fate - gut · sporogenes (8053, strain mca 3679), klebsiella aerogenes (418, type 1, strain 240),...

Gut, 1969, 10, 477-483

Fate of trypsin and chymotrypsin in the humansmall intestine

D. M. GOLDBERG,' R. CAMPBELL, AND A. D. ROY

From the University Departments of Surgery and Pathological Biochemistry,Western Infirmary, Glasgow

The present consensus regarding the fate of thepancreatic proteases trypsin and chymotrypsin inthe human intestinal tract rests largely uponexperiments by Borgstrom, Dahlqvist, Lundh, andSjovall (1957), who measured their activity in juiceobtained by intubation of the small intestine.The results indicated a progressive fall in activityfrom the pylorus towards the ileocaezal valve.Support for their conclusion that enzyme degrad-ation takes place in the small intestine has comefrom Pelot and Grossman (1962), who demon-strated rapid inactivation of pancreatic enzymes inthe rat.

Studies in this laboratory have shown binding oftrypsin and chymotrypsin to human intestinal cells(Goldberg, Campbell, and Roy, 1968a), and havedemonstrated that the spectrophotometric assay ofLundh (1957), as used by Borgstrom et al (1957),was incapable of estimating bound enzymes. Analternative possibility was that the enzymes becamebound to the surface of cells during transit throughthe intestine and to components of intestinaldebris. Measurement of the daily output of trypsinin subjects on ileal drainage (Roy, Campbell, andGoldberg, 1967) gave data which, when comparedwith the trypsin concentration of human pancreaticjuice (Haverback, Dyce, Bundy, and Edmondson,1960), suggested that most of the enzyme reachedthe terminal ileum intact.

It therefore seemed desirable to examine the fate ofpancreatic enzymes in the human small intestine,considering all the factors that can interact with theenzymes from the point of their release into theduodenum. Such a study is the subject of the presentreport.

MATERIALS AND METHODS

The following materials were employed:-ILEAL FLUID This was obtained from patients with anestablished ileostomy as previously described (Roy et al,'Present address: Department of Chemical Pathology, The RoyalHospital, Sheffield, 1.

1967). Where output was quantitated the patients wereon a controlled diet 24 hours before the start of theexperiment, and this was continued throughout thecollection period. Homogenates of the ileostomy con-tents were prepared in 0.15 M NaCI, and supernatantswere separated after centrifugation at 45,000g perminute. These steps, together with the titrimetric assayof trypsin and chymotrypsin by procedures slightlymodified from those of Haverback, Dyce, Gutentag, andMontgomery (1963), have already been described (Royet al, 1967).

PANCREATIC JUICE This was obtained during sphincter-otomy on a subject with recurrent cholelithiasis. Thepancreas was normal at operation. Juice was collectedinto ice from a cannula draining the pancreatic duct fora period of 30 min after injection of 90 units of pan-creozymin (Boots Pure Drug Co. Ltd., Nottingham,England). This was activated by adding bovine pancreatictrypsin to a final concentration of 1 ftg/ml and CaCI2 toa final concentration of 0.005 M. It was then incubated at4°C until no further increase in trypsin or chymotrypsinactivity took place (approximately 56 hours) after whichit was divided into aliquots and stored at -20°C. Wewere unable to obtain any further samples of pancreaticjuice during the period spanned by this investigation.

GASTRIC JUICE This was obtained following histaminestimulation in normal individuals. All samples werechecked for the presence of acid and were filteredthrough gauze. Bile-stained samples were rejected.

BILE This was obtained from subjects on T-tube drain-age after choledochotomy, and was cultured to ensuresterility and filtered through gauze.

INTESTINAL HOMOGENATES Sections of small intestinewere obtained at several operations. These comprisedgastrojejunostomy when a small sample of jejunum wasobtained; resection of caecal carcinoma when a 12 cmsection of terminal ileum was available; and resection ofcolonic neoplasm adherent to small bowel when variablelengths of small bowel segments were available. All thematerial was histologically normal.The sections were splitlongitudinally, washed four to six times with cold 0.25 Msucrose until no more faecal material could be removed,clamped at the corners, and the mucosa was gently

477

on February 19, 2020 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.10.6.477 on 1 June 1969. D

ownloaded from

http://gut.bmj.com/

D. M. Goldberg, R. Campbell, and A. D. Roy

scraped with the back of a scalpel blade. It was foundmicroscopically to consist almost exclusively of intestinalepithelium. Homogenization was carried out in cold025 M sucrose using a motor-driven teflon pestle(Sireica, New York) until the cells, as gauged by lightmicroscopy, had been adequately ruptured. Nitrogen wasdetermined by a microKjeldahl procedure (Prunty,McSwiney, and Hawkins, 1959).

BACTERIA Cultures of E. coli and Bacteroides isolatedfrom human ileal contents were harvested anaerobicallyon tryptone soya broth (Oxoid, London). The followingorganisms obtained from the Torry Research Institute,Aberdeen, Scotland, were also harvested as above:Streptococcus faecalis (775, strain Tissier), Clostridiumsporogenes (8053, strain MCA 3679), Klebsiellaaerogenes (418, type 1, strain 240), Proteus vulgaris(4175, strain Lehmann NCT 4175), Lactobacillis acid-ophilus (1723, strain Ruttger 4B, grown in milk medium),Staphylococcus aureus (6571, strain 3 R 7089).

Suspensions were made in 0-15 M NaCl, and aliquotsruptured under refrigeration using the Soniprobe type1130 (Dawe Instruments, Acton, London) at 4-8 ampsfor three one-minute periods.

ENZYME SOLUTIONS The enzyme standards used in thiswork were: trypsin ex bovine pancreas, 2 x crystallized,salt free; a-chymotrypsin ex bovine pancreas, 3 xcrystallized, salt free, both from Koch-Light Laboratories,Colnbrook, Bucks, England. They were dissolved in0.005 N HCl, 0.005 M with respect to CaCl2. We havefound it necessary to add crystalline bovine serumalbumin (British Drug Houses Ltd., Poole, Dorset,England) to the chymotrypsin standards in a final con-centration of 1% (w/v) to prevent loss of activity duringstorage at -20°C. These solutions were used in experi-ments on the behaviour of bovine enzymes. In theexperiments on recovery from intestinal instillation, theenzymes were dialysed for 48 hours against frequentchanges of distilled water at 4°C before being made upin 0.15 M NaCI. Protein was precipitated during dialysisbut did not affect the activity which was checked beforeand after administration to the patient.

RESULTS

EFFECT OF INTESTINAL SECRETIONS When solutionsof crystalline enzymes were incubated at 37°C fortwo hours with human gastric juice, virtually all the

trypsin and chymotrypsin activities were lost(Table I). One-third of the chymotrypsin and 15% ofthe trypsin failed to survive 15 minutes' incubationwith gastric juice; 0.1 N HCl was without effect ontrypsin, but inactivated 38% of the chymotrypsinover a two-hour period. Bile was without pronouncedeffect on either enzyme.

EFFECT OF AUTOINCUBATION When homogenates ofileostomy contents were incubated at 370C, therewas a steady decrease in chymotrypsin activity to74% after six hours (Table II). A more rapid fall intrypsin activity took place under these conditions,but this seemed to level off so that at four and sixhours after incubation about 37% of the originalactivity was lost.

TABLE IIEFFECT OF INCUBATION AT 370c ON TRYPSIN AND

CHYMOTRYPSIN ACTIVITIES OF HUMAN ILEALHOMOGENATES1

IncubationTime (hr)

246

Trypsin

83-2 ± 2-962-6 ± 2-162-9 ± 4.0

Chymotrypsin

91-3 i 3-483-1 + 2-974.0 ± 6.0

'Mean ± SE of four experiments. Data as percentage of zero timecontrol.

EFFECT OF INTESTINAL CELL PREPARATIONS Theaddition of crystalline enzymes to homogenates ofileal (three samples) and jejunal (three samples)mucosa in the proportion of 10 ,ug enzyme plus2 mg homogenate nitrogen per millilitre of mixturegave no immediate change in activity, indicating theabsence of specific inhibitors. When these mixtureswere incubated at 37°C for periods up to two hours,at the pH used for the enzyme determinations(pH 8-2, trypsin; pH 7-8, chymotrypsin), thedifferences never exceeded the reproducibility ofreplicate analyses (± 5%). Thiseliminates destructionby intracellular proteases and is in agreement withthe very low proteolytic activity at neutral andalkaline pH previously found in intestinal cellpreparations (Goldberg, McAllister, and Roy, 1967).

TABLE IEFFECT OF INTESTINAL SECRETIONS ON E

TRYPSIN AND CHYMOTRYPSIN1Trypsin

Gastric juiceN/10 HCIBile

70 ± 1-799.9 ± 0195-8 ± 1-7

'Mean i SE of four experiments. Equal volumes cand enzyme solutions (1 mg/ml) incubated at 37°CData as percentage of zero time control.

EFFECT OF NEOMYCIN The total daily output ofBOVINE trypsin and chymotrypsin was measured on three orfour consecutive days in patients with ileal drainage.Chymotrypsin Neomycin sulphate, in a dose of 2 g, qid, was then

given. After 24 hours, daily measurements of the11 i 0-7 output of trypsin and chymotrypsin were made for

6212 182 the same number of days as previously, while106.2 ±- 18-5neomycin therapy was continued. There was a fall of

of test secretion 5 to 10% in the chymotrypsin output in thefor two hours.homogenate and in the supernatant of the ileal

478



j.com/


ownloaded from

http://gut.bmj.com/

Fate of trypsin and chymotrypsin in the human small intestine

TABLE IIIAFFECT OF NEOMYCIN (8 G/DAY) ON OUTPUT OF TRYPSINAND CHYMOTRYPSIN IN SUBJECTS ON ILEAL DRAINAGE'

Trypsin Chymotrypsin

Homogenate Supernatant Homogenate Supernatant

Control 222 ± 56 143 ± 26 354 ± 63 173 + 40Test 116 28 80 17 321 61 165 ± 39

'Data as mg per 24 hours, mean ± SE for seven subjects. In four,test and control periods were four days; in three, test and controlperiods were three days.

drainage material after neomycin, but these changeswere insignificant (Table III). By contrast, neomycinlowered the trypsin output in homogenate andsupernatant by over 30 %. Analysis of the meanvalues for each subject before and after neomycinby the paired t test showed these changes to bestatistically significant (homogenate, to = 3-43;P<0.02; supernatant, to = 2-94; P<0-05). Thedistribution of each enzyme between the soluble andinsoluble phases in ileal material was unaltered byneomycin. This is best expressed by considering theoutput in the supernatant as a percentage of that ofthe whole homogenate. The mean values before andafter neomycin were trypsin 6444% and 68.9%;chymotrypsin 48.8% and 51.5 %.

EFFECT OF BACTERIA When mixtures were madecontaining in final concentration 20 jig of trypsin orchymotrypsin and 2 mg bacterial nitrogen permillilitre at a pH of 8.2 and 7-8 respectively, noactivity was lost after 30 min at 37°C. All organismswere tested twice as intact and twice as rupturedpreparations. No activity was lost when the enzymeswere added to the broths in which the organismswere grown. Trypsin-like or chymotrypsin-likeenzymes could not be detected in any of the organ-isms studied, either whole, ruptured, or extra-cellularly in the broth.

RECOVERY OF ENZYMES A series of experiments wasundertaken to test the recovery of enzymes instilledinto the intestine. Patients on ileal drainage hadtheir enzyme output determined over four con-secutive days. A radioopaque polythene tube of0-20 cm external diameter and 010 cm bore with abrass weight sutured to its tip was passed overnightinto the second or third part of the duodenum. Theposition of the tube was checked radiologically. Ifsatisfactory, a solution of bovine pancreatic trypsinand chymotrypsin cooled in ice was then admin-istered by tube. This took place over four hourswithout interference with the patient's dietaryregime. The position of the tube was checked

radiologically before withdrawal. The first twosubjects were given 100 mg of both trypsin andchymotrypsin dissolved in 250 ml 0.15 M NaCl.Patients 3 to 6 were given 200 mg of both enzymesin 450 ml 0 15 M NaCl and patients 7 to 9 receivedthe same quantity in 50 ml 0.15 M NaCl. Patients 7and 8 were in addition given neomycin sulphate in adose of 2 g, qid, during the basal and recoveryperiods (Table IV). In a further four subjects theexperiment had to be abandoned after completionof the control collection because of difficulties inobtaining a satisfactory position of the tube. Theileal drainage material was collected for 48 hoursafter the start of enzyme administration and theoutput of enzymes in the homogenate determined.

TABLE IVRECOVERY OF TRYPSIN AND CHYMOTRYPSIN FROM SUBJECTSON ILEAL DRAINAGE AFTER INTRADUODENAL INSTILLATION'Subject Trypsin Chymotrypsin Protocol

234567

+ 105+ 112+ 28- 318- 30- 210- 28

+ 90+ 131+ 58+ 116- 2+ 21- 48

8 - 27 + 233

9 + 72 + 156

100 mg in 250 ml100 mg in 250 ml200 mg in 450 ml200 mg in 450 ml200 mg in 450 ml200 mg in 450 ml200 mg in 50 mlNeomycin in test andcontrol periods200 mg in 50 mlNeomycin in test andcontrol periods200 mg in 50 ml

'Data give change in output for each subject in mg for the 48-hourperiod of test based upon four-day control period. All measurementson homogenate of ileal contents.

From the mean value obtained during the four-daybasal period the enzyme recovery was calculated asthe increment over the expected amount during the48-hour collection period. In those patients given100 mg of each enzyme recovery was complete. Thepatients given 200 mg of each enzyme failed toproduce such a satisfactory recovery, and showedconsiderable variation. This variance could not beassociated with any specific factors. Neither thevolume in which the enzymes were administered norprior sterilization of the gut with neomycin sulphateexercised any obvious effect upon recovery. Onlytwo generalizations can be drawn from the data:that the output of trypsin was decreased instead ofincreased, and that the recovery of added chy-motrypsin averaged 38 %.

MEASUREMENT OF ENZYME CONSTANTS Attempts weremade to compare the identity of the enzymes ofileal fluid with those of pancreatic origin. Aliquotsof a single sample of human pancreatic juice wereavailable for this purpose. When this material was

479



j.com/


ownloaded from

http://gut.bmj.com/


03r

02 t

E

X 04-a

_- ,

_< .- -,"' '

-100

TRYPSIN

Human

CHYMOTRYPSIN

-

X

x-

-20 20i (Moles /mI.)

FIG. 1. Lineweaver-Burk plot of Michtrypsin and chymotrypsin.

exhausted bovine pancreatic enzymes were used-> instead.

The relationship between substrate concentrationand reaction velocity was studied, and the data usedto calculate the Michaelis constant according tothe re-iprocal plot of Lineweaver and Burk (1934)

Human ileal * as shown in Figure 1. The value for human pan-n pancreatic X-X creatic trypsin (1 30 x 10-3) compared well with

that of ileal fluid (12.5 x 10-3), the differences beingwithin the limits of experimental error. The valuefor human pancreatic chymotrypsin (8.3 x 10-3)was substantially different from that of ileal fluid(23.8 x 10-3).The relationship between temperature and reaction

velocity was studied in order to calculate the energyof activation for the enzymes according to theArrhenius plot (Fig. 2). The values for bovinepancreatic and ileal fluid trypsin were 13,500 and

100 180 12,800 cals per mole respectively, a difference of thismagnitude being within the limits of experimental

aelis equation for error. The value for bovine pancreatic chymotrypsinfor (8,500 cals per mole) was substantially different from

that of ileal fluid (13,200 cals per mole).

10 TRYPSIN Bovine pancreatic 3--oHuman ileal *_-

0.05-.

0~~~~~~~

-0.5

-10,:t i1 0 CHYMOTRYPSINIcC L....

4.0-

3-0

c 20-

10

0O

x--x Human Pancreatic*-- HumsnIleal

6:0 70 8-0pH

90 10i0

I X 103TABS

FIG. 2. Arrhenius plot showing relationship betweentemperature and velocity for trypsin and chymotrypsin.

FIG. 3. The pH-activity curves for trypsin.

The pH activity curves were studied in theuniversal buffer system of Davies (1959). Excellentagreement was obtained for trypsin of pancreatic andileal fluid (Fig. 3). Reproducible curves for chy-motrypsin could not be obtained. Several experi-ments were conducted with pancreatic and ilealfluid, but spontaneous decomposition of the sub-strate at pH values outside the range 6.5 to 8-0 led

L-

9

480



j.com/


ownloaded from

http://gut.bmj.com/

Fate of trypsin and chymotrypsin in the human small intestine

to very high blanks and uncertainty concerning thepossibility of subsidiary peaks. Three samples ofileal fluid showed the main peak of activity at threedifferent pH values, 6.5 70, and 9.0 comparedwith a value of 7-5 for pancreatic chymotrypsin.

DISCUSSION

The inactivation of pancreatic enzymes by humangastric juice agrees with previous studies on trypsinand lipase (Heizer, Cleaveland, and Iber, 1965). Thestability of trypsin in acid and bile has already beenrezorded (Elmslie, 1965). Our results show rapidinactivation of chymotrypsin by gastric juice, partialinactivation by acid, and stability in bile. The findingthat the major part of trypsin and chymotrypsin isstill active after six hours at 37°C is at variance withprevious reports claiming rapid inactivation overperiods as short as 30 min (Lundh, 1958; Wohlman,Kabacoff, and Avakian, 1962). Previous workinvolved intestinal juice obtained by intubation.This would exclude cell debris which, by binding ofthe enzymes at a site other than the active centre,contribute to the preservation of the enzymes in anactive configuration by a form of particle stabil-ization (Goldberg et al, 1968a).The autoincubation experiments give some indi-

cation of the likely contribution of bacterial andintestinal cell proteolysis to enzyme degradation.More direct experiments have shown these to be oflittle importance. Our failure to demonstrate anychange in activity when the enzymes were incubatedwith preparations of fresh small intestinal epitheliumis of considerable interest. Human small intestinalmucosa may be unique in being able to bind trypsinand chymotrypsin without inactivation, and con-trasts with the mucosa of the colon which inactivatesthe enzymes and shows no capacity to bind them inthe fresh state (Goldberg et al, 1968a). Theseobservations may prove helpful in contributing to anunderstanding of the processes involved in proteindigestion and cell protection.The effect of neomycin on enzyme output is

surprising in view of previous claims that sterilizingthe gut in the rat (Borgstr6m et al, 1959) and in man(Haverback et al, 1963) increases the faecal outputof pancreatic enzymes. Neomycin in final con-centrations up to 50 mg/ml had no effect upon theenzyme activity of ileal contents in vitro. Thereduction in trypsin output which followed admin-istration of the drug could be due to the followingfactors:

1 Removal of bacteria which produce trypsin-likeenzymes, which is unlikely, since Grossman (1962)has shown that bacteria do not produce suchenzymes in the dog, and we have been unable to

detect trypsin or chymotrypsin activity in any strainexamined so far.

2 Liberation of proteolytic enzymes from deadbacteria, which has been eliminated by directmeasurement of proteolytic activity (Goldberg,McAllister, and Roy, 1968b).

3 Changes in the intestinal cell lining, which areknown to occur as a consequence of neomycintherapy (Jacobson, Prior, and Faloon, 1960). It isconceivable that the altered epithelium has a greateravidity for trypsin.

4 Direct effect upon the pancreas: it is possiblethat small quantities of the drug following systemicabsorption might have such an effect. This wouldadd a further explanation for the malabsorptionassociated with the use of the drug (Jacobson,Chodos, and Faloon, 1960; Jacobson and Faloon,1961). The fact that trypsin and chymotrypsin arenot affected in parallel need not preclude a directeffect upon the pancreas, since recent dietaryexperiments have shown that the rates of synthesisof individual pancreatic enzymes respond differently,both in degree and in direction, to various stimuli(Snook, 1965; Reboud, Marchis-Mouren, Pasero,Cozzone, and Desnuelle, 1966). Evidence is availablewhich points to a deficiency of lipase induced byneomycin (Rogers, Vloedman, Bloom, and Kalser,1966). It has also been established in bacterialsystems that neomycin causes amino-acid sub-stitutions in peptide chains leading to impairedsynthesis of certain enzymes (Gorini, 1966).

In view of our findings with neomycin, theattempts to characterize the enzymes in ileal fluidare relevant. These studies could not be carried outexclusively using human pancreatic enzymes. Tothat extent, a cautious interpretation of experimentswith enzymes of bovine origin is indicated. It maybe seen (Figs. 1 to 3) that the enzyme constants ofileal fluid trypsin agree with those for pancreatictrypsin, whereas differences with chymotrypsin areapparent. This latter anomaly may be explained byconversion of chymotrypsin to a variety ofmolecular forms during contact with high trypsinconcentration in the course of intestinal transit(Desnuelle, 1960), and several peaks of chymotrypsinhave been identified by Sephadex chromatographyof rat intestinal contents (Snook and Meyer, 1964).The process of activation used for the humanpancreatic juice in this work produces oc-chy-motrypsin only (Rovery, Poilroux, Yoshida, andDesnuelle, 1957), and a-chymotrypsin of bovineorigin was used in other experiments. Thus thedifferences noted do not signify that ileal fluidchymotrypsin is not of pancreatic origin.So far we have defined gastric juice and auto-

degradation as the main factors leading to loss of

481



j.com/


ownloaded from

http://gut.bmj.com/


enzyme activity in the human small intestine. Sincepepsin can only function at an acid pH it is unlikelythat gastric juice would exercise an effect in theabsence of regurgitation of intestinal contents.Assuming a transit time of four to six hours from theduodenum to the terminal ileum, an approximationthat corresponds with data obtained using markers inour subjects, one might expect that at least 60% oftrypsin and chymotrypsin would reach the terminalileum intact. The results of the intubation experi-ments are therefore surprising.

It is difficult to reconcile the complete recoveryobtained in two subjects when 100 mg of eachenzyme was administered with the poor recoveryfound in seven subjects given 200 mg. The dailyoutput before administration of the enzyme appearedsufficiently consistent to allow application of thesevalues for the calculation of enzyme recovery. Wehave been unable to find any factors such as faultypositioning of the tube or regurgitation into thestomach which might explain this poor recovery. Inview of claims that pancreatic enzymes may beabsorbed into the blood stream (Kabacoff, Wholman,Umhey, and Avakian, 1963), we have tested for thispossibility in two patients by sampling blood at fourhours and eight hours after the start of admin-istration, but no increment over the zero time controlcould be detected in the trypsin or chymotrypsinactivity of whole blood and serum.

This leaves a number of alternative explanationsfor the poor recoveries: (1) The bulk of theadministered enzymes were destroyed. This requiresdismissal of the results in the first two patients asdue to chance. (2) Added enzymes are adsorbed tomucosal cells in situ. (3) The operation of a feedbackmechanism controlling the level of intestinalenzymes. This suggestion has attractive implications.It ts conceivable that some factor which is stimulatedby pancreatic enzymes operates to diminish pan-creatic flow in a manner analogous to the inhibitionof gastric acid by acid stimulation of the duodenum.The daily output of trypsin and chymotrypsin inthese subjects averaged 208 mg and 320 mg respec-tively. Administration of 200 mg would double thedaily trypsin output, but since this was given overfour hours there could be a 12-fold increase inenzyme concentration. It has been shown that feed-ing of trypsin inhibitor to the rat and chick resultsin pancreatic hypertrophy and increased enzymeproduction and secretion, especially of trypsin(Chernick, Lepkovsky, and Chaikoff, 1948; Lyman,1957; Booth, Robbins, Ribelin, and De Eds, 1960;Haines and Lyman, 1961; Khayambashi andLyman, 1966). The fact that this response is seenafter a single feed of inhibitor (Lyman andLepkovsky, 1957; Lyman, Wilcox, and Monsen,

1962) would fit in with the concept of a feedbackmechanism linking the rate of pancreatic secretionwith intestinal trypsin concentration inversely.We have compared the output from our subjects

with recent indirect estimates of pancreatic secretion.The consistently higher output of chymotrypsinaccords with the fact that the human pancreas hasa higher content of chymotrypsinogen than oftrypsinogen (Figarella, 1966). Worning and Mullertz(1966) have reported the enzyme concentrations ofhuman duodenal contents during stimulation by atest meal in healthy subjects. On the basis of a meantrypsin concentration which we estimate at 200,ug/ml and a mean volume of 188 ml/90 min, atheoretical daily output may be about 700 mg.

Petersen, Myren, and Foss (.1967) have studied theprotein content of pancreatic juice. Applying theirmean value of 100 mg/100 ml, and resting volumeof 26 ml/20 min yields a figure of 1.9 g proteinsecreted daily by the human pancreas. Cook,Lennard-Jones, Sherif, and Wiggins (1967) havemeasured trypsin activity in duodenal aspiratesfrom healthy subjects after stimulation by a test meal,and report a mean value of 5 m-equiv/min during atwo-hour period. We estimate this to represent anoutput of approximately 300 mg/day. AlthoughCook et al (1967) state their recovery to varybetween 15 and 25 %, the rates measured duringtheir studies would represent maximal pancreaticoutput over short periods, much lower values beingapplicable to basal conditions.Although we have been unable to reach a firm

conclusion on the question that prompted theseinvestigations, it is to be hoped that publication ofthe present data will stimulate further research inthis field. There is an urgent need for reliableinformation on the daily enzyme output of thehuman pancreas. Studies on the recovery of labelledenzymes would also assist in circumventing theuncertainties of the present techniques.

SUMMARY

The fate of trypsin and chymotrypsin in the humansmall intestine has been studied using in-vitromethods to assess the interaction of gastrointestinalsecretions and cells upon bovine pancreatic enzymes,and in-vivo methods to assess bacterial degradationand recovery of enzymes administered by intubationto subjects on ileal drainage under controlled dietaryand metabolic conditions.

Gastric juice produced rapid loss of activity ofboth enzymes which was almost complete by twohours. Bile was without effect on either enzyme, but0'1 N HCI inactivated chymotrypsin by 38% aftertwo hours. Autoincubation of homogenates of

482



j.com/


ownloaded from

http://gut.bmj.com/

Fate of trypsin and chymotrypsin in the human small intestine 483

ileostomy material brought about a decrease of 37%in trypsin and 26% in chymotrypsin after six hours,but homogenates of ileal and jejunal epitheliumwere without effect on bovine pancreatic enzymes.The output of trypsin from subjects on ileal

drainage was significantly reduced by neomycinsulphate but chymotrypsin output was unaffected.Calculation of Michaelis constants, activation energy,andpH optima gave good agreement between trypsinof pancreatic and ileal sources, but discrepancieswere noted with chymotrypsin. Added bovine pan-creatic enzymes were completely recovered in twosubjects given 100 mg ofeach by duodenal intubation.When 200 mg of each was given to seven subjectsrecovery of chymotrypsin averaged 38 %, and trypsinoutput appeared to be suppressed. Absorption ofenzymes into the blood stream could not be detectedin two subjects. These results raise the possibilitythat neomycin may alter pancreatic secretion and arecompatible with feedback inhibition of the pancreasby intestinal trypsin. The relationship between pan-creatic enzymes and ileal output requires directstudy of pancreatic secretions.

We are indebted to Professor A. W. Kay for advice andguidance during this work.

REFERENCES

Booth, A. N., Robbins, D. J., Ribelin, W. E., and De Eds, F. (1960)Effect of raw soybean meal and amino acids on pancreatichypertrophy in rats. Proc. Soc. exp. Biol. (N. Y.), 104, 681-683.

Borgstrom, B., Dahlqvist, A., Gustafsson, B. E., Lundh, G., andMalmquist, J. (1959). Trypsin, invertase and amylase contentof feces of germfree rats. Ibid., 102, 154-155.

, Lundh, G., and Sjovall, J. (1957). Studies of intestinaldigestion and absorption in the human. J. clin. Invest., 36,1521-1536.

Chernick, S. S., Lepkovsky, S., and Chaikoff, I. L. (1948). A dietaryfactor regulating the enzyme content of the pancreas: changesinduced in size and proteolytic activity of the chick pancreasby the ingestion of raw soybean meal. Amer. J. Physiol.,155, 33-41.

Cook, H. B., Lennard-Jones, J. E., Sherif, S. M., and Wiggins, H. S.(1967). Measurement of tryptic activity in intestinal juice as adiagnostic test of pancreatic disease. Gut, 8, 408-414.

Davies, M. T. (1959). A universal buffer solution for use in ultra-violetspectrophotometry. Analyst, 84, 248-251.

Desnuelle, P. (1960). Chymotrypsin. In The Enzymes, 2nd ed., editedby P. D. Boyer, H. Lardy, and K. Myrback, vol. 4, pp. 93-118.Academic Press, New York.

Elmslie, R. (1965). The effect of bile on the activation of trypsinogenand the activity of trypsin in pancreatic juice. Brit. J. Surg.,52, 465-470.

Figarella, C. (1966). Composition et stabilite de l'equipementenzymatique du pancreas de 1'homme et de divers animaux.Bull. Soc. Chim. biol. (Paris), 48, 97-115.

Goldberg, D. M., Campbell, R., and Roy, A. D. (1968a). Binding oftrypsin and chymotrypsin by human intestinal mucosa.Biochim. biophys. Acta (Amst.). 671, 613-615.McAllister, R. A., and Roy, A. D. (1967). The proteolvticactivity of human intestinal contents. Proc. Ass. clin. Biochem.,4, 202-205.

* , ,- (1968b). Studies on human intestinal proteolysis.Scand. J. Gastroent., 3, 193-201.

Gorini, L. (1966). Antibiotics and the genetic code. Sci. Amer. 214,no. 4, 102-109.

Grossman, M. I. (1962). Fecal enzymes of dogs with pancreaticexclusion. Proc. Soc. exp. Biol. (N. Y.), 110, 41-42.

Haines, P. C., and Lyman, R. L. (1961). Relationship of pancreaticenzyme secretion to growth inhibition in rats fed soybeantrypsin inhibitor. J. Nutr., 74, 445-452.

Haverback, B. J., Dyce, B., Bundy, H., and Edmondson, H. A. (1960).Trypsin, trypsinogen and trypsin inhibitor in human pancreaticjuice; mechanism for pancreatitis associated with hyper-parathyroidism. Amer. J. Med., 29, 424-433.-, Gutentag, P. J., and Montgomery, D. W. (1963). Measure-ment of trypsin and chymotrypsin in stool. A diagnostic testfor pancreatic exocrine insufficiency. Gastroenterology, 44,588-597.

Heizer, W. D., Cleaveland, C. R., and Iber, F. L. (1965). Gastricinactivation of pancreatic supplements. Bull. Johns Hopk.Hosp., 116, 261-270.

Jacobson, E. D., Chodos, R. B., and Faloon, W. W. (1960). Anexperimental malabsorption syndrome induced by neomycin.Amer. J. Med., 28, 524-533.

, and Faloon, W. W. (1961). Malabsorptive effects of neomycin incommonly used doses. J. Amer. med. Ass., 175, 187-190.

-, Prior, J. T., and Faloon, W. W. (1960). Malabsorptive syndromeinduced by neomycin: morphologic alterations in the jejunalmucosa. J. Lab. clin. Med., 56, 245-250.

Kabacoff, B. L., Wohlman, A., Umhey, M., and Avakian, S. (1963).Absorption of chymotrypsin from the intestinal tract. Nature(Lond.), 199, 815.

Khayambashi, H., and Lyman, R. L. (1966). Growth depression andpancreatic and intestinal changes in rats force-fed amino aciddiets containing soybean trypsin inhibitor. J. Nutr., 89, 455-464.

Lineweaver, H., and Burk, D. (1934). The determination of enzymedissociation constants. J. Amer. chem. Soc., 56, 658-666.

Lundh, G. (1957). Determination of trypsin and chymotrypsin inhuman intestinal content. Scand. J. clin. Lab. Invest., 9,229-232.

(1958). Intestinal digestion and absorption after gastrectomy.Acta. chir. scand., suppl. 231.

Lyman, R. L. (1957). The effect of raw soybean meal and trypsininhibitor diets on the intestinal and pancreatic nitrogen in therat. J. Nutr., 62, 285-294.and Lepkovsky, S. (1957). The effect of raw soybean meal andtrypsin inhibitor diets on pancreatic enzyme secretion in therat. Ibid., 62, 269-284.Wilcox, S. S., and Monsen, E. R. (1962). Pancreatic enzymesecretion produced in the rat by trypsin inhibitors. Amer. J.Physiol., 202, 1077-1082.

Pelot, D., and Grossman, M. I. (1962). Distribution and fate ofpancreatic enzymes in small intestine of the rat. Ibid., 202,285-288.

Petersen, H., Myren, J., and Foss, 0. P. (1967). Total nitrogen contentof human duodenal juice before and after intravenous injectionof secretin. Scand. J. Gastroent., 2, 1-6.

Prunty, F. T. G., McSwiney, R. R., and Hawkins, J. B. (1959).A Laboratory Manual of Chemical Pathology, p. 165. PergamonPress, Oxford.

Reboud, J. P., Marchis-Mouren, G., Pasero, L., Cozzone, A., andDesnuelle, P. (1966). Adaptation of rate of biosynthesis ofpancreatic amylase and chymotrypsinogen to starchrich orprotein-rich diets. Biochim. biophys. Acta. (Amst.), 117, 351-367.

Rogers, A. I., Vloedman, D. A., Bloom, E. C., and Kalser, M. H.(1966). Neomycin-induced steatorrhea. A preliminary reporton the in vivo hydrolysis of a long-chain unsaturated fat.J. Amer. med. Ass., 197, 185-190.

Rovery, M., Poilroux, M., Yoshida, A., and Desnuelle, P. (1957).Sur Ia degradation du chymotrypsinogene par la chymotrypsine.Biochim. biophys. Acta. (Amst.), 23, 608-620.

Roy, A. D., Campbell, R., and Goldberg, D. M. (1967). Effect of dieton the trypsin and chymotrypsin output in the stools of patientswith an ileostomy. Gastroenterology, 53, 584-589.

Snook, J. T. (1965). Dietary regulation of pancreatic enzyme synthesis.secretion and inactivation in the rat. J. Nutr., 87, 297-305.and Meyer, J. H. (1964). Effect of diet and digestive processes onproteolytic enzymes. Ibid., 83, 94-102.

Wohlman, A., Kabacoff, B. L., and Avakian, S. (1962). Comparativestability of trypsin and chymotrypsin in intestinal juice. Proc.Soc. exp. Biol. (N. Y.), 109, 26-28.

Worning, H., and Mullertz, S. (1966). pH and pancreatic enzymes inthe human duodenum during digestion of a standard meal.Scand. J. Gastroent., 1, 268-283.



j.com/


ownloaded from

http://gut.bmj.com/

fate - gut · sporogenes (8053, strain mca 3679), klebsiella aerogenes (418, type 1, strain 240),...

Documents