Gut, 1990, 31, 1318-1323

Pancreatic secretory trypsin inhibitor ingastrointestinal mucosa and gastric juice

T C Freeman, R J Playford, C Quinn, K Beardshall, L Poulter, J Young, J Calam

AbstractWe studied the distribution of pancreaticsecretory trypsin inhibitor (PSTI) in theepithelia of the gastrointestinal tract anddetermined whether PSTI is secreted intogastric juice. PSTI was measured by a specificradioimmunoassay in biopsy specimens takenfrom the upper (n=8) and lower (n=7) gastro-intestinal tract of patients with normal endo-scopies. PSTI was present in the stomach,small intestine, and colon. Concentrations(1ig/g protein) were highest in the stomach,and significantly higher in the antrum (1240,670-1700, median and range) than in thegastric body (370, 350-570) (p

Pancreatic secretory trypsin inhibitor in gastrointestinal mucosa andgastricjuice

Trypsin inhibitor activity 0

C.00.0

0 10

Figure 1: Elution profile ofpancreatic secretory t?ypsininhibitor (PSTI) extractedfrom human pancreatic juicefrom a Pharmacia Mono Scolumn, equilibrated withammonium acetate (0 I M,pH 3 5) and eluted with agradient runfrompH 3 5 to4 5. Elution ofthe PSTIfrom the column wasobserved as optical density at280 nm and trypsin inhibitoractivity.

plentiful fluids. The mean (SD) weight of biopsyspecimens was 10 6 (3 0) mg.

*4*5 Biopsy specimens were immediately frozen inliquid nitrogen where they remained untilextraction. They were extracted on ice by homo-genisation in 200 p1 of Tris buffer (10 mM, pH773) for 1 minute. Extracts were centrifuged at15 850 g for 1 minute and supernatants frozen on

4.0 ~4 solid CO2 and stored at -20°C before radio-immunoassay for PSTI and measurement ofprotein concentration by a modification ofLowry's method.'2

a3.5

Fraction No

with '25I by the chloramine T method" andtracer, 1500 cpm/tube, was incubated with anti-serum T4 (final dilution 1:500 000) together withPSTI standards (0-01-50 ng/ml) or samples in1 ml ofsodium phosphate buffer (0 5 M, pH 7 3)containing 0-15% bovine serum albumin (Sigma)and 0 02% sodium azide. Incubation was at 4°Cfor 3 days and separation was achieved by addingto each tube at 40C, 100 pl of ethylenediaminetetra-acetate (EDTA) (0 1 M, pH 7 3), 100 RI of2% rabbit serum in assay buffer, 100 ptl ofsecond antibody (goat antirabbit antiserum, typeR 0881, Sigma) diluted 1:5 in assay buffer, and700 p1l of 6% polyethylene glycol 6000 inalbumin free assay buffer. The tubes were mixedand incubated at 40C for 40 minutes before beingcentrifuged at 3500 g and 40C for I5 minutes.The supernatant was aspirated into separatetubes and both tubes counted.

COLLECTION AND EXTRACTION OF ENDOSCOPICBIOPSY SPECIMENSUpper gastrointestinal biopsy specimens werecollected during routine endoscopy, usingFB25K forceps (Keymed, Southend-on-Sea,Essex), from eight patients, three men and fivewomen, mean age 46 years (range 32-61 years).These patients were under investigation fordyspepsia, but endoscopy and other investiga-tions proved normal and the final diagnosis wasnon-ulcer dyspepsia. Paired endoscopic biopsyspecimens were normal on histological examina-tion. No patients took any drugs within two daysofthe examination. The mean (SD) weight of thebiopsy specimens was 6 8 (2 0) mg.Lower gastrointestinal biopsy specimens were

collected during routine endoscopy, usingFG15L forceps (Keymed), from seven patients,four men and three women, mean age 47 years(range 26-65 years). These patients were underinvestigation for abdominal pain or disturbanceof bowel habit, but endoscopy and other investi-gations showed no abnormality and the finaldiagnosis was the irritable bowel syndrome.Paired endoscopic biopsy specimens were normalon histological examination. None ofthe patientstook any drugs during the two days beforeexamination except for a bowel preparation,which comprised a low residue diet, two sachetsof Picolax (Ferring, Feltham, Middx), and

GASTRIC JUICEGastric juice was collected during routinepentagastrin tests on 18 patients, 13 men and fivewomen, in whom duodenal ulcers had been seenat endoscopy within seven days of study. Theirmean age was 49 years (range 25-77 years). Nonetook any drugs in the two days before the study.

Gastric juice was collected from the last ofthree 10 minute basal collections and afterstimulation with pentagastrin 0-6 rig/kg perhour for at least 80 minutes. Juice (2 ml) wascollected directly from the aspiration tube, andimmediately neutralised by mixing with 3 ml0-17 M sodium bicarbonate on ice. Samples werethen frozen at - 20°C before assay.

Gastric juice samples were analysed forbilirubin with a RA-1000 analyser (TechnicalInstrument Corporation), using Techniconmethod, number SM-0179887. Tryptic activitywas determined by thepH stat method using Na-p-tosyl-L-arginine methyl ester (Sigma) as sub-strate.'3

CHROMATOGRAPHY OF GASTROINTESTINAL PSTIPSTI-LI in gastric juice and extracts of biopsyspecimens taken from the colon and gastricantrum were analysed by reversed phase highpressure liquid chromatography on a 4 6x250 mm, C-8 Dynamax column (12 lim, 150 A,Rainin), eluted with a gradient of 16-30% aceto-nitrile in 0 1d% triflouroacetic acid. Eluates werelyophilised before radioimmunoassay. Thesystem had been previously calibrated with pan-creatic PSTI.

STUDIES OF THE STABILITY OF PSTI IN GASTRICJUICEPentagastrin stimulated gastric juice wasobtained from two subjects with duodenalulcers. Tris-HCl was added to a final concentra-tion of 10 mM, to stabilise the pH during thestudy. Portions (10 ml) of each juice wereadjusted to pH 2-0, pH 4 0, pH 6-0, and pH 7*4by the addition ofNaOH. Pure human PSTI wasthen incubated with each portion at an initialconcentration of 60 ng/ml at 370C. At the timesshown in Figure 4, 250 p1 samples wereremoved, immediately neutralised by addition ofan equal volume of 0- 17 M sodium bicarbonate,frozen on solid CO2, and stored at -200C untilradioimmunoassay. In control studies PSTI wasincubated as already described, but in Tris-HCIbuffer at pH 2-0 and 4 0.The stability of PSTI in unbuffered gastric

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Freeman, Playford, Quinn, Beardshall, Poulter, Young, Calam

Figure 2: The inhibition ofbinding ofpancreaticsecretory trypsin inhibitor(PSTI) tracer to antiserumT4 by human PSTI andother substances.

2

a)c)

14.

..

m

o L0.1

juice, pH 1 2, was also tested in the presence andabsence of pepstatin (Sigma) 200 ,ug/ml withincubation for 1 hour at 37°C.

CONCENTRATION OF PSTI IN PANCREATIC JUICEPancreatic juice was collected from postopera-tive pancreatic drains from three patients (oneman, two women), two ofwhom had undergonepancreatic surgery for pancreatic tumours andone ofwhom had chronic pancreatitis. The juicewas frozen and stored at -20°C until the con-centration of PSTI was determined by radio-immunoassay.

IMMUNOHISTOCHEMISTRYSections 2 [tm thick were cut from samples ofnormal oesophagus, stomach, and small andlarge intestine. Immunoperoxidase stainingwas performed using a routine peroxidase-antiperoxidase procedure.14 Briefly, sectionswere dewaxed, rehydrated, and trypsinised at37°C for 10 minutes to unmask antigenic sites.'5Endogenous peroxidase was blocked usingmethanolic hydrogen peroxide for 30 minutes,and the sections rinsed in phosphate bufferedsaline and incubated with normal swine serum(Dako Ltd, High Wycombe, Bucks) for 15minutes. They were then incubated with theprimary antibody, T4, overnight at 4°C, rinsed,and incubated with swine antirabbit immuno-globulin (Dako) for 30 minutes at room tempera-ture. After rinsing in phosphate buffered salinethe sections were incubated with peroxidase-antiperoxidase complex (Dako) for 30 minutesat the recommended dilution and rinsed again.The sections were then developed with 3,3'-diaminobenzidine tetrahydrochloride (AldrichLtd, Gillingham, Dorset) for 5 minutes and thencounterstained lightly with haematoxylin.Finally, the sections were dehydrated andmounted using pertex (Histolab and Cytolab,Hemel Hempstead, Herts). Negative controlswere obtained by substituting normal rabbitserum for the PSTI specific antiserum.

STATISTICAL ANALYSISFor statistical analysis Wilcoxon's rank sum testwas used and results are expressed as median andrange; p

Pancreatic secretory trypsin inhibitor in gastrointestinal mucosa andgastricjuice

15000

Figure 3: Rates ofgastricsecretion ofpancreaticsecretory trypsin inhibitor(PSTI) before and afterstimulation withpentagastrin. The horizontallines indicate the medians.*p

Freeman, Playford, Quinn, Beardshall, Poulter, Young, Calam

B

I-

.4

Paafin ......

Figure 6: Paraffin sections ofgastrointestinal mucosa stained with an indirect immunoperoxidase method using an anti-PSTI antibody, (A) normal gastric mucosa(original magnification x31 5, inset x 236) showingfoveolar cell positivity in gastric pits; (B) normal duodenal mucosa (original magnification x50, inset x 193)showing Paneth cell positivity; (C) normal colonic mucosa (original magnification x31-5, inset x96) showing non mucus cell positivity in colonic crypts.

duodenum, and colon but undetectable in theoesophagus. Tissue concentrations were greatestin the gastric antrum. PSTI-LI was found to bein a specific cell type in each region of the gut.We have shown for the first time that PSTI issecreted into the lumen of the stomach. PSTI israpidly destroyed by acid pepsin but stable at theneutral pH found in the gastric mucus layer.

In a previous study Shibata et al measuredPSTI-LI in cadaveric small intestine and surgic-ally resected stomach,6 and found concentrationsover an order of magnitude lower than in thepresent study. The lower concentrations thatthey reported may have been due to loss ofPSTIthrough hydrolysis by' mucosal enzymes beforeextraction and a higher proportion of sub-mucosal tissues in samples.The cellular distribution of gastrointestinal

PSTI-LI reported in the present study is con-sistent with the main findings of two othergroups.78 The weak PSTI-LI seen by' others,however, in the goblet cells of the colon and inother gastric cell types, including chief cells, wasnot observed in the present study.We are not awaie of any previous report of the

secretion ofPSTI into gastric juice. The concen-trations of PSTI-LI in gastric juice did not risesignificantly during the infusion of pentagastrin,although there was a significant increase in theoutput of gastric PSTI. Pentagastrin has beenreported to increase gastric secretion of carbo-hydrate from the same cell type in the cat.16

In the present study PSTI was shown to berapidly destroyed by pepsin in gastric juice atacid pH, but stable if the juice was neutralised.PSTI probably exerts its protective effect inthe gastric mucus layer which is kept at a neutralpH due to gastric secretion of bicarbonate."7Hydrolysis of PSTI by pepsin is probablyresponsible for the rapid loss of PSTI-LI inacidic extracts of gastric mucosa that wasreported by Shibata et al.6

The results ofthe present analytical studies areconsistent with the results of others who haveshown that pancreatic PSTI exists in multipleforms.23 These differ chiefly in the degree ofdeamination ofthe asparagine residues which areunusually abundant in PSTI. Fraction III and II2had the predicted mass of the molecule whereasfractions I and III had a molecular mass consist-ent with monodeaminated PSTI. In addition,Kikuchi et al found a form of PSTI in pancreaticjuice which had five amino acids missing fromthe N-terminal, which was not found here.2The presence of PSTI in all regions of the gut,

and its secretion into the lumen, suggest thatPSTI may protect the whole gut from proteolyticenzymes. Gastric PSTI is presumably importantduring episodes of duodenogastric reflux whichoccur in health,'8 occur more frequently in somediseases such as gastric ulcer,'9 and occur more orless constantly after some forms of gastricsurgery.'9 Reflux of duodenal juice may raiseintragastric pH to levels at which pancreaticenzymes are active but PSTI is stable. Concen-trations of PSTI measured in pancreatic juice inthe present study were similar to those reportedby others3 and approximately 1000 times higherthan concentrations found in gastric juice. Con-centrations of PSTI in the gastric mucus-bicarbonate layer, however, may be considerablyhigher than those found in the lumen of thestomach. In addition, the concentration of tryp-sin entering the mucus layer may be diminishedby dilution and by peptic destruction of trypsinin the lumen of the stomach.20 It is interestingthat gastric mucosal PSTI is most abundant inthe antrum, which is most exposed to refluxedenzymes. Colonic PSTI may protect the colonicepithelium from pancreatic enzymes whichremain active in colonic contents.2' PSTI alsoinhibits elastase and chymotrypsin4 as well astrypsin.

Recent work has shown that PSTI is a growth

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Pancreatic secretory trypsin irhilbitur ia grsitrointestinal mucosa andgastricjuice 1323

fa&u(r'J as well as a protease inhibitor. We showedtB it hmnan PSTI stimulates growth of AR4-2Jcells derived from a rat pancreatic acinar celltuYolajr.72 Others have shown that human PSTIstini-iiiat.-s growth of human fibroblasts23 andnu^ncn 2ndthelial cells.24 The growth stimulat-ing effect of PSTI may be a consequence of itsseqtuenlce homology with epidermal growthfactor. Rais;ed intragastric PSTI concentrationscould co-i-tribute to trophic effects seen in theprolonged absence of gastric acid.26

Gastroirn-estinal PSTI may provide an import-ant and hitherto unrecognised protective mecha-nism. Fuirtqer studies are required to determinethe lactors which control the secretion of gastro-int2stinla! PSTI and its possible role in theconti-ol of gastrointestinal growth.

\V'e thank tie Wellcome Trust for financial support; the MedicalRe earch Council for funding RP as an MRC training fellow; andDr S Lcvi and Sister Francis-Reme and the nursing staff of thegastric clir.ic for help in the collection of biopsy samples andsamnpkls of gastric juice.

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