and the U test gave the same result, only the t test is quoted. RESULTS Fifty per cent proximal gut resection Effects on pancreatic secretion Table 1 shows that compared with sham operated rats, a 5 % proximal resection induced after one week, a slight decrease of body weight, a 26 % increase in basal bicarbonate concentration of the pancreatic juice (P < -1), and a 43% increase in the basal bicarbonate output (P < -1). Differences in the concentration and output of protein of the basal pancreatic secretion were not significant. Secretin and CCK-PZ injections resulted in significant increases over basal values (P <.1, paired t test) in bicarbonate and protein secretion in both sham operated and resected rats. Although basal values differed between sham operated and resected rats, the results obtained following secretin and CCK-PZ injections were similar in both groups of animals. Four weeks after resection, body weights, basal pancreatic flow rate, bicarbonate, and protein concentration and output of sham operated and resected animals were not different. Effects on secretin, gastrin and somatostatin Table 2 shows that 1 week following a 5% proximal gut resection, the mean basal plasma secretin concentration (pg/ml.) was four times higher in resected rats ( ) than in sham operated rats ( ) (P < -1), and much higher than in normal rats (P < 1). However, after 4 weeks, there was no significant difference in basal plasma secretin concentration between resected an sham operated animals. No significant differences were observed between sham operated, resected and normal animals in basal serum gastrin concentration, and in somatostatin contents of both small intestinal mucosa and pancreas 1 or 4 weeks after intestinal surgery.

5 EXOCRINE PANCREAS AFTER ENTERECTOMY 75. C) Qia do a COP -_. -.Q ri;. 2._ q ;._ C; r. c3 s. r_ CL; o. V E o o - * 6 ;. - o 11 M - do o6 t le _t ' C1. 7 em o di.o.. = 1- o> = 4 64 N I t-1 1 if om o~n N r ~ Q Q bc * m en m _ N _ mc -+1+l ow t- I- +- N _ rn N = N g --q V * - o C) s * ++ - q$. (1) (1) e r c 3 g c d - e,, -o ed -..O, = x c: = m l (= = m l w * +-+-+e 4+~ *,- - 4Q (-

6 76 M. D. GELINAS, C. L. MORIN AND J. MORISSET Effects on pancreatic secretion The results of a similar study done 1 week, 4 weeks and 6 months after removal of 75 % of the proximal intestine are compared with those of sham operated rats in Table 3. Body weights were similar in resected and sham operated animals 4 weeks and 6 months following surgery. In the resected groups, 1 week and 4 weeks after surgery, pancreatic bicarbonate outputs were increased significantly by 78 % TABLE 2. Secretin, gastrin and somatostatin levels 1 and 4 weeks after a 5 % proximal intestinal resection Sham Resected Normals (6)t (5) (6) Basal plasma secretin (pg/ml.) 1 week T * 4 weeks t 1-8+ Basal serum gastrin (pg/ml.) 1 week weeks Somatostatin (ng) in total pancreas 1 week weeks Somatostatin (ng) in total mucosa 1 week ±125 4 weeks * P < 1 between sham operated and resected animals 1 week after surgery. t Figures in parentheses indicate the number of animals at each period. T Three values below the detection limit of 1-8 pg/ml. Five values below the detection limit of 1-8 pg/ml. (P < 1) and 5 % (P < 5), respectively. While these differences were due to both an increase in flow rate and bicarbonate concentration in the experimental groups, at 1 week only the bicarbonate concentration was significantly higher (47 %, P < 1), whereas at 4 weeks only the flow rate was statistically increased (27 %, P < -1). Differences in the concentration and output of proteins in the basal secretion at all periods of study were not significant and are not shown in Table 3. At 6 months following surgery the mean values for basal bicarbonate concentration and bicarbonate output were higher by 28 and 44 % respectively in the resected rats; however, those differences were not statistically significant due to a high dispersion of results as reflected by a large standard error. Stimulation with exogenous secretin and CCK-PZ resulted in increases over basal values (P < '1, paired t test) for the studied variables in both sham operated and resected animals: the only exception being in the protein concentration and output of the resected group, where stimulation failed to provide significant increases (results not shown). Fig. 1 demonstrates the significant inhibiting effect of an i.p. injection of a jejunoileal mucosa homogenate on the volume (-3 %, P < 5), bicarbonate concentration (-33 %, P < 1) and bicarbonate output (-54 %, P < 5) in seven 75 % resected rats compared to their own pre-injection values (paired t test). Pre- and

7 EXOCRINE PANCREAS AFTER ENTERECTOMY 77 -*t,i - = (M _r _ > 1-6. & cq cq C1)._._o.- * ) -o o. E. Cv o r- = (M * --4d = cq. co,: Cm m Im = Cb U6 ce C= r N N oz _ = C-oz =: N me m o4 m C14 v 4- *) C)._- a) ^ D:b >, _ m em N en m Cm m em c km v * e Q TD. 4) bl * ca Ca._.=4 z $.4 C. -C (1) (D j U C.) (1) m i 4) F..4 E --ll Ca E - 't - --,= ,. X t- M t- ITZ (1) 4'. C.) (1) m (1) E ---O ce.= tn. m t- llc. Q (1) m (1) $ E ce..-cl X Lm m t- C; C) a P4 X e cue.4., ce Q

8 78 M. D. GELINAS, C. L. MORIN AND J. MORISSET (7) (7) 4 2 *4 (6) 3- ~~~~15 E i2 Z lo1 BHi BHi BHm BHi BHi BHm BHi BHi BHm Volume HCO3 concentration HCO3 output Fig. 1. Effect of an i.p. injection of a jejunoileal mucosa (Hi) or abdominal muscle (Hm) homogenate on basal (B) pancreatic flow rate (volume), HC3- concentration and HCO3- output, 1 week after a 75 % proximal intestinal resection. Results are expressed as means (±s.e.) in sham operated (open bars) and resected rats (hatched bars). Figures in parentheses indicate the number of animals. TABLE 4. Pancreatic tissue 4 weeks after a 75% proximal intestinal resection (experimental group) per 1/sg DNA % change % change from from Mean + S.E. controlt Mean + S.E. controlt Final body weight (g) % -- Pancreas wet weight (mg) T12% Tl9% per g pancreas DNA (mg) % Protein (mg) %** III% Amylase (,smole/min) %* %** Lipase (,umole/min) %** 398& % Chymotrypsinogen (,umole/min) %** %** ** P<.1, * P<*5. t Indicate percentage change in six experimental animals compared with values from six rats taken as 1%. Not significant. post-mucosa injection values were similar in six sham operated animals. The effect of the i.p. injections of the mucosal homogenate was such that there were no longer any differences in pancreatic secretion between sham operated and resected rats. No significant changes were observed in seven other 75 % resected animals following the i.p. injection of a similarly prepared homogenate of abdominal muscles. Biochemical data on pancreatic tissue Pancreatic tissue was obtained 4 weeks (Table 4) and 6 months after 75 % resections; only the results after 4 weeks are shown here. At both periods, body weights, pancreas wet weights and DNA contents of sham operated and resected

9 EXOCRINE PANCREAS AFTER ENTERECTOMY 79 animals did not differ significantly. Decreases in tissue protein and enzyme levels per gram of pancreatic tissue were found in the resected animals in comparison with sham operated animals 4 weeks following resection: protein by 29 % (P < 5), amylase by 34% (P < 1), lipase by 21 % (P < -5) and chymotrypsinogen by 35 % (P < 2). Amylase (P < -1) and chymotrypsinogen (P < 5) contents expressed per 1#tg DNA were also decreased. Six months following resection, only the chymotrypsinogen content was reduced (41 %, P < -5) in the resected group. DISCUSSION This study showed that in rats, 5 and 75 % proximal intestinal resections induced an increase in basal pancreatic bicarbonate output after 1 week, due primarily to higher bicarbonate concentration. This hypersecretion of bicarbonate was accompanied by a 4-fold increase in basal plasma secretin concentration 1 week after intestinal resection. Increases in pancreatic bicarbonate secretion and in plasma secretin were transient, since one month after 5 % resection no differences were seen between sham operated and resected animals. Furthermore, the magnitude and duration of the increase in bicarbonate secretion were related to the extent of the resection being more pronounced and lasting longer in animals with larger resections. Four weeks after a 75 % resection, the bicarbonate output was still significantly higher in resected animals. This study also showed a decrease in certain digestive enzymes of the pancreatic tissue in 75 % resected rats, namely amylase, lipase, and chymotrypsinogen 4 weeks after resection. The tissue chymotrypsinogen level was still much lower 6 months after resection; this finding may explain the resected animals' lack of responsiveness in pancreatic protein secretion after hormonal stimulation. To our knowledge the present findings in partially enterectomized rats have not yet been reported. In comparison with studies measuring basal pancreatic juice in normal conscious rats, values for sham operated rats in the present study were similar in terms of flow rate, bicarbonate and protein concentrations (De Waele et al. 1974; Cavarzan, Teixeira, Sarles, Palasciano, Tiscornia, 1975; Chariot, Roze, LaTour, Souchard, Vaille & Debray, 1976). Some findings of the present study differ from work done by Baxter (1931) in rabbits and Grossman (1958) in rats who showed a marked reduction of the basal pancreatic secretions after gut resection. However, those experiments were performed, either the day of the enterectomy or 2 days later while our experiments were done 1, 4 weeks and 6 months after resection. Furthermore, in these two studies, the entire small bowel was removed, thus eliminating most of the secretin mechanism (Ramirez, Hubel & Clifton, 1966) and CCK-PZ activity (Schneeman & Lyman, 1977). In our study, the duodenum and the first 6 cm of the jejunum were intact. This, as well as the time interval after enterectomy, very likely accounts for the differences in results. In addition to the time interval after enterectomy, the differences observed by Niessen et al. (1974), Debas & Yamagishi (1977) and Seal et al. (1979) could be due to differences in species and state of consciousness, these two factors being known to influence largely basal pancreatic secretions (Chariot et al. 1976). The complex interaction between secretion of the gut, stomach and pancreas are very likely to be disturbed by removal of a part of the small bowel. Alteration of the

10 8 M. D. GELINAS, C. L. MORIN AND J. MORISSET gut nervous system may be responsible for the observed increase in volume and bicarbonate of the basal pancreatic secretion (Fleischer & Kasper, 1974); however, our experiment with mucosal homogenate injection argues against this explanation. Furthermore, hyperplasia or hypertrophy of the pancreas could not account for the results since no significant increases in pancreas weights, cell number (DNA/g pancreas), or total protein per cell (protein/,sg DNA) in the pancreatic tissue were found at different times after enterectomy. In addition, gastrin does not mediate the observed changes since no differences in serum gastrin were observed after resection; this has been previously reported (Oscarson, Veen, Williamson, Ross & Malt, 1977). It is tempting to speculate that following intestinal resection, the increase in volume and bicarbonate of the basal pancreatic secretion is induced by increased secretin concentration as observed, which is due to the loss of a jejunoileal inhibitors) and/or to gastric hypersecretion that could itself be secondary to the loss of jejunoileal inhibitors) (Osborne etal. 1966; Ruderman& Nagy, 1974). The inhibitory phenomenon observed in resected animals after mucosa homogenate injection, but not after muscle injection, shows that the results are due to some specific factor(s) present in the jejunoileal mucosa rather than to a non specific factor present in all tissues. Furthermore, the inhibitory phenomenon appears to be saturated in sham operated animals and suggests the presence of a limited number of receptor sites for a hormonal inhibitor controlling basal pancreatic secretion. Since somatostatin is present in the gastrointestinal mucosa and pancreas (Rufener, Dubois, Malaisse-Lagae & Orci, 1975), and has been found to inhibit basal pancreatic exocrine secretion in conscious rats (Chariot et al. 1978), we analysed the somatostatin content of the whole small bowel mucosa and pancreas at different times after resection. However, no difference was found between transacted and resected animals in either tissue, showing that the early adaptive growth that occurs in the gut mucosa after resection (Hanson et al. 1977; Obertop et al. 1977), restores rapidly the level of somatostatin in the gut and that a deficit in somatostatin is unlikely to explain the observed results. Although we do not know which mucosal factor is involved in the increased levels of secretin and basal pancreatic secretion following resection, the relationship between the magnitude of resection and the response in pancreatic secretion supports the existence ofmucosal inhibitorss. Furthermore, the adaptive intestinal hyperplasia and hypertrophy following proximal resection in rats may account for the transience of the pancreatic hypersecretion and hypersecretinemia, the inhibitors) being gradually synthesized within the proliferative response. Our observations are consistent with Debas & Yamagishi's hypothesis (1977), that resection of the jejunoileum removes an entero-pancreotone. Pancreotone, the polypeptide material extracted from ileal and colonic mucosa of pigs and cats (Harper, Hood, Mushens & Smy, 1979) may be the inhibitor of pancreatic secretion lost with resection, since part of the ileum was removed in our experiments. The observed hypersecretion in secretin, volume and bicarbonate of the basal pancreatic secretion may serve as useful adaptive mechanisms to counterbalance the gastric hypersecretion associated with extensive resection, and possibly to influence the adaptation of the remaining gut. This study was supported by the Medical Research Council of Canada (MA-332) and by the Fondation Justine Lacoste-Beaubien. The authors wish to express their gratitude to Dr W. Y. Chey for plasma secretin determinations,

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