in human duodenal aspirate'

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1 Gut, 197, 11, The interreltionships of pncretic enymes in humn duodenl spirte' D M GOLDBERG ND K G WORMSLEY From the Royl Hospitl nd Royl Infirmry, Sheffield SMMRY The interreltionships of proteolytic enymes nd mylse hve been studied in the duodenl spirte obtined from subjects with norml nd bnorml pncretic function during stimultion with secretin nd pncreoymin While the reltionship of trypsin to chymotrypsin ws independent of stimulus nd presence of pncretic disese the rtio of proteolytic enymes to mylse rose when the degree of stimultion of the pncres ws incresed Ptients with recent cute pncretitis nd- with chronic pncretitis tended to hve more severe impirment of secretion of proteolytic enymes thn of mylse In routine tests of pncretic function both proteolytic nd non-proteolytic enymes should be mesured, both becuse n bnorml rtio my be of dignostic significnce nd becuse the two different groups of enymes provide mutul checks of the secretory cpcity of pncretic enymes Since the secretion of digestive enymes is function of the pncres, estimtion of enyme secretory cpcity provides n importnt nd specific index of the stte of the pncretic secretory cells However, there is no unnimity bout the type nd number of pncretic enymes which must be estimted in order to provide stisfctory criterion of the secretory cpcity of pncretic enymes In the present study, we hve investigted the interreltionships mong some of the pncretic enymes in the duodenl spirte obtined during tests of pncretic function in order to determine the most stisfctory mesure for routine investigtionl use Methods The subjects of the present study comprised 16 helthy norml individuls (eight medicl students nd eight ptients without limentry complints, TPlese ddress ny requests for reprints to K G Wormsley, Clinicl Investigtion nit, Mryfield Hospitl, Dundee DD4 7TL Received for publiction 13 pril 197 with inguinl herni or vricose veins); 2 ptients with chronic duodenl ulcer; 13 ptients who were tested either between three nd six weeks or more thn four months fter ttcks of cute pncretitis; 1 ptients with chronic pncretitis; seven ptients with nicteric pncreticcrcinom ll subjects were tking norml diets t the time of their tests In ech subject, one or more enymes were estimted in duplicte or by more thn one method Techniques of intubtion hve been described previously (Wormsley, 1968) mylse ws estimted using the methods of Norby (Lgerlof, 1942) nd of Bernfeld (Bernfeld nd Studer- Pech, 1947); proteolytic ctivity by the method of Gowenlock (1953); trypsin nd chymotrypsin by the titrimetric method of Hverbck, Dyce, Gutentg, nd Montgomery (1963) using tosyl rginine methyl ester s substrte for trypsin, nd cetyl tyrosine ethyl ester s substrte for chymotrypsin Enyme estimtions were crried out within two to three dys ofthetestonduodenl spirte which hd been diluted with n equl volume of glycerol nd kept froen (-2 C) The stbility of rndom smples ws tested over the course of one month Ech subject hd two tests, during which pn- Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

2 86 D M Goldberg nd K G Wormsley cretic secretion ws stimulted by continuous intrvenous infusion of secretin (25 or 2 C/kg-hour), or combintion of secretin (25 C/kg-hour) plus pncreoymin (16 CHR /kg-hour) The hormones were obtined from the GIH Lbortory, Krolinsk Institute, Stockholm, Sweden Ech test lsted 5 minutes, excepting five studies which were continued for two hours Two to three weeks were llowed to elpse between tests The vlues of enyme ctivity hve been expressed s concentrtions of individul 1- minute smples or s men concentrtions of pooled smples comprising the duodenl spirte secreted during the whole test or during the finl 3 minutes of ech test The significnce of the differences in the distribution of the rtios of the enymes to ech other hs been ssessed by mens of the Mnn-Whitney '' test (Siegel, 1956) Results REPRODCIBILITY The reproducibility of ll methods ws very good (Figs 1 nd 2) There ws no decrese in the ctivity of the mesured enymes during the course of one month The errors in the titrimetric method of estimting trypsin nd chymotrypsin hve been described previously (Goldberg, Cmpbell, nd Roy, 1969) 1- u 3 C, o W > 1' E u E : n *u * -~~~~ -m TRYPSIN CONCENTRTION (/ML) TEST 1 Fig 1 Reproducibility of the mesurement of proteolytic ctivity (Gowenlock method) Ech point represents duplicte estimtions on one smple of duodenl spirte (denoted by 'Test 1' nd 'Test 2' respectively) m CORRELTION BETWEEN THE DIFFERENT METHODS OF MESREMENT The correltion between the two methods of mesuring mylse ws excellent (Fig 3) greement between totl proteolytic ctivity (Gowenlock method) nd trypsin concentrtion (Hverbck method) ws similrly good (Fig 4) CORRELTION BETWEEN TRYPSIN ND CHYMOTRYPSIN The correltion between trypsin (Hverbck et l, 1963) nd chymotrypsin ws excellent nd independent of stimulnt or the condition of the pncres (Fig 5) The rtio of trypsin to chymotrypsin rnged from 15 to 3 in different subjects with norml pncres, but not by more thn 5 within this rnge in the sme individul under different conditions of stimultion or during duplicte tests lthough the rnge of rtios ws considerbly greter in ptients with pncretic disese (8 to 46) the difference from norml ws not significnt (p> 3) OTPTS OF PROTEOLYTIC ENZYMES ND OF MYLSE Ptients with pncretic disese secreted less proteolytic enymes (Gowenlock method) thn norml (Fig 6) except some months fter n cute ttck of pncretitis, when the secretion of proteolytic enymes hd reverted within norml limits (Tble nd Fig 6) The pttern of -1 W 9 > 8 X Z 4 3 ; 2 > MYLSE CONCENTRTION mm/ml TEST 2 Fig 2 Reproducibility of the mesurement of mylse ctivity by the method of Bernfeld (significnce ofpoints s in Figure 1) Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

3 The interreltionships ofpncretic enymes in humn duodenl spirte * * * o * 5 9 E MYLSE CONCENTRTION (BERNFELD METHOD)(mM/ML) Fig 3 Comprison of ctivity of mylse by the methods of Norby nd Bernfeld Ech point denotes the two vlues of ctivity of one smple of duodenl spirte N 3 o 2 E I_ co, W 1 X W o' 1 Z:D Z 8 9-,v 6-4 W u 2- * * * *o 1 : ' ' qe - %e - ton W * ~~ 3 m 1 o * Secretin + Pncreoymin o * Secretin 25 C/Kg-Hour Secretin 2 C/Kg-Hour m: - i 1 2 i TRYPSIN CONCENTRTION Fig 4 Comprison of the proteolytic ctivity (Gowenlock method) nd tryptic ctivity (ph stt method of Hverbck) Ech point denotes the two vlues of ctivity of one smple ofduodenl spirte ', L o tto L - d CHYMOTRYPSIN CONCENTRTION(pg/ML) Fig 5 Correltion between concentrtions of trypsin nd chymotrypsin Ech point represents the vlues of one smple of duodenl spirte N = spirte from subjects with norml pncres; = spirte from ptients with bnorml pncres Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

4 862 D M Goldberg nd K G Wormsley 6 > > 3 cl 2 P CP PC cl *: is > 1 5 m N P CP PC Fig 6 Outputs ofproteolytic ctivity (trypsin nd mylse) during SO-minute infusions of secretin plus pncreoymin N = norml nd duodenl ulcer subjects; P = cute pncretitis; CP = chronic pncretitis; PC = pncretic crcinom The open symbols represent results from ptients tested within six weeks ofn cute ttck; the closed symbols, tests more thn four months fter n ttck of cute pncretitis,i 4-3 1o 2 O 4 %o ;4 c 2 W ~ioo t- 4 T p N 13 P 1 : I:! r I T T CP Fig 7 Concentrtions nd outputs ofproteolytic ctivity (trypsin nd mylse) during the first 1 minutes of tests depicted in Figure 6 T = trypsin; = mylse PC 1- B: 2C ; ::5 * LI T I K' * 8 Cli N P CP 5 : m L S 3 PC T TTT S S Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

5 863 the concentrtion or output of proteolytic enymes (Figs 8 nd 9) The rtio ws lso dependent on the type of being lowest with the low dose rtes of secretin, significntly greter (P < 1) when pncreoymin ws dded, nd highest (p< 1) with the high dose rtes of secretin (Figs 8, 9, nd 1) The rtio of proteolytic enymes to mylse did not chnge during two-hour infusions of secretin (Fig 11) The rtio of proteolytic ctivity to mylse ws not different (p> 4) between norml subjects nd ptients with duodenl ulcer, nd these two groups hve therefore been tken together for the purpose of cl- The interreltionships ofpncretic enymes in humn duodenl spirte Ptient Trypsin1 mylse* Rtio of Trypsin Trypsin' mylse' Rtio of Trypsin (K/5 min) (MS5 min) to mylse (K/S min) (MISC min) to mylse * Tble Seril responses ofptients fter ttcks ofcute pncretitis (-25 C/kg-hour secretin + 16 CHR /kg-hour pncreoymin) 'Estimted by the method of Gowenlock (1953) 'Estimted by the method of Bernfeld (Bernfeld nd Studer-PNch, 1947) norml vlues of mylse output (Bernfeld method) showed distribution which ws skew towrds the lower end of the rnge, so tht there ws some overlp between the mylse of individuls outputs of norml subjects nd of ptients with pncretic disese (Fig 6) There ws considerble overlp between the outputs of proteolytic enymes nd of mylse during the first 1 minutes of the tests in norml subjects nd in ptients incresed with n increse in with pncretic disese (Fig 7) but the differences were significnt (p < O1) stimulus, CORRELTION BETWEEN PROTEOLYTIC CTIVITY ND MYLSE There ws no simple reltionship between proteolytic ctivity (Gowenlock method) nd the N Secretin + Pncreoymin Secretin 25 C/KG-Hour Secretiii 2C/KG-Hour cultion nd illustrtion 3 I F- o 2~ O o Discussion no JDono 4 * S 1-1~ 2 o It RTIO OF TRYPSIN TO MYLSE Fig 8 Reltionship between the rtio ofproteolytic ctivity (trypsin to mylse) in duodenl spirte nd the concentrtion ofproteolytic enymes (symbols s in Figure 5) concentrtion of mylse(bernfeld method) in the duodenl contents obtined during different conditions of stimultion nd in different groups For the purpose of presenttion, the results from ech individul hve therefore been expressed s the rtio of proteolytic ctivity to the concentrtion of mylse during ech test The rtio of proteolytic ctivity to mylse The bnorml rtio of proteolytic ctivity to mylse of ptients fter n ttck of cute pncretitis returned to norml within six months, when enyme secretory cpcity reverted to norml (Tble) The rtio of proteolytic ctivity to mylse tended to be low in ptients with cute nd chronic pncretitis nd pncretic crcinom (Fig 1) The results of the present study hve shown tht the techniques used for estimting the ctivity of the proteolytic enymes nd of mylse give reproducible results The reltionship of the two proteolytic enymes (trypsin nd chymotrypsin) to ech other ws not dependent on the mgnitude of the enyme output, type of secretory stimulus, or pncretic disese Good correltion between trypsin nd chymotrypsin in duodenl spirte hd been recorded previously (Zieve, Silvis, Mulford, nd Blckwood, 1966; Ribet, Pscl, nd Vysse, 1968) under experimentl conditions which differed from those used in the present study, lthough Rothmn (1967) hd noted tht pncreoymin stimulted the secretion Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

6 864 ) 'O ) E H 1: 4 D M Goldberg nd K G Wormsley '7t 6 6 t -5 OW 4 Z 3 > WHOLE TEST (5 MINTES) o o * O o 8O * *- 1d ** C C E ^ Fig 9 Reltionship between the rtio of proteolytic ctivity (trypsin to mylse) in duodenl spirte nd the output ofproteolytic enymes in response to S-minute infusions or during the finl 3 minutes of ech infusion N nd s in Fig 5 S = secretin 25 or 2- C/kg-hour; S+P = secretin (25 C per/kg-hour) + pncreoymin (16 CHR /kg-hour) SECRETIN+PNCREOZYMIN N P CP PC t 6t ql Fig 1 Rnge of rtios ofproteolytic ctivity (trypsin to mylse) in helth nd disese Ech point represents the result from one subject in response to the SO-minute infusion indicted t the top of ech of the three columns (symbols s in Figure 6) i d FINL 3 MINTES CO& c o o * O so * S S RTIO OF TRYPSIN TO MYLSE SECRETIN 25 C/KG-HOR N P CP PC So 5 :o 8 * S,P N o S 25 S SECRETIN 2 C/KG-HOR I N P CP PC I prl S i Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

7 865 The interreltionships ofpncretic enymes in humn duodenl spirte o LLQ-<: 91,,8 7 C4 u Secretini 2 C/Kg -Hour , 1 1 1' 1 MINTE PERIODS Fig 1 1 Rtio ofproteolytic ctivity (trypsin to mylse) during two-hour infusions of secretin (-25 C/kg-hour in three subjects nd 2- C/kghour in two subjects) Ech point represents the vlue of the rtio in the pproprite 1-minute smple of duodenl spirte of trypsin, without prllel secretion of chymotrypsin, in n experimentl preprtion of isolted pncres Most of the controversy bout the interreltionships of humn pncretic enymes hs concerned the rtio of proteolytic enymes to mylse (nd to lipse) Lgerlof (1942) noted liner reltionship between the concentrtion of trypsin nd mylse fter injection of secretin, confirming Bbkin's hypothesis tht pncretic enymes were secreted 'in prllel' 'Prllel' secretion of pncretic enymes ws lso reported in response to injections of secretin (Burton, Evns, Hrper, Howt, Oleesky, Scott, nd Vrley, 196) nd of pncreoymin (Burton et l, 196; Creutfeldt, 1964; Rick, 1965; Srles, Figrell, Preelin, nd Souville, 1966; mmnn, Tgwercher, Kshiwgi, nd Rosenmund, 1968) On the other hnd, Guth, Komrov, Shy, nd Style (1956) found sttisticlly significnt dy-to-dy devitions from 'prllelism' in the enyme response to food in dogs, while Worning nd Mullert (1966) described considerble vrition between the concentrtions of different enymes in response to test mels in mn, minly ttributble to vrition between individuls Srles et l (1966) noted tht lipse disppered from humn duodenl contents during prolonged infusions of secretin, without chnge in the ctivities of the other enymes, while shm feeding elicited different type of dissocition, with mrked stimultion of the secretion of lipse, less of trypsin, nd virtully no chnge in the output of mylse further type of dissocition ws described by Ribet et l (1968), who noted n increse in the rtio of trypsin to lipse when pncreoymin ws dded to n infusion of secretin, similr to the increse in the rtio of proteolytic enymes to mylse in response to pncreoymin found in the present study The secretion of pncretic enymes hs lso been found to devite from 'prllelism' in ptients with pncretic disese Lgerlof (1939) suggested tht in ptients with cute pncretitis the concentrtion of mylse might be bnormlly low while trypsin remined within defined norml limits Similr findings were reported in ptients with other types of pncretic disese (Burton et l, 196) nd it ws suggested tht mylse secretion ws the most stisfctory index of pncretic disese, since trypsin secretion could be norml in ptients with pncretic disorders On the other hnd, Hershfield, Lind, nd Hildes (1968) found tht isolted, defective secretion of mylse in response to injections of secretin nd pncreoymin could not be considered relible indiction of disese of the pncres Similrly, Gibbs (195) showed tht the secretion of mylse ws sometimes grossly defective in norml infnts, whose secretion of trypsin ws norml, while Choi, Goldstein, Wirts, nd Menduke (1967) recommended the mesurement of trypsin becuse trypsin secretion could be bnorml when mylse secretion remined norml in ptients with pncretic disese In much erlier study, Dimond, Siegel, Gll, nd Krlen (1939) hd lredy suggested tht the prctice of estimting only one enyme ws not n dequte mesure of totl pncretic cpcity to secrete enymes, becuse they found frequent dissocition between trypsin nd mylse secretion The present study hs shown tht the reltionship between mylse nd proteolytic enymes is not constnt but cn chnge mrkedly both in pncretic disese nd if the stimulus to secretion is chnged by the ddition of pncreoymin or by incresing the dose rte of secretin It is possible tht the chnge in the rtios of the enymes in pncretic disese is relted, in prt, to chnges in food intke or bsorption nd to ltertion in the reltive vilbility of the bsic constituents of food mterils, since Grossmn, Greengrd, nd Ivy (1942) nd Ben bdeljlil nd Desnuelle (1964) hve demonstrted tht selective chnges in the secretion of pncretic enymes could be brought bout by experimentl chnges in the composition of the diet Chnges in nutrition could not explin the trnsient chnges in the rtios of the pncretic enymes fter ttcks of cute pncretitis, nor the chnges elicited by different pncretic stimulnts The findings of the present study suggest tht the output of proteolytic enymes provides better index of enyme secretory cpcity thn does output of mylse, since the norml pncres responds to incresed stimultion with greter increse in the secretion of proteolytic enymes thn of mylse In ddition, the secretion of proteolytic enymes in response to pncretic Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright

8 866 D M Goldberg nd K G Wormsley stimulnts given by continuous intrvenous infusion provides stisfctory criterion for the seprtion of norml from bnorml pncretic function However, since the vlue of the rtio of proteolytic to non-proteolytic enymes my be useful in the dignosis of disese, it is preferble to mesure both types of enyme in routine clinicl studies The estimtion of mylse lso provides check on the informtion derived from the mesurement of trypsin nd gurds ginst the possible misinterprettion of isolted defects in the secretion, or filure of ctivtion, of the precursors of the proteolytic enymes The uthors wish to thnk Miss C xelson nd Mr D Clrke for expert technicl ssistnce reserch grnt from the Cystic Fibrosis Reserch Trust to KGW nd grnt for equipment from the Bord of Governors, nited Sheffield Hospitls to DMG re grtefully cknowledged References mmnn, R W, Tgwercher, E, Kshiwgi, H, nd Rosenmund, H (1968) Dignostic vlue of fecl chymotrypsin nd trypsin ssessment for detection of pncretic disese mer J dig Dis, 13, Ben bdeljlil,, nd Desnuelle, P (1964) Sur l'dption des enymes exocrines du pncr6s l composition du r6gime Biochim biophys ct (mst), 81, Bernfeld, P, nd Studer-P6ch, H (1947) Sur les enymes mylolytiques (II) L'ffinit6 entre enyme et substrtum pour quelques -mylses Helv chim ct, 3, Burton, P, Evns, D G, Hrper,, Howt, H T, Oleesky, S, Scott, J E, nd Vrley, H (196) test of pncretic function in mn bsed on the nlysis of duodenl contents fter dministrtion of secretin nd pncreoymin Gut, 1, Choi, H J, Goldstein, F, Wirts, C W, nd Menduke, H (1967) Norml duodenl trypsin vlues in response to secretinpncreoymin stimultion with preliminry dt in ptients with pncretic disese Gstroenterology, 53, Creutfeldt, W (1964) Funktionsdignostik bei Erkrnkungen des exokrinen Pnkres Vehr dtsch Ges inn Med, 7, Dimond, J S, Siegel, S, Gll, M B, nd Krlen, S (1939) The use of secretin s clinicl test of pncretic function mer J dig Dis, 6, Gibbs, G E (195) Secretin tests with bilumen gstroduodenl dringe in infnts nd children Peditrics, 5, Goldberg, D M, Cmpbell, R, nd Roy, D (1969) Studies on the binding of trypsin nd chymotrypsin by humn intestinl mucos Scnd J Gstroent, 4, Gowenlock, H (1953) The estimtion of tryptic ctivity in duodenl contents Biochem J, 53, Grossmn, M I, Greengrd, H, nd Ivy, C (1942) The effect of dietry composition on pncretic enymes mer J Physiol, 138, Guth, P H, Komrov, S, Shy, H, nd Style, C Z (1956) Reltionship between protein nitrogen, proteolytic, mylolytic nd lipolytic enymes in cnine pncretic juice obtined under vrious conditions of stimultion mer J Physiol, 187, Hverbck, B J, Dyce, B J, Gutentg, P 3, nd Montgomery, D W (1963) Mesurement of trypsin nd chymotrypsin in stool dignostic test for pncretic exocrine insufficiency Gstroenterology, 44, Hershfield, N B, Lind, J F, nd Hildes, J (1968) Clinicl correltions with pncretic function tests Cnd med ss J, 98, Lgerlof, H (1939) The secretin test of pncretic function Qurt J Med, 8, Lgerlof, H (1942) Pncretic function nd pncretic disese studied by mens of secretin ct med scnd, Suppl 128 Ribet,, Pscl, J-P, nd Vysse, N (1968) Recherche de l cpcit6 enymtique mxim du pncres exocrine humin norml sous perfusion continue de s6cr6tine et de pncr6oymine Biol et Gstroent, 2, Rick, W (1965) Zur Pthologie der Enymsekretion des Pnkres ct Gstro-ent belg, 28, Rothmn, S S (1967) 'Non-Prllel trnsport' of enyme protein by the pncres Nture (Lond), 213, Srles, H, Figrell, C, Preelin, G, nd Souville, C (1966) Comportement diff6rent de I lipse, de l'mylse, et des enymes proteolytiques pncretiques pres differents modes d'excittion du pncr6s humin Bull Soc Chim biol (Pris), 48, Siegel, S (1956) Nonprmetric Sttistics for the Behviourl Sciences McGrw-Hill, New York Wormsley, K G (1968) Response to secretin in mn Gstroenterology, 54, Worning, H, nd Mullert, S (1966) ph nd pncretic enymes in the humn duodenum during digestion of stndrd mel Scnd J Gstroent, 1, Zieve, L, Silvis, S E, Mulford, B, nd Blckwood, W D (1966) Secretion of pncretic enymes I Response to secretin nd pncreoymin mer J dig Dis, 11, Gut: first published s 11136/gut on 1 October 197 Downloded from on 25 September 218 by guest Protected by copyright