pancreatic function Faecal chymotrypsin: a reliable index of exocrine
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1 Archives of Disese in Childhood, 1988, 63, Fecl chymotrypsin: relible index of exocrine pncretic function G A BROWN, D SULE, J WILLIAMS, J W L PUNTIS, I W BOOTH, AND A S McNEISH Institute of Child Helth, University of Birminghm, Birminghm SUMMARY Simultneous mesurements of duodenl nd fecl chymotrypsin were mde in 30 children ged 3 weeks to 14 yers Apprent chymotrypsin secretion rtes mesured fter stimultion with pncreozymin were compred with the men fecl chymotrypsin concentrtion derived from three stool specimens collected t rndom within 72 hours of the intrduodenl test In the 25 children who responded to pncreozymin stimultion the men fecl chymotrypsin concentrtion ws significntly positively correlted with the pprent chymotrypsin secretion rte Correltion using single specimen stools collected t rndom ws pprecibly poorer In the five children with undetectble or only trces of chymotrypsin in the duodenum fter stimultion, the men fecl chymotrypsin concentrtions were only 3-10% of the lower limit of the reference intervl In second group of 46 children with cystic fibrosis proved by swet tests nd clinicl evidence of mlbsorption, the chymotrypsin concentrtion mesured in single stool specimen collected t rndom ws unequivoclly subnorml in ech cse Fecl chymotrypsin mesurement is rpid, simple, chep, redily repeted, non-invsive test of high specificity nd sensitivity Fecl chymotrypsin should be mesured before contemplting intrduodenl tests of pncretic function In routine clinicl prctice pncretic exocrine Subjects nd methods function is most ccurtely ssessed by mesuring the products of pncretic secretion in fluid from the To investigte the correltion between enzyme upper smll intestine1 Tests requiring intubtion of ctivities in feces nd duodenl fluid we studied 30 the intestine re, however, techniclly difficult, children who were hving intrduodenl pncretic expensive, nd invsive nd there hve been mny function tests for investigtion of gstrointestinl ttempts to find n lterntive, prticulrly for the symptoms Their ges rnged from 3 weeks to 14 investigtion of children One pproch-the yers (medin 21 months) nd weights rnged from mesurement of pncretic proteolytic enzymes in 1-8 to 35 kg (medin 86) Of five children who feces using pncretic enzyme specific substrteshs been vilble for mny yers2 but there is still pncreozymin nd secretin two hd Shwchmn- filed to secrete enzyme fter stimultion with lck of concensus bout its vlue Mesurement of Dimond syndrome, one hd hd resection of fecl chymotrypsin hs been recommended by pncres for nesidioblstosis, one hd pncretic some workers3 4 but other peditric gstroenterologists discourge it,56 nd firm theoreticl bsis known One child with prtil insufficiency hd pseudocyst, nd in one the etiology ws un- is required to justify further use of the test We hve inflmmtory bowel disese with mlnutrition therefore sought to vlidte mesurement of fecl The remining 24 children were considered to chymotrypsin by investigting its performnce in hve norml pncretic function fter investigtion two wys: firstly by correlting the results with the ctivities of chymotrypsin nd trypsin in the duodenum fter stimultion with pncreozymin nd known to hve pncretic exocrine insufficiency we To investigte the bility of the test in ptients secretin, nd secondly by mesuring its bility to studied 46 children with cystic fibrosis proved by identify the defect in known cses of pncretic rised concentrtions of electrolytes in the swet, exocrine insufficiency nd clinicl evidence of mlbsorption 785
2 786 Brown, Sule, Willims, Puntis, Booth, nd McNeish INTRADUODENAL PANCREATIC FUNCTION TESTS After fsting nd sedtion with chlorpromzine the children were intubted with 12 FG nsoduodenl tube tht hd been modified by removing the end nd dding side holes The tip of the tube ws plced t the duodenojejunl flexure under rdiogrphic control Contmintion of duodenl fluid with gstric fluid ws prevented by frequently spirting the stomch through nsogstric tube Full detils of the technique re given by Tripp et l7 The pncretic function tests were done by technique similr to tht of Hdorn8 Fluid ws collected by suction over two 10 minute bsl secretion periods Pncreozymin ws given intrvenously (2 U/kg body weight) nd further two 10 minute fluid collections were then mde This ws followed immeditely by n intrvenous injection of secretin (2 U/kg body weight), nd fluid collection ws continued for further three 10 minute periods Ech fluid specimen ws plced in vcuum flsk with solid crbon dioxide immeditely fter collection In the lbortory, fluids were kept t -20 C Chymotrypsin nd trypsin were mesured within 24 hours of the completion of the test The duodenl fluid ws nlysed without ny preprtion except centrifugtion t 3000 g for 10 minutes t 4 C to remove insoluble mteril Enzyme ctivities were mesured in ech 10 minute fluid specimen Men concentrtions were clculted for the five specimens collected fter stimultion with pncreozymin Secretion rtes were clculted by dding the content of the sme five specimens nd expressing the result s proportion of body weight The smpling procedure my hve resulted in the loss of some prt of the enzyme secreted, so secretion rtes were designted 'pprent secretion rte' STOOL COLLECTION AND PREPARATION For the 30 children in the first prt of the study, three specimens of stool weighing 1-2 g were collected t rndom on three seprte dys within 72 hours of the intrduodenl pncretic function test Stool nlyses were done on smples diluted 1/7 w/w About0-5 g stool ws first homogenised 1/3 w/w with wter using Potter-Elvehjem pttern glss nd Teflon homogeniser with 0 1 mm clernce for mximum dispersion of stool solids This homogente ws diluted with n equl volume of 0-01 M TRIS buffer, ph 7-8, to produce finl stool dilution of 1/7w/w Chymotrypsin ctivity is dsorbed to the solid phse of fecl homogentes, so cre ws tken to prepre extremely fine homogentes nd to mix them thoroughly before smpling for nlysis For the 46 children with cystic fibrosis nd clinicl mlbsorption single stool specimen ws collected t rndom before strting replcement of pncretic enzymes, or fter withdrwl of enzymes replcement for minimum of 72 hours before stool collection ENZYME MEASUREMENTS Chymotrypsin (EC 34211) nd trypsin (EC 34214) were mesured by kinetic, potentiometric methods,2 in which the rte of hydrolysis of enzyme specific, synthetic substrtes (cetyl tyrosine ethyl ester nd benzoyl rginine ethyl ester for chymotrypsin nd trypsin, respectively) ws recorded with n utomtic titrtor nd recorder (Rdiometer, Copenhgen) The chymotrypsin nd trypsin concentrtions were determined using bovine chymotrypsin (Sigm C4129) nd trypsin (Sigm T8642) for the reference stndrds The precision of the fecl chymotrypsin nlysis ws determined by duplicte nlysis of 30 fecl smples covering the rnge ig/g stool The coefficient of vrition ws 58% The recovery of 25 ig bovine chymotrypsin dded to 14 seprte fecl homogentes rnged from 90* % with men recovery of 992% The bovine chymotrypsin reference stndrd hd n ctivity of 0*17 U/tg, the unit being the ctivity tht hydrolysed 1 [tmol of cetyl tyrosine ethyl ester in one minute under the conditions described for the method Results DUODENAL FLUID ENZYMES Of the 30 children studied, 25 responded to the pncreozymin stimultion Twenty four hd pprent trypsin secretion rtes bove the lower limit of the control rnge tht ws defined by Hdorn8 nd confirmed in this deprtment (fig 1), nd one ws below In the remining five tests, the pprent trypsin secretion rtes were grossly subnorml with no enzyme ctivity detectble in three nd trces only in two The pprent chymotrypsin secretion rtes were segregted in the sme wy The five children who filed to produce trypsin response fter stimultion with pncreozymin lso filed to produce chymotrypsin response The child tht produced mesurble but subnorml pprent trypsin secretion rte lso hd subnorml pprent secretion rte of chymotrypsin of only 30% of the lower limit (men minus 2 SD) of the 24 norml responders The results of nlysis of duodenl fluid therefore rnged from grossly subnorml through prtil insufficiency to the physiologicl rnge, thereby providing wide
3 1800- C x D c 1200 N l ooo c 800-0' 200 ch I 600 *: sa 1* Fecl chymotrypsin: relible index of exocrine pncretic function I % -600 to' - 0 4~00 00 I * O Trypsin Chymotrypsin Fecl chyffotrypsin Intrduodenl secretion rtes Fig 1 Apprent rtes ofsecretion for trypsin nd chymotrypsin in 30 intrduodenl tests compred with men fecl chymotrypsin concentrtions Segregtion ws identicl by ll three mesurements A =men minus 2 SD for control subjects8: B=men minus 2 SD of dt presented but omitting the highest vlue tht ws more thn 3 SD bove the men; men (indicted by n open squre); C=lower limit ofobserved rnge from 150 stool specimens collected t rndom from 25 helthy infnts *=ptients with norml response to pncreozymin stimultion, nd O=ptients with pncretic insufficiency rnge for identifiction of correltion between enzyme ctivities in duodenl juice nd feces * r 1800 FAECAL CHYMOTRYPSIN CONCENTRATIONS The men fecl chymotrypsin concentrtions were segregted in the sme wy s the ctivities in duodenl juice (fig 1) In the five ptients with bsent or only trce of duodenl chymotrypsin nd trypsin fter stimultion, fecl chymotrypsin ws lso extremely low or bsent The highest men chymotrypsin in this group ws 12,ug/g stool, which ws only 10% of the lower limit of our reference rnge (120,ug/g stool) Of the 25 children with mesurble duodenl enzyme ctivities, 24 hd men fecl chymotrypsin concentrtions of 120,ug/g stool or greter The one child in this group whose results were low (40 Rg/g stool, 33% of the lower limit of our reference rnge) hd the subnorml but mesurble pprent secretion rtes of trypsin nd chymotrypsin lredy mentioned CORRELATION BETWEEN DUODENAL ENZYME ACTIVITIES AND FAECAL CONCENTRATIONS In the 25 children who produced mesurble enzyme ctivities in the duodenum fter stimultion, men fecl chymotrypsin concentrtion showed highly significnt positive correltion with pprent secretion rtes of chymotrypsin (fig 2) The Spermn rnk correltion coefficient ws 0-63 (p<0001) When the pprent secretion rtes of chymotrypsin were correlted with chymotrypsin concentrtions from single stool specimens collected t rndom (the initil stool from ech triplicte), the correltion only just reched significnce (r=0-43, p<0 05, >0-025) Significnt positive correltions were lso shown between men fecl chymotrypsin concentrtion nd men duodenl chymotrypsin concentrtion, nd lso between men fecl chymotrypsin concentrtion nd the pprent secretion rtes of trypsin, but the degree of ssocition mesured by the Spermn rnk correltion coefficient vlues ws less for both correltions (correltion coefficients 0-45 nd 0*48 respectively, p<001) Using the lower limit of the rnge of control pprent secretion rtes for trypsin or chymotrypsin nd the lower limit of the fecl chymotrypsin reference rnge to define the boundry between norml nd bnorml pncretic function, none of E o >, r4 C 0 _ c L CL_ >0 E w * U- - E 500- C LA m rs=0 63 p<0001 X- 0 I I I I I I Meon foecl chymotrypsin (pg/g stool ) Fig 2 Correltion ofpprent secretion rtes of chymotrypsin with men fecl chymotrypsin concentrtions * 1500
4 788 Brown, Sule, Willims, Puntis, Booth, nd McNeish the children produced inconsistent results from either source PATIENTS WITH PANCREATIC INSUFFICIENCY In the 46 children with cystic fibrosis nd clinicl mlbsorption fecl chymotrypsin ws bsent in 16, nd in the remining 30 concentrtions were 60% (72 [ig/g stool) or less of the lower limit of our reference rnge The medin concentrtion ws 6 ig/g stool, or 5% of the lower limit of the reference rnge Discussion In the study correlting duodenl enzyme ctivities nd fecl concentrtions, men fecl chymotrypsin concentrtions derived from three seprte stool specimens collected t rndom within 72 hours of the intrduodenl test were highly significntly correlted with the pprent secretion rtes of chymotrypsin The correltion pplied whether pncretic function ws compromised or within the physiologicl rnge, with the result tht the segregtion in the pprent secretion rtes of chymotrypsin between norml pncretic function nd pncretic insufficiency ws reproduced identiclly by the men fecl chymotrypsin concentrtions In the 46 children with cystic fibrosis nd clinicl evidence of mlbsorption fecl chymotrypsin concentrtions were without exception grossly subnorml Vrious mesures of correltion hve been reported in dults between: duodenl output of chymotrypsin nd output in 24 hour stool collections9; duodenl output of trypsin nd chymotrypsin concentrtions in stool specimens collected t rndom'(; nd duodenl concentrtion of chymotrypsin nd fecl concentrtion" None, however, correlted chymotrypsin secretion rtes with chymotrypsin concentrtion in stool specimens collected t rndom-the only specimens tht re redily vilble in infnts nd young children There hve been no correltion studies reported in children It ws necessry to use multiple fecl nlyses to chieve the degree of ssocition between duodenl nd fecl mesurements tht we hve shown Using only the results from the first of the three stool nlyses for correltion produced significntly lower degree of ssocition Homogeneity of chymotrypsin distribution hs been climed both within the individul stool12 nd between three stool specimens collected t rndom on seprte dys11 This my be wht usully hppens, but our experience hs been tht the occurrence of comprtively unrepresenttive results from nlysis of single stool specimen is sufficiently frequent to mke multiple nlyses essentil Four of the 75 individul stool nlyses in the correltion study differed by more thn 50% (medin vlue=14%) from the men vlue of their relevnt triplictes, nd the lrgest devition ws 84% Such differences re quite lrge enough to give rise to misinterprettion of single results t the lower limit of the reference rnge Other workers hve lso emphsised this point, recommending the use of two'( or more13 stool specimens collected t rndom Moeller'3 found three of 22 fecl chymotrypsin results from nine dults with pncretic exocrine insufficiency within their control rnge By repet testing nd using men vlues ll flse negtives were eliminted We recommend the use of three specimens collected t rndom If ll three stool specimens produce chymotrypsin concentrtions significntly greter thn the lower limit of the reference rnge, pncretic insufficiency t the time of the investigtion cn be excluded This would not be true for specific lipse, co-lipse, or trypsinogen deficiencies but these conditions re extremely rre In the 46 children with cystic fibrosis nd cliniclly recognised mlbsorption, fecl chymotrypsin concentrtions were unequivocily subnorml, nd single stool specimens were sufficient to identify the gross pncretic insufficiency chrcteristic of the disese The children were selected for the present study becuse they hd mlbsorption nd it ws not considered ethiclly justifible to confirm the degree of pncretic insufficiency by intubtion nd hormone stimultion Controversy persists over the efficiency of fecl chymotrypsin mesurement s method of ssessing pncretic function becuse of confusion over the function nd cpbility of the test The test my not detect the initil stges of insufficiency in developing disese It cnnot differentite between norml pncretic function nd chronic pncretitis where secretion (though invribly ultimtely compromised) my initilly be norml, nd pncretic crcinom where reduced secretion (if it occurs) depends on the site nd nture of the lesion The reputtion of the test hs suffered becuse it does not resolve these 'dult' problems In ddition to the difference in the clinicl questions being sked, there my be other resons for greter specificity in children Men whole gut trnsit times re shorter in children, the trnsit time of the neonte14 being bout qurter of tht of the dult'5 The shorter trnsit time reduces the opportunity for bcteril'6 nd enzymtic'7 degrdtion in trnsit, mking the test more direct nd incresing its specificity Reducing dult whole gut trnsit time with purgtives to bout tht of the neonte improves the duodenl fecl chymotrypsin correltion9
5 In ddition to the high sensitivity nd specificity, ssessing pncretic function by fecl chymotrypsin mesurements hs much to recommend it in children It is completely non-invsive, does not entil orl dosing nd requires little coopertion on the prt of the ptient, except for stool retention The quntity of feces needed is too smll to give offence in or outside the lbortory or to present the ltter with problems of microbiologicl continment of potentilly hzrdous mteril The procedure is simple, chep, nd rpid compred with other methods of mesuring pncretic function These fetures mke the test repetble s often s necessry so tht confirmtion by repet testing is lwys possible nd longitudinl studies requiring frequent repet tests re lso possible A further dvntge of chymotrypsin is its stbility t mbient tempertures in the United Kingdom Little loss of ctivity occurs fter it hs been stnding t 18 C for 72 hours, so tht specimens my be sent by post with confidence nd without ny specil provision other thn secure pcking nd voiding weekends Intrduodenl testing will continue to be necessry to identify the rre, specific deficiencies of pncretic enzymes, but in most children in whom pncretic exocrine functions is questioned the nswer should be sought first by mesuring fecl chymotrypsin concentrtions References DiMgno EP Dignosis of chronic pncretitis: re noninvsive tests of exocrine pncretic function sensitive nd specific? Gstroenterology 1982;83: Hverbck BJ, Dyce BJ, Gutentg PJ, Montgomery DW Mesurement of trypsin nd chymotrypsin in stool: dignostic test for pncretic exocrine insufficiency Gstroenterology 1963;44: Brbero GJ, Sibeng MS, Mrino JM, Seibel R Stool trypsin nd chymotrypsin: vlue in the dignosis of pncretic insufficiency in cystic fibrosis Am J Dis Child 1966;112: Fecl chymotrypsin: relible index of exocrine pncretic function Scott MS, Mrzni MD, Mggiore G, De Gicomo C, Melzi d'eril GV, Mortti R Fecl chymotrypsin: new dignostic test for exocrine pncretic insufficiency in children with cystic fibrosis Clin Biochem 1985;18: Pcker SM, Mill PJ, Tripp JH Investigtory techniques In: Hrries JT, ed Essentils of peditric gstroenterology Edinburgh: Churchill Livingstone, 1977: Tripp JH, Muller DPR The gstrointestinl trct In: Clyton BE, Round JM, eds Chemicl pthology nd the sick child Oxford: Blckwell Scientific Publictions, 1984: Tripp JH, Cndy DCA Mnul of peditric gstroenterology Edinburgh: Churchill Livingstone, Hdorn B The exocrine pncres In: Anderson CM, Burke V, eds Peditric gstroenterology Oxford: Blckwell Scientific Publictions, 1975: Sle JK, Goldberg DM, Thjodleifsson B, Wormsley KG Trypsin nd chymotrypsin in duodenl spirte nd feces in response to secretin nd cholecystokinin-pncreozymin Gut 1974;15:132-8 "' Durr HK, Otte M, Forell MM, Bode JC Fecl chymotrypsin: study on its dignostic vlue by comprison with the secretincholecystokinin test Digestion 1978;17:404-9 Lmi F, Cllegri C, Miglioli M, Brbr L A single fecl chymotrypsin test in the dignosis of pncretic insufficiency: correltion with secretin-cholecystokinin nd NBT-PABA tests Am J Gstroenterol 1984;79: Kspr P, Newmnn U The distribution of chymotrypsin within the feces nd description of new device for the preprtion of stool smples Clin Chem 1984;30: Moeller DD, Dewey Dunn G, Klotz AP Dignosis of pncretic exocrine insufficiency by fecl chymotrypsin ctivity Am J Dig Dis 1973;18: Rubltelli FF, Lrgjolli G Effect of light on gut trnsit time in jundiced newborns Act Peditr Scnd 1973;62:146-8 '5 Cnn PA, Red NW, Brown C, Hobson N, Holdsworth CD Irritble bowel syndrome: reltionship of disorders in the trnsit of single solid mel to symptom ptterns Gut 1983;24: Borgstrom A, Genell S, Ohlsson K Elevted fecl levels of endogenous pncretic endopeptidses fter ntibiotic tretment Scnd J Gstroenterol 1977;12: Lyer P, Go VLW, DiMgno EP Fte of pncretic enzymes during smll intestinl borl trnsit in humns Am J Physiol 1986;251:G Correspondence to Mr GA Brown, Institute of Child Helth, Frncis Rod, Birminghm B16 8ET Accepted 22 Februry 1988 Arch Dis Child: first published s /dc on 1 July 1988 Downloded from on 2 April 2019 by guest Protected by copyright
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