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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
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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
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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
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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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