Gut, 1989, 30, 1143-1149

Influence of treatment with pancreatic extracts onpancreatic enzyme secretionJ MOSSNER, H P WRESKY, W KESTEL, J ZEEH, U REGNER,AND W FISCHBACH

From the Medizinische Poliklinik, University of Wurzburg, Wurzburg, Federal Republic of Germany

SUMMARY We have evaluated the effects of porcine pancreatic extracts on human pancreaticsecretion. Ten male volunteers were intubated with a 4-lumen jejunal tube to collect gastric andduodenal secretions separately via the first and third tube, to infuse PEG 4000 distal the pylorus viathe second tube and to apply porcine pancreatic extracts via the fourth tube distal the ligament ofTreitz. Pancreatic extracts were given four times at 40 minute intervals; the first two as activeenzymes and subsequently as heat denatured proportions. Secretin was continuously infusedintravenously (0.5 Elkg bw/h) to achieve minimal pancreatic flow. Lipase, amylase, trypsin,chymotrypsin, volume, and bicarbonate were measured in duodenal contents in eight pooled 15minute fractions. Three subjects who received HEPES-Ringer buffer instead of pancreatic enzymesserved as controls. Plasma cholecystokinin (CCK) was measured using a sensitive bioassay. Bothactive and heat denatured pancreatic extracts caused a small but significant increase in amylase andchymotrypsin secretion. Basal plasma CCK values were 0.85 (0.05) pM. After intrajejunalinstillation of either active or heat denatured pancreatic extracts plasma CCK rose to 3.25 (0.30) pMand to 3-28 (0.36) pM respectively. In a second group of five volunteers, plasma CCK concentrationswere measured after a test meal. On day 1, volunteers received a liquid fat and protein rich meal andon day 2, the same test meal containing porcine pancreatic extracts. In both cases, a similar increasein plasma CCK was observed. We conclude that therapy with pancreatic extracts stimulatepancreatic enzyme secretion. This may be mediated through release of CCK.

Regulation of pancreatic enzyme secretion bypancreatic proteases in the duodenum via a negativefeedback has been shown in a number of studies inrats,'-3 chicken,4 and pigs.5 In rats this negativefeedback control is clearly mediated through CCK.'The findings in man are more controversial. In theabsence of nutrients, pancreatic secretions did notexert a negative feedback on human pancreaticsecretions.' Furthermore, in other studies, inhibitionof intraduodenal trypsin did not stimulate pancreaticsecretion.""' Other groups, however, reported feed-back regulation of human pancreatic secretion bytrypsin. `' Studies on patients with chronic pan-creatitis seemed to further support the hypothesisthat negative feedback regulation exists in man. Inthis disease, which sooner or later leads to a decreaseof pancreatic protease secretion, raised plasma CCKAddress for correspondence: Priv Doz Dr med Joachim Mossner, MedizinischePoliklinik, University of Wurzburg, Klinikstr 8, 87(N) Wurzburg, FRG.

Accepted for publication 8 December 1988.

concentrations have been reported. '5' Treatmentwith pancreatic enzymes caused a reduction of painwhich was considered to be the result of lowering theintraductal pressure by intraluminal trypsin.'7Others, however, could neither confirm raised CCKconcentrations in advanced chronic pancreatitis norcould they confirm a lessening of pain when patientswere treated with pancreatic enzymes.`' 1`

In the present study, we investigated the effects ofporcine pancreatic extracts on pancreatic enzymesecretion and on plasma CCK of healthy volunteers.The aim was to clarify whether commercially avail-able capsules of pancreatic enzymes, given in adosage known to be effective in pancreatic insuffici-ency, are able to inhibit pancreatic secretion.

Methods

MATERIALSThe following chemicals were used: N-2-

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hydroxyethyl-piperazine-N'-2-ethanesulfonic acid(HEPES), ethylenediaminetetraacetic acid, partiallypurified lyophilised enterokinase, porcine pancreaticlipase (type IV-S), porcine pancreatic amylase (typeVI), porcine pancreatic trypsin (type IX), soybeantrypsin inhibitor (type I-S), cholecystokinin octa-peptide (CCK8), gum arabic, bovine plasma albumin(fraction V) from Sigma Chemical, St Louis, MO;chromatographically purified collagenase (typeCLSPA) from Cooper Biomedical, Wiesbaden,FRG; polyethylene glycol 4000 (PEG), trichloro-acetic acid, zincsulfate, phenol red, indigocarmin,acetonitrile from Merck, Darmstadt, FRG; Bio-Rad protein assay from Bio-Rad Laboratories,Richmond, CA; octadecylsilylsilica (SEP-Pak C-18)cartridges from Water Associates, Milford, MA;synthetic secretin (SekretolinR) from Hoechst,Frankfurt, FRG; aprotinin (TrasylolR) from Bayer,Leverkusen, FRG; Whatman 42 ashless filters fromW&R Balston Lim, UK; and minimal Eagle'smedium amino acid supplement from Biochrom KG,Berlin, FRG. PanzytratR20000 was a gift fromNordmark Arzneimittel GMBH, Uetersen, FRG.All other reagents had the highest purity gradecommercially available.

S U B J E CI STwenty healthy male volunteers, 19-26 years partici-pated in the studies. All subjects were within 10% oftheir ideal body weight. None were taking anymedication, or had any history of gastrointestinaldiseases. The studies were approved by the Commis-sion for Ethics at the University of Wiirzburg.

INTRADUODEFNAL/JEJUNAI PERFUSIONSTU DI lSAll studies were performed after an overnight fast. Afour lumen polyvinyl tube was placed under fluoro-scopic control. Gastric secretions were continuouslyaspirated through the first lumen with the tubesituated at the gastric antrum using an automaticpump (Oehler & Braun, Stuttgart, FRG). Poly-ethylene glycol 4000 (15.0 g/l; 3 ml/min) was infusedthrough the second lumen just distal the pylorus.Duodenal secretions were aspirated through thethird tube by continuous suction (suction pressurevarying from 0 to 400 mmHg) with the aspirationholes of the tube 15-25 cm distal the PEG infusionsite. Pancreatic extracts were given distal the liga-ment of Treitz through the fourth tube 30 cm distalthe last duodenal aspiration site.A new preparation of acid protected commercially

available porcine pancreatic enzymes was applied ina dosage commonly used for treatment of pancreaticinsufficiency"' (PanzytratR20000, capsules withmicrotablets, containing per capsule according to the

information provided by the manufacturer, triacyl-glycerollipase 20000 Ph Eur-U, amylase 18000 PhEur-U, proteases 1000 Ph Eur-U). For our studies,three capsules PanzytratR20000 were homogenisedby UltraturaxR in 20 ml of a buffer containing 10mM HEPES, 140 mM NaCI, 4.7 mM KCI, 1 mMNaHPO4, 113 mM MgCI,, 1 28 mM CaCl2, adjustedto pH 7-4. According to our own measurements, theactivities of various pancreatic enzymes per 20 mlbuffer were as follows: trypsin 28.62 (1.63) U (incomparison, 20 mg of pure porcine trypsin type IXobtained from Sigma Chemicals had an activity of33.83 (0.55) U); chymotrypsin 9-31 (0.40) U; lipase198.25 (27.54) U; amylase 55900 (7738) U (mean(SE) for n=eight separate determinations).

Potential retroduodenal reflux of porcine pan-creatic enzymes perfused intrajejunally was excludedby mixing the extracts with indigocarmin. Refluxcould, therefore, be monitored visually when the dyeappeared in the duodenal aspirations. The experi-ments were started between 7 and 8 am and a correctpositioning of the tube was achieved at 11 am (1) h(time 0). Polyethylene glycol 4000 was continuouslyperfused (15 g/l physiological saline, 3 mI/min)starting at time 0. Duodenal contents were recoveredby constant suction, collected on ice, and pooled at 15minute intervals. Thirty minutes later a continuousintravenous low dose infusion of secretin was startedto achieve minimal pancreatic flow (SekretolinR, 05U/min/kg body weight). At the same time threecapsules of PanzytratR20000 homogenised in 20 mlHEPES-Ringer buffer were perfused through thefourth lumen within five minutes. After a further fiveminutes, duodenal contents were collected for 2x 15min (time 55 and 70 minutes). This schedule wasrepeated three times: pancreatic extracts perfused attimes 70, 110, and 150 minutes, duodenal contentscollected 80-110, 120-150, 160-190 minutes. Attimes 110 and 150, however, instead of perfusingactive enzymes, the same amount of heat denaturedPanzytrat 20000 solutions was used. In anothergroup of volunteers the same procedure wasrepeated, but instead of starting with active enzymesheat denatured enzymes were given first, at times 30and 70 minutes. A third group of three volunteersreceived instead of pancreatic extracts just HEPES-Ringer buffer (controls). In all three groups 20 mlblood were collected at times -5, 10, 25, 35, 50, 65,75, 90, 105, 115, 130, 145, 155, 170, 185 minutes fordetermination of CCK. Some volunteers had to beexcluded due to reflux, non compliance or lowrecovery of PEG. A total of 13 completed the studies.

IN FI U ENCE OF FOODAN) PANCREATIC EXTRACTS

ON PlASMA CCKFive volunteers received after an overnight fast a

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Influence oftreatment with pancreatic extracts on pancreatic enzyme secretion

mixed liquid meal (6 ml/kg bw) made of cream, milk,eggs, cacao and sugar (41.5% fat, 17% protein,41.5% carbohydrate w/w) which they had to drinkwithin two minutes. Twenty millilitres blood weredrawn 15 and one minute before and 7 5, 15, 30,45, and 60 minutes after feeding for determinationsof CCK. This procedure was repeated with thesame subjects the day after but with an additionalapplication of microtablets from six capsulesPanzytratR20000.

CHOLECYSTOKININ BIOASSAY

Cholecystokinin was extracted from plasma using amodification of methods described by Liddle et al.'Aprotinin was added to plasma samples (final con-centration 400 U/mI). Plasma was diluted with ice-cold 2% trifluoroacetic acid (TFA) (1:4). CCK wasextracted by adsorption on SEP-PAK cartridgespreviously washed with 10 ml acetonitrile and 10 ml0 1% TFA. The cartridges were then washed againwith 10 ml 0.1% TFA and the CCK was eluted with2 ml of acetonitrile/0-1% TFA (1:1). The eluateswere collected in flat bottomed incubation vials anddried under a nitrogen stream at 45°C and stored at-70°C. These vials were subsequently used forincubation with 1 ml of acini suspensions.

PREPARATION OF ISOLATED PANCREATIC ACINI

Pancreatic acini were prepared from male SpragueDawley rats (150-250 g) by digestion of pancreatictissue (08-1.3 g) with purified collagenase at 37°C inan agitating water bath as described in the litera-ture.25'4 After digestion the acini were dissociatedby up-and-down suction through polypropylenepipettes with restrictive orifices, filtered through a150-[tm nylon cloth, and further purified by centrifu-gation through a 4% albumin gradient. Thereafter,the acini were suspended in a buffer containing 40mM Tris (hydroxymethyl) aminomethane, 103 mMNaCl, 1 mM NaHPO4, 47 mM KCl, 1 28mM CaCI2,0*56 mM MgC12, 11.1 mM glucose, 0.1 mg/mIsoybean trypsin inhibitor, enriched with minimalEagle's medium amino acid supplement, and 5 mg/mlbovine plasma albumin. This buffer was equilibratedwith 100% 02 and adjusted to pH 7-4. The averageacinar protein concentration was in the range of 0-2-0.5 mg/ml.

Isolated acini were then incubated with eitherplasma extracts or various concentrations of CCK8for 30 min at 37°C and the release of amylasemonitored. Amylase release, expressed as percent-age of total amylase activity, was compared with adose response curve for CCK8 in order to calculatethe CCK content of plasma expressed as CCK8equivalents. Preparations of rat pancreatic acini haveshown the typical biphasic dose response curve to

CCK8. The threshold dose was between 1-3 pM andmaximal secretion was seen at 30-100 pM.

ASSAYSAmylase activity for bioassays of CCK was assayed inpancreatic homogenates and supernatants usingprocion yellow as substrate.'5

Protein was measured according to the method ofBradford using the Bio-Rad reagent.tmMeasurement of pancreatic enzymes in duodenal

contents: trypsin activity was assayed using anenzymatic colorimetric test with benzoylarginine-p-nitroanilide as the substrate (test combination,Boehringer Mannheim, FRG).`7 Chymotrypsinactivity was assayed using a colorimetric enzymeassay with N-(3-carboxypropionyl)-phenylalanine-p-nitroanilide as the substrate (Boehringer,Mannheim, FRG).'` Lipase and amylase activitywere assayed using a nephelometric method withpurified olive oil as the substrate for lipase andamylopectin for amylase"' (Perkin-Elmer Model 91Amylase-Lipase-Analyzer, Perkin-Elmer, OakBrook, Ill). Bicarbonate was measured t'ia backtitration with 0 1 M NaOH with phenol red asindicator. Polyethylene glycol was measuredturbidometrically according to the methods des-cribed by Hyden et all' and Malawer et al.`

Statistical analysis was performed using analysis ofvariance, paired, and unpaired t test with the accept-ance criteria of p=0.05. All results were expressed asmean (SE).

Results

EFFECT OF PORCINE PANCREATIC EXTRACT'S ONHUMAN PANCREATIC SECRETIONCommercially available porcine pancreatic extracts,used for treatment of human pancreatic insufficiency,were applied in the upper jejunum and secretion ofbicarbonate and endogenous pancreatic digestiveenzymes measured. When compared with perfusionof the upper jejunum with Hepes-Ringer bufferalone, pancreatic enzymes caused a small butstatistically significant increase of amylase andchymotrypsin secretion but not of trypsin and lipase(Fig. 1). The proteases contained in the enzymecapsules did not cause any decrease of endogenousnon-food stimulated enzyme secretion when com-pared with controls. When pancreatic extracts wereheat denatured before their jejunal application, asmall but significant increase of secretion of amylaseand chymotrypsin was again seen but none for trypsinand lipase (Fig. 1). When the effect of active enzymeson pancreatic secretion was compared with the effectof inactive ones in the same volunteer, many subjectsshowed a lower enzyme secretion after the applica-

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Trypsin Lipase30- T 1500-

.E 100E 20- jo

10- :D| 500

Chymotrypsin

r-

DE

Bicarbonate

3000- 9E

2000. 6

0

1000. ~~~E 3-

0

100-

80-

60-

U/1 5min

40-

Amylase Duodenal volume

40 90-~

20 .-

XE 30-

0 0

O Control

3 Active

* Inactive

Fig. 1 Influence ofpot cine pancreatic extracts onpancreatic secretion. Ten male volunteers receivedfour timespancreatic extracts intrajejunally, the first two times as activeenzymes followed by the application ofthe same amount ofheat denatured extrcacts or vice versa, and duodenal contentswere collected to measure output ofpancreatic enzymes.Tlhree volunteers received HEPES-Ringer buffer instead ofpancreatic extracts (control). The mean offour 15 minduodenalffractions per subject was formed after eitller activeor inactive extrtacts, as well asfrom eight 15-min fractions percontrol subject. Bars show the mean (SE) of the data from allsubjects. Asterisk displays a statistically significant differencebetween control and both active and inactive extracts.

tion of active enzymes. Trypsin for example, was

lower in eight of 10 subjects after the application ofactive enzymes as compared with inactive ones (Fig.2). Chymotrypsin was lower in eight, amylase in six,and lipase in five subjects (data not shown). Becauseof considerable variations in pancreatic secretionbetween different subjects, however, neither themeans of enzyme activities in each 15 min fraction(data not shown) nor of the pooled 15 min fractions(Fig. 1) showed any statistically significant differ-ences.

EFFECT OF PANCREATIC DIGESTIVE ENZYMES ON

PLASMA CCK

Basal plasma CCK, determined at times -5, 10, and

20-

o

Trypsin

Active InactiveFig. 2 Influence ofactive versus inactive porc.ine pancreaticextracts on trypsin output. The mean ofthe trypsin activityfrom four 15 min duodenalfractions from subject.s of Figure1 is compared intraindividually after the application of eithleractive or inactive extracts.

25 min, was 0-85 (0.05) pM (mean (SE); CCK wasdetermined at each time point in duplicate) insubjects who received later pancreatic extracts(n=10) and 1-33 (0.17) pM in volunteers whoreceived HEPES-Ringer buffer (n=3). The differ-ence was not statistically significant. Application ofHEPES-Ringer buffer did not cause any statisticallysignificant changes in plasma CCK (1.31 (0.11) pM)(Fig. 3). Both active as well as heat denaturedporcine pancreatic enzymes, when applied intra-jejunally, however, caused a small but statisticallysignificant and comparable increase in plasma CCK(Fig. 3). Integrated plasma CCK was 3-25 (0.30) pMafter active enzymes and 3-28 (0.36) after inactiveones.

PLASMA CCK AFTER INGESTION OF FOOD WITH

OR WITHOUT SIMULTANEOUS APPLICATION OFPANCREATIC ENZYMESIngestion of food caused a rapid and sustainedincrease of plasma CCK (Fig. 4). The addition of six

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pmol2-

EQ

C~D

Control Active InactiveFig. 3 Influence ofactive and inactive pancreatic extr acts onplasma CCK. Plasma CCK was measured via bioassay. Barsshow the mean (SE) of integrated plasma CCK values of thlethirteen subjects from Figure 1. Asterisk displays a

statistically significant difference between contr-ol, and both2active and inactive.

capsules of PanzytratR20000 to the liquid meal didnot cause a different pattern of plasma CCK (Fig. 4).

Discussion

Pain in patients suffering from chronic pancreatitismay be caused by a rise of pressure in pancreaticducts.` A reduction of pressure via an inhibiton ofpancreatic secretion could, therefore, reduce pain.There are now many studies supporting the hypo-thesis of negative feedback regulation of humanpancreatic secretion by proteases. 12'-4 With theapplication of proteases, therefore, one should beable to inhibit pancreatic secretion. Patients withpancreatic insufficiency are usually treated withporcine pancreatic extracts which contain a mixtureof digestive enzymes. At present, acid protectedtablets with high amounts of lipase are preferred inthe treatment of pancreatic insufficiency.' In thisstudy we investigated whether acid protected porcinepancreatic extracts in a dosage known to be success-ful in the treatment of pancreatic insufficiency, giveneither alone or together with a meal, are able toinhibit pancreatic secretion. In an experimentaldesign in man, however, one can not apply porcinepancreatic enzymes orally or perfuse intraduodenallyand measure secretion of endogenous pancreaticenzymes simultaneously. In the studies reported bySlaff et al,'' therefore, enzyme therapy was discon-tinued the day before pancreatic secretion wasmeasured. In this design, however, one can notmeasure acute effects of pancreatic extracts on pan-

8-

6-

4-

2-

n-

Plasma CCK

-15 0 7-5 15 30 45 60Time (min)

Fig. 4 Effect offood c.ontaining active pancreatic extr actson plasma CCK. Five subjects received at times 0 a liquidmeal. Values slhow the mean (SE) ofplasma CCK at varioustimes (interrupted line). Same subjects received on day two asimilar liquid meal containing six capsules ofpancreaticextracts (Panzytrat R20000) (uninterrupted line).

creatic secretion. In the study reported by Owyang etal only pure enzymes, and not a combination ofdigestive enzymes, were perfused intraduodenally. Itmay be possible that the amount of other proteinspresent in pancreatic extracts overwhelms a potentialinhibitory effect of proteases. We, therefore, per-fused the upper jejunum with pancreatic extractswhich enabled us to measure the effect on pancreaticsecretion. Both active pancreatic extracts and heatdenatured enzymes caused a small increase in plasmaCCK and an increase in secretion of chymotrypsinand amylase when compared with controls.Comparing the effect of active extracts on pancreaticsecretion with inactive ones, it was not possible toshow a statistically significant inhibition of pancreaticenzyme secretion. We next added those extracts tofood. With this experimental design it is not possibleto measure endogenous secretion because pancreaticsecretions cannot be diverted. Therefore, plasmaCCK was measured assuming that the level of CCKdisplays a strong correlation to pancreatic secretion.The addition of active pancreatic extracts to food didnot cause a lower increase in plasma CCK. Thus, wecould not show feed back regulation of enzymesecretion with doses of pancreatic extracts commonlyused in clinical medicine.

Perfusing the duodenum with trypsin, Owyang et alclearly showed an inhibition of phenylalanine, oleicacid, and meal stimulated chymotrypsin and lipaseoutput.' The amount of trypsin they used to inhibitpancreatic secretion, however, was rather high, 150-300 mg bovine type III S, within one hour." Thetrypsin activity per tablet of porcine pancreaticextracts available in West Germany ranges between800 and 2000 FIP Units. According to our own

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measurements with regards to Panzytrat R20000,which contains 1000 FIP, one capsule correspondsapproximately to 6 mg trypsin (porcine type IX,Sigma). According to the data of Owyang et al 25-50capsules per meal would have to be used to achieveinhibition of secretion. In the study reported by Slaffet al, protease specific suppression of pancreaticexocrine secretion was also seen only with very highdoses of trypsin.' It may be assumed that trypsinbinds to food. Thus high levels of trypsin wereneeded in the studies reported by Owyang et al" andSlaff et al to overcome the binding of trypsin to foodso that sufficient trypsin is present to exert negativefeedback. Proteases in very high concentrations inthe presence of food and in may be lower con-centrations in the absence of food may be capable,therefore, to inhibit human pancreatic secretion.Pancreatic extracts, however, given in a dosagecommonly used in clinical medicine do not inhibit,but rather stimulate pancreatic secretion. This maybe because of the high protein content of enzymecapsules. We doubt, therefore, that pain in chronicpancreatitis can be treated via an inhibition ofpancreatic secretion by the application of pancreaticextracts commercially available at present.

This study was supported by a grant of the DeutscheForschungsgemeinschaft (Mo 372/2-1). Some of theresults are part of the MD thesis of H P Wresky. Partsof the study have been published as abstract.34 Theauthors thank Mrs S Stieber, Mrs L Kneis, and Mrs GGebert for expert technical assistance.

References

1 Green GM, Lyman RL. Feedback regulation of pan-creatic enzyme secretion as a mechanism for trypsininhibitor-induced hypersecretion in rats. Proc Soc ExpBiol Med 1972; 140: 6-12.

2 Ihse I. Lilja P, Lundquist 1. Trypsin as a regulator ofpancreatic secretion in the rat. Scand J Gastroenterol1979; 13: 873-80.

3 Miyasaka K, Green GM. Effect of partial exclusion ofpancreatic juice on rat basal pancreatic secretion.Gastroenterology 1984; 86: 114-9.

4 Chernick SS, Lepkovsky S, Chaikoff IL. A dietaryfactor regulating the enzyme content of the pancreas:changes induced in size and proteolytic activity of thechick pancreas by ingestion of raw soybean meal. Am JPhysiol 1948; 155: 33-41.

5 Corring T. Mechanisme de la secretion pancreatiqueexocrine chez le porc: regulation par retro inhibition.Ann Biol Anim Biochim Biophys 1973; 13: 755-6.

6 Folsch UR, Cantor P, Wilms HM, Schafmayer A,Becker HD, Creutzfeldt W. Role of cholecystokinin inthe negative feedback control of pancreatic enzymesecretion in conscious rats. Gastroenterology 1987; 92:499-58.

7 Louie DS. May D, Miller P. Owyang C. Cholecysto-kinin mediates feedback regulation of pancreaticenzyme secretion in rats. Am J Phtysiol 1986; 250: 252-9.

8 Lee PC, Newman BM, Praissman M, Cooney DR,Lebenthal E. Cholecystokinin: a factor responsible forthe enteral feedback control of pancreatic hypertrophy.Pancreas 1986; 1: 335-40.

9 Krawisz BR. Miller LJI DiMagno EP. Go VLW. In theabsence of nutrients, pancreatic-biliary secretions in thejejunum do not exert feedback control of humanpancreatic or gastric function. J Lab Clin Med 1980; 95:13-8.

lt) Dlugosz JI Folsch UR, Creutzfeldt W. Inhibition ofintraduodenal trypsin does not stimulate exocrinepancreatic secretion in man. Digestion 1983; 26: 197--204.

11 Hotz JI Ho SB, Go VLW, DiMagno EP. Short-terminhibition of duodenal tryptic activity does not affecthuman pancreatic, biliary, or gastric function. J LabClin Med 1983; 101: 488-95.

12 Slaff J, Jacobson D, Tillman CR, Curington C, ToskesP. Protease-specific suppression of pancreatic exocrinesecretion. Gastroenterology 1984; 87: 44-52.

13 Owyang C, Louie DS, Tatum D. Feedback regulationof pancreatic enzyme secretion. Suppression ofcholecystokinin release by trypsin. J Clin Invest 1986;77: 2042-7.

14 Ihse 1, Lilja P. Lundquist 1. Feedback regulation ofpancreatic enzyme secretion by intestinal trypsin inman. Digestion 1977; 15: 303-8.

15 Funakoshi A. Nakano I, Shinozaki H, Tateishi K,Hamaoka T, lbayashi H. High plasma cholecystokininlevels in patients with chronic pancreatitis havingabdominal pain. Amn J Gastr-oenterol 1986; 81: 1174-8.

16 Schafmayer A, Becker HD, Werner M, Folsch UR,Creutzfeldt W. Plasma cholecystokinin levels in patientswith chronic pancreatitis. Digestion 1985; 32: 136-9.

17 Isaksson G, Ihse 1. Pain reduction by an oral pancreaticenzyme preparation in chronic pancreatitis. Dig Dis Sci1983; 28: 97-102.

18 Cantor P. Petronijevic L, Worning H. Plasma chole-cystokinin concentrations in patients with advancedchronic pancreatitis. Panc reas 1986; 1: 488-93.

19 Halgreen H, Pedersen TN, Worning H. Symptomaticeffect of pancreatic enzyme therapy in patients withchronic pancreatitis. Scand J Gastroenterol 1986; 21:104-8.

20 Lankisch PG. Lembcke B, Kirchhoff S. Hilgers R,Creutzfeldt W. Treatment of pancreatogenicsteatorrhea: a comparison of two acid-protected enzymepreparations. Dtscli Med Wochenschlr 1988; 113: 15-7.

21 Liddle RA. Goldfine ID, Williams JA. Bioassay ofplasma cholecystokinin in rats: effects of food, trypsininhibitor, and alcohol. Gastroenterology 1984; 87:542-9.

22 Liddle RA, Goldfine ID, Rosen MS. Taplitz RA,Williams JA. Cholecystokinin bioactivity in humanplasma. J Clin Invest 1985; 75: 1144-52.

23 Williams JA. Korc M, Dormer RL. Action ofsecretagogues on a new preparation of functionallyintact, isolated pancreatic acini. Am J P/liysiol 1978; 235:517-24.

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Influence oftreatment with pancreatic extracts on pancreatic enzyme secretion 1149

24 Sankaran H, Iwamoto Y, Korc M, Williams JA,Goldfine ID. Insulin action in pancreatic acini fromstreptozotocin-treated rats. II. Binding of '25l-insulin toreceptors. Am J Physiol 1981; 240: 63-8.

25 Jung DH. Preparation and application of procion yellowstarch for amylase assay. Clin Chim Acta 1980; 100:7-10.

26 Bradford M. A rapid and sensitivc method for thequantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Anal Biochem1976; 72: 248-54.

27 Erlanger BF, Kokowsky N, Cohen W. The preparationand properties of two new chromogenic substrates oftrypsin. Arch Biochem Biophys 1961; 95: 271-8.

28 Nagel W, Willig F, Peschke W, Schmidt FH. Uber dieBestimmung von Trypsin und Chymotrypsin mitAminosaure-p-Nitroaniliden. Hoppe Seylers Z PhysiolChem 1965; 340: 1.

29 Zinterhofer L, Wardlaw S, Jatlow PJ, Seligson D.Nephelometric determination of pancreatic enzymes. IILipase. Clin Chim Acta 1973; 44: 173-8.

30 Zinterhofer L, Wardlaw S. Jatlow PJ, Seligson D.Nephelometric determination of pancreatic enzymes. IAmylase. Clin Chim Acta 1973; 44: 5-12.

31 Hyden S. A turbidometric method for the determinationof higher polyethylene glycols in biological materials.Kungl Lantbrukshogsk Ann 1956; 22: 139-45.

32 Malawer SJ. Powell DW. An improved turbidometricanalysis of polyethylene glycol utilizing an emulsifier.Gastroenterology 1967; 53: 25().

33 Wolfson P. Surgical management of inflammatory dis-orders of the pancreas. Surg Gynecol Obstet 1980; 151:689-98.

34 Mossner J, Wresky H, Zeeh J, Regner U. Influence oftreatment with pancreatic extracts on plasma chole-cystokinin (CCK) and pancreatic enzyme secretion.Pancreas 1988; 3: 609.

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