659

pancy between our study and the other three studiescould be due in part to the relatively higher survival-ratein our control patients. The 3-year survival-rate of ourcontrol patients was 56% and ranged from 35 to 40% inthe other three studies. Finally, if the results of our

study and those of the other three are pooled, the dif-ference in the survival-rates of patients with shunts andcontrol patients is not significant.12 These pooled resultsstrongly suggest that the benefit of therapeutic porta-caval shunt in terms of longevity is either absent or verylimited.Thus, therapeutic portacaval shunt in cirrhosis does

not affect, or only slightly affects, the survival-rate andmay induce chronic hepatic encephalopathy in a signifi-cant number of patients. For these reasons, portacavalshunt can no longer be accepted as a suitable treatmentfor cirrhotic patients with gastrointestinal bleeding.However, this pessimistic conclusion is valid only for pa-tients selected according to current criteria and for theconventional end-to-side or side-to-side portacavalshunt. Our study and the other reported studies of

therapeutic shunt were carried out before endoscopy hadbeen extensively used to recognise the source of bleedingin cirrhotic patients; this means that some patients withnon-variceal bleeding have had shunts. Hsemodynamicfindings were not considered in selecting patients forportacaval shunt; the knowledge of portal venous pres-sure and liver blood-flow could aid the better selectionof patients. Finally, our discouraging conclusion shouldnot be extended to recently described shunting methods,such as combination of portacaval shunt and arterialisa-tion of the portal vein" and selective distal splenorenalshunt.14 However, controlled studies should be carriedout soon to assess these new surgical methods, to avoidthe thirty-year delay which was needed for the correctevaluation of the conventional portacaval shunt.This work was presented in part at the 9th Meeting of the European

Association for the Study of the Liver, Hemsedal, Norway, 1974. Wethank all those members of the staff who participated in the care ofthe patients included in this study, especially Dr P. Clot, Dr P. Dela-gousie, Dr B. Estenne, Dr F. Fekete, Dr B. Goyer, Dr Giuli, and DrM. Huguier who performed portacaval shunting, and Dr C. Atterburyfor help in the preparation of this manuscript.Requests for reprints should be addressed to B.R., Hopital Beaujon,

92110 Clichy, France.REFERENCES

1. Grace, N. D., Muench, H., Chalmers, T. C. Gastroenterology, 1966, 50,684.

2. Conn, H. O., Lindenmuth, W. W., May, C. J., Ramsby, G. R. Medicine, Bal-timore, 1972, 51, 27.

3. Jackson, F. C., Perrin, E. B., Felix, W. R., Smith, T. Ann. Surg. 1971, 174,672.

4. Resnick, R. H., Iber, F. L., Ishihara, A. M., Chalmers, T. C., Zimmerman,H., Boston Inter-Hospital Liver Group. Gastroenterology, 1974, 67, 843.

5. Mikkelsen, W. P. Archs Surg. 1974, 108, 302.6. Rueff, B., Benhamou, J. P. Clins Gastroenterology, 1975, 4, 425.7. Prandi, D., Rueff, B., Roche-Sicot, J., Sicot, C., Maillard, J. N., Benhamou,

J. P., Fauvert, R. Am. J. Surg. (in the press).8. Schwartz, D., Flamant, R., Lellouch, J. L’Essai Thérapeutique chez

l’Homme; p. 218. Paris, 1970.9 Siegel, S. Non Parametric Statistics for the Behavioral Sciences; p. 96. New

York, 1956.10. Snedecor, G. W., Cochran, W. G. Statistical Methods; p. 329. Ames, Iowa,

1967.11. Conn, H. O. Am. J. Gastroenterol. 1973, 59, 207.12. Conn, H O. Gastroenterology, 1974, 67, 1065.13 Maillard, J. N., Rueff, B., Prandi, D., Sicot, C. Archs Surg. 1974, 108, 315.14 Warren, W. D., Salam, A. A., Huston, D., Zeppa, R. ibid. p. 306.15 Garceau, A. J., Donaldson, R. M. Jr., O’Hara, E. T., Callow, A. D.,

Muench, H., Chalmers, T. C., Boston Inter-Hospital Liver Group. Gas-troenterology, 1968, 54, 1057.

16. Jackson, F. C., Pernn, E. B., Smith, A. G., Dagradi, A. E., Nadal, H. M.Am. J. Surg. 1968, 115, 22.

17 Resnick, R. H., Ishihara, A., Chalmers, T. C., Schimmel, E. M. Gastroen-terology, 1968, 54, 1057.

18 Conn, H. O., Lindenmuth, W. W. New Engl. J. Med. 1965, 272, 1255.

ENTEROTOXIGENIC ESCHERICHIA COLI ANDREOVIRUS-LIKE AGENT IN RURAL

BANGLADESH

ROBERT W. RYDERBacterial Diseases Division, Bureau of Epidemiology, Centerfor Disease Control, Public Health Service, Atlanta, Georgia

30333, U.S.A.

DAVID A. SACK

Department of Medicine, Johns Hopkins University School ofMedicine, Baltimore, Maryland, U.S.A.

ALBERT Z. KAPIKIAN

Laboratory of Infectious Diseases, National Institutes ofAllergy and Infectious Diseases, National Institutes of Health,

Bethesda, Maryland, U.S.A.

JAMES C. MCLAUGHLIN JYOTSNAMOY CHAKRABORTYA. S. M. MIZANUR RAHMAN

Cholera Research Laboratory, Dacca, Bangladesh

MICHAEL H. MERSON JOY G. WELLSBacterial Diseases Division, Bureau of Epidemiology, Center

for Disease Control, Atlanta, Georgia, U.S.A.

Summary 48 patients admitted to a rural Bangla-desh hospital with dehydration second-

ary to diarrhœa were examined for infection caused byreovirus-like agent (R.L.A.) or enterotoxigenic Escheri-chia coli (E.T.E.C.). The diagnosis of R.L.A. infection wasestablished by electron microscopy of stool filtrates andby a fourfold or greater rise in serum complement-fixingantibodies to the Nebraska calf diarrhœa virus. Evi-dence of infection by heat-labile-toxin (L.T.)-producingE.T.E.C. was sought by stool culture and serological test-ing using the adrenal-cell tissue-culture system. Infec-tion by heat-stable-toxin (S.T.)-producing E.T.E.C. wassought by stool culture using the infant mouse test. 12patients, all less than two years old, had evidence ofR.L.A. infection, accounting for illness in 55% of the 22patients who were less than two years old. None of these22 children had evidence of E.T.E.C. infection. R.L.A.

diarrhœa lasted five to six days, often led to seriousdehydration, and was associated with vomiting andfever. 11 cases of E.T.E.C. diarrhœa were detected,accounting for 56% of the cases of diarrhœa in the 18patients who were more than ten years old. Diarrhœacaused by E.T.E.C. was sudden in onset, shorter in

duration, and caused pronounced dehydration. In a

community survey E.T.E.C. was isolated with equal fre-quency in the stools of control and case family members.The data suggest that E.T.E.C. is a common cause ofadult diarrhœa in Bangladesh, while R.L.A. is a commoncause of diarrhœa in children.

Introduction

ENTEROTOXIGENIC Escherichia coli (E.T.E.C.) and reo-virus-like agent (R.L.A.) are major causes of acute undif-ferentiated diarrhcea in many parts of the world.1-8During December, 1974, and January, 1975, we investi-gated human diarrhoeal illness caused by these agents inrural Bangladesh where diarrhoea not caused by com-monly recognised enteric pathogens (e.g., Vibriocholera, salmonella, shigella, &c.) is frequent.9

660

Materials and MethodsBackground

Matlab Hospital, the field hospital of the Cholera ResearchLaboratory is located about 40 miles from Dacca in one of themost densely populated areas of Bangladesh. Patients are

weighed on admission and daily thereafter. All patients aretreated on cholera cots which permit liquid stool to be collectedin calibrated containers. In hospital, patients receive oraland/or parenteral fluid rehydration. Stool cultures are platedon MacConkey’s, salmonella-shigella, and Monsur agars andincubated at 37° overnight. Suspect shigella or vibrio coloniesare identified by agglutination with group and type specificantiserum.

Study DesignPatients included in this study had the following charac-

teristics : (1) they were in hospital with severe dehydrationcaused by diarrhoea; (2) they had become ill less than seventy-two hours before admission; (3) they had not received antece-dent antibiotic therapy; (4) they had no leucocytes in theiradmission stool specimen;1O (5) they had stool cultures nega-tive for V. cholera, shigella, and salmonella during the firsttwo days of their hospital stay; and (6) they were the first pa-tient admitted each day who satisfied the above criteria. Allstudy patients remained in hospital and had daily rectal swabstaken for at least six days; none of them received antibiotics.The clinical course of 15 patients concurrently in hospital withcholera and receiving oral tetracycline was observed for com-parison. Blood specimens were drawn from patients by vene-puncture on admission and during convalescence ten to four-teen days later.We defined diarrhoea as loose stools occurring more

frequently than normal. Clinical and bacteriological surveil-lance for diarrhoea in each patient’s family ("case family") andan adjacent "control family" was conducted by a trained fieldteam within forty-eight hours of the patient’s admission to hos-pital. This team visited each case and control family on threealternate days inquiring about the presence of diarrhoea,recording demographic data, collecting water samples, andobtaining rectal swabs from each family member. Rectal swabsobtained in the field were collected with a sterile swab andplaced in Cary-Blair medium for transport to the laboratory.Serum samples were also collected from individuals in controlfamilies of specified ages who had not had diarrhoea in the pre-vious week.

Laboratory StudiesBacteriology.-From each MacConkey’s agar plated on

admission, 10 typical E. coli colonies were selected and inocu-lated onto nutrient agar slants for further study; this is a reli-able method of selecting E. coli.1’ A pool of these 10 colonieswas also made. 10 E. coli colonies from subsequent stool cul-tures from patients, and all cultures from family contacts andcontrols were pooled and separate nutrient agar slants were in-oculated from each pool. All slants were incubated at 370Covernight and then held at room temperature.

TABLE I-DIARRHOEAL ILLNESS IN FAMILY CONTACTS AND CONTROLS

TABLE II—CHARACTERISTICS OF STUDY PATIENTS

Enterotoxin assays.- The 10 E. coli isolates from each pa-tient’s admission stool culture and the pooled cultures isolatedfrom these patients, their family contacts, and controls (a totalof 1142 cultures) were tested for heat-labile-enterotoxin (L.T.)production by the Y 1-adrenal-cell system using the miniculturetechnique.12 In addition, E. coli isolates from drinkingbathing, and cooking water sources were also tested. 5 E. coliisolates from each patient’s admission culture were also

assayed for heat-stable-enterotoxin (s.T.) production using theinfant mouse assay. 13 A ratio of intestinal weight to remainingbody-weight of 0083 or greater was regarded as positive." AllE.T.E.C. isolates were serotyped using 143 E. coli antisera in 19pools. The antibiotic sensitivity of all E.T.E.C. isolates wasdetermined using standard disc sensitivity methods." L.T.anti-toxin titres from hospital patients and neighbourhood controlswere assayed in the adrenal-cell system." Paired sera were notavailable from 6 study patients.

Virology.—R.L.A. infection was established by: (1) examina-tion of stools by immune electron microscopy using convales.cent-phase sera or human immune serum globulin as a sourceof antibody;’16 (2) a fourfold or greater rise in serum comple-ment-fixing antibodies using the Nebraska calf diarrhea virus(N.C.D.V.) as a substitute antigen.’

Stools from patients at admission were prepared as a 2%suspension in veal infusion broth containing 0.5% bovineserum albumin. Each suspension was shaken with glass beadsand centrifuged at 500 g for an hour and the sediment dis-carded. The faecal suspension was successively filtered througha 30 m and a 022 m Millipore filter before streptomycinand tetracycline were added to produce final concentrations of1000 g/ml and 33 g/ml, respectively. Paired sera and/orstools were not obtained from 6 patients.

TABLE III -FREQUENCY OF E.T.E.C. AND R.L.A. BY AGE IN 48 PATIENTS IN HOSPITAL WITH DIARRHOEA

661

Results

During the study period (December, 1974, to Jan-uary, 1975) 423 patients were admitted to the hospitalwith diarrhoea; stool cultures from 242 (57%) of thesepatients were negative for shigella, salmonella, or V.

choleræ. 48 (20%) of these 242 individuals were in-cluded in the investigation. The mean age of these 48patients was twelve; the median age was two years. Thefrequency of diarrhoea in family contacts and controlswas almost identical within each age-group (table i).

Laboratory StudiesThere was evidence of diarrhoea caused by E.T.E.C. in

11 (23%) patients (L.T.[+]), 10 of whom were more thanten years old (table II). E.T.E.C. was found in stools of 8patients, while 3 additional patients had a fourfold risein antibody to L.T. (table III). None of the E.T.E.C. iso-lates belonged to a currently recognised enteropatho-genic serotype. Using pooled specimens, enterotoxigenicorganisms were no more common in family contacts ofL.T.(+) patients than of L.T.(-) patients. E. coli fromstools of 3 (36%) of 84 family contacts of L.T.(+) pa-tients and none of 71 controls were enterotoxigenic,while stools from 4 (1.8%) of 219 family contacts ofL.T.(-) patients and 3 (15%) of 201 controls containedL.T.-producing organisms. Only 1 of these 10 L.T.(+) in-dividuals had diarrhoea at the time of culture. E. coli iso-lates from 1 of 39 contaminated water sources wereenterotoxigenic. Pooled cultures from patients withdiarrhoea were a satisfactory method for screening iso-lates, since no individual isolates were positive whenpools were negative. Of the 8 cases with enterotoxigenicpools from admission cultures, all but one contained 9or 10 of 10 enterotoxigenic single-colony isolates; in thisone pool only 4 of 10 isolates produced L.T. All E.T.E.C.produced s.T. and L.T. Cases continued to shed E.T.E.C.in their stools for an average of 3 - 6 days after diarrhoeaceased. E.T.E.C. isolated from 3 of these patients demon-strated multiple antibiotic resistance; the remainderwere multiply sensitive.6 (75%) of 8 patients with enterotoxigenic organisms

and 3 (88%) of 35 other patients had a fourfold in-crease in antibody titre to L.T. enterotoxin. Initial anti-toxin titres of individuals with diarrhoea caused byE.T.E.C. resembled those in patients with diarrhoeacaused by other agents and symptom-free controls

(table iv). Younger patients in all groups tended to havehigher titres. In the 8 L.T.(+) patients there was no cor-

TABLE IV-ANTIBODY TITRES AGAINST L.T. IN PATIENTS AND

SYMPTOM-FREE COMMUNITY CONTROLS

*Obtained 10-14 days after initial titre.tlnitial and second titres are significantly different by the Mann-Whitney U Test(p<0.001).

relation between antitoxin titre and length of stool car-riage of the organism or severity of illness.

There was evidence that diarrhoea was caused byR.L.A. in 12 (55%) of the 22 patients who were less thantwo years old and from whom sera and/or stool filtrateswere collected and tested (tables n and III). None of the20 patients who were more than two years old and fromwhom sera and/or stool filtrates were available hadR.L.A. infection. R.L.A. was seen in the stools of 7 (44%)of 16. children who were less than two years old; 3 ofthese had a fourfold or greater increase in complement-fixing antibody titre. 5 additional cases were confirmedserologically. Stool filtrates from only 2 of these caseswere examined for R.L.A.; both were negative.

Clinical Results

Vomiting, abdominal cramps, and raised temperaturewere more common in patients with R.L.A. than in pa-tients with E.T.E.C. (table ii). Although patients withE.T.E.C. waited a shorter time (mean nine hours) beforecoming to the hospital than did patients with R.L.A.

(mean forty-six hours), E.T.E.C.-positive patients weremore dehydrated (mean admission serum-protein 11.1g/dl) on arrival than patients with R.L.A. (mean admis-sion serum-protein 9.8 gjdl) table v). Because of thispronounced dehydration E.T.E.c. patients required morefluid replacement (131.9 ml/kg) than did patients withR.L.A. (90.9 ml/kg). However, patients with R.L.A. had

TABLE V--CHARACTERISTICS REFLECTING THE SEVERITY OF ILLNESS OF STUDY PATIENTS, MATLAB HOSPITAL, DECEMBER, 1974-JANUARY, 1975

’All patients with cholera received 5 days of tetracycline while in hospital.ffnterquarttte range.

662

diarrhoea for a longer period after admission to hospitaland passed larger volumes of liquid stool before theirdiarrhoea ceased than did patients with E.T.E.C. E.T.E.C.patients were as dehydrated on admission as cholera pa-tients but required less fluid replacement.

Discussion

We used the adrenal-cell tissue-culture system to

detect L.T. E. coli enterotoxin,12 the infant mouse assayto detect s.T. E. coli enterotoxin," and electron micro-scopic examination of stool filtrates’6 to detect R.L.A. Inaddition, we used acute and convalescent phase sera todetect an increase in antibody titre to L.T.’s and R.L.A.complement-fixing antibodies.8

11 cases of E.T.E.C. diarrhoea were detected, accountingfor 56% of the cases of diarrhoea in patients agedmore than ten who were included in the study. 6 (73%) of8 bacteriologically confirmed cases had a fourfold rise intitre of antibodies to L.T. Only 3 of 3 5 -patients withoutL.T. E. coli in their stools had a fourfold rise. The in-

sensitivity of bacteriological methods currently used toidentify E.T.E.C. may explain our inability to find entero-toxigenic organisms in stools from these 3 individuals. Asignificant response in antibody titre to L.T. E.T.E.C. hasbeen demonstrated in Bengali patients with diarrhma.17However, in studies of travellers’ diarrhoea, Americansin Mexicoi8 and Kenya’ in whom L.T. E.T.E.C. diarrhoeadeveloped did not have a fourfold serological response.Differences in the illness or quantity of antigen mayexplain these findings. Alternatively, an anamnestic re-sponse resulting from prior infection may be responsible.Our data conflict with the data of Nalin et al.20 who

tested E. coli from a similar group of patients using theChinese hamster ovary-cell technique21 to detect L.T.

and dog intestinal loops22 to detect s.T. They isolatedL.T.(+) organisms from 33% of all inpatients and s.T.(+)organisms from 33% of L.T.(-) cases. Differences in

technique, interpretation of data, or specificity of theassays used may explain these disparities.The spectrum of disease caused by E.T.E.C. ranges

from a severe cholera-like syndrome with copious rice-water diarrhoea in India3 to a mild illness of shortduration with loss of small amounts of fluid reported inBrazil’ and parts of the U.S.2 These same studiesshowed that E.T.E.C. strains are important causes ofadult diarrhoea in the Indian subcontinent and diarrhoeain children in the Americas. The difference in age-speci-fic attack-rate between these regions may be related tothe higher cholera antitoxin titres found in children liv-ing in cholera endemic areas23 (i.e., the Indian subcon-tinent) which may protect against E.T.E.C. infection. L.T.is immunologically similar to cholera toxin and is neu-tralised by cholera antitoxin.17 24

Our finding of E.T.E.C. in stools of only 3 of 84 familycontacts of the 8 patients with E.T.E.C. should be inter-preted with caution as our methods of bacteriologicalsurveillance were not entirely satisfactory. Alternate-dayculturing for six days may not have been frequentenough to detect toxigenic organisms in family contacts.Pooling of 10 E. coli isolates on a single slant and testingthese pools for enterotoxin production may have led tocolicin inhibition or bacterial overgrowth of enterotoxi-genic organisms. Because colony-counts of E.T.E.C. in

symptom-free persons may be very low, our picking only10 colonies per stool sample may have caused us to miss

the relatively few enterotoxigenic organisms present. Wemay also have conducted this investigation during atime of year when disease caused by E.T.E.C. is uncom-mon.

12 patients, all less than two years old, had R.L.A,

diarrhoea accounting for 55% of the diarrhoea seen inthe 22 patients aged less than two who were studied.Only 3 of 7 patients with R.L.A. in their stool had a four-fold or greater increase in complement-fixing antibodiesto N.C.D.v. This lack of efficiency in detecting serologicalresponses in virus-positive patients may have been dueto the young age of the patients studied and/or to theuse of the N.c.D.v. as a substitute complement-fixingantigen for the human agent. Had we been able to (a)examine multiple filtrates of stools from all patients, (b)use the human antigen, or (c) test the sera by the im-munofluorescent-antibody technique we might havedetected additional cases of R.L.A.

R.L.A. diarrhoea is found almost exclusively in youngchildren6-8 21 and can cause severe dehydration.25 Wefound that R.L.A. diarrhoea lasted five to six days andwas associated with fever, vomiting, and a large fluidloss because of persistent diarrhoea. Parenteral rehy-dration was often required. We did not find f&aelig;cal leuco-cytes. This accords with previous results26 and indicatesthat the virus does not invoke an inflammatory re-

sponse.1o This finding may be useful in diagnosis.We are grateful to Jean Froelich, Adrian George, Debra G. Snyder,

Patsy Bellamy, and the staff of the Matlab Hospital for expert techni-cal assistance. Acknowledgement is made to the Gerontology ResearchCenter, National Institute of Child Health and Human Developmentfor facilities extended under its guest scientist programme. This studywas supported in part by U.S. Army contract DADA 17-73-C-3055and by the Cholera Research Laboratory which is supported by theGovernments of the People’s Republic of Bangladesh, the U.S.A., theU.K. and Australia.

Requests for reprints should be addressed to R. W. R.

REFERENCES

1. Guerrant, R. L., Moore, R. A., Kirschenfeld, P. M., Sande, M. A. New Engl.J Med. 1975, 293, 567.

2. Sack, R. B., Hirschhorn, N., Brownlee, I., Cash, R. A., Woodward, W.E.,Sack, D. A. ibid. 1975, 292, 1041

3. Sack, R. B., Gorbach, S. L., Banwell, J. G., Jacobs, B., Chatterjee, B. D.,Mitra, R. C. J. infect. Dis. 1971,123, 378.

4. DuPont, H. L., Formal, S. B., Hornick, R. B., Snyder, M. J., Libanoti, J. P.,Sheahan, D. G., LaBrec, E. H., Kalas, J. P. New Engl. J. Med. 1971,285, 1.

5. Gorbach, S. L., Kean, B. H., Evans, D. G., Evans, D. J., Bessudo, D. ibid.1975, 292, 933.

6. Davidson, G. P., Bishop, R. F., Townley, R. R. W., Holmes, I. H., Ruck,B. J. Lancet, 1975, i, 242.

7. ibid. p. 257.8. Kapikian, A. Z., Cline, W. L., Mebus, C. A., Wyatt, R. G., Kalica, A. R.,

James, H. D., VanKirk, D., Chanock, R. M., Kim, H. W. ibid. p. 1056.9. Lindenbaum, J., Greenough, W. B., Benenson, A. S., Oseasohn, R., Rizvi,

S., Saad, A. ibid. 1965, i, 1081.10. Harris, J. C., DuPont, H. L., Hornick, R. B. Ann intern. Med. 1972, 76,

697.11. Morris, G. K., Merson, M. H., Sack, D. A., Wells, J. G., Martin, W. T.,

DeWitt, W. E., Feeley, J. C., Sack, R. B., Bessudo, D. J. clin. Microbiol.(in the press).

12. Sack, D. A., Sack, R. B. Infect, Immun 1975, 11, 334.13. Dean, A. G., Ching, Y. C., Williams, R. G., Harden, L. B. J. infect. Dis.

1972, 125, 407.14. Bauer, A. W., Kirby, W. M., Sherris, J. C., Turck, M. Am. J. clin. Path.

1966, 45, 493.15. Donta, S. T., Sack, D. A., Wallace, R. B., DuPont, H. L., Sack, R. B. New

Engl. J. Med. 1974, 291, 117.16. Kapikian, A. Z., Kim, H. W., Wyatt, R. G., Rodriguez, W. J., Ross, S.

Cline, W. L., Parrott, R. H., Chanock, R. M. Science, 1974, 185, 104917. Sack, R. B., Jacobs, B., Mitra, R. J. infect. Dis. 1974, 129, 330.18. Merson, M. H., Morris, G. K., Sack, D. A., Wells, J. G., Feeley, J. C., Sack.

R. B., Creech, W. B., Kapikian, A. Z., Gangarosa, E. J. New Engl JMed. (in the press).

19. Sack, D. A. Unpublished.

663

DR RYDER AND OTHERS: REFERENCES&mdash;continued20. Nalin, D. R., McLaughlin, J. C., Rahaman, M., Yunus, M., Curlin, G. Lan-

cet, 1975, ii, 1116.21 Guerrant, R. L., Brunton, L. L., Schnaitman, T. C., Rebhun, L. I., Gilman,

A. G. Infect. Immun. 1974, 10, 320.22. Nalin, D. R., Bhattacharjee, A. K., Richardson, S. H. J. infect. Dis. 1974,

130, 595.23. Benenson, A. S., Saad, A., Mosley, W. H., Ahmed, A. Bull. Wld Hlth Org.

1968, 38, 287.24. Smith, N. W., Sack, R. B. J. infect. Dis. 1974, 127, 164.25. Shepherd, R. W., Truslow, S., Walker-Smith, J. A., Bird, R., Cutting, W.,

Darnell, R., Barker, C. M. Lancet, 1975, ii, 1082.26 Echevema, P., Blacklow, N. R., Smith, D. H. ibid. p. 1113.

DIAGNOSIS OF PANCREATIC DISEASE BY ASYNTHETIC PEPTIDE

A New Test of Exocrine Pancreatic Function

CONSTANTINE ARVANITAKIS NORTON J. GREENBERGER

Department of Medicine, Kansas University School ofMedicine, Division of Gastroenterology, Kansas City, Kansas

66103, U.S.A.

Summary N-benzoyl-L-tyrosyl-p-aminobenzoicacid, a synthetic peptide, is specifically

cleaved by the pancreatic endopeptidase chymotrypsin;the released p-aminobenzoic acid (P.A.B.A.) is absorbedand excreted in the urine. To evaluate its diagnosticvalue, the urinary excretion of P.A.B.A. after oral admin-istration of this peptide was examined in patients withpancreatic disease (chronic pancreatitis with pancreaticinsufficiency and pancreatic carcinoma). Recovery ofP.A.B.A. in the urine was significantly lower in patientswith pancreatitis (40%) and pancreatic carcinoma (56%)than in the control group (75%) (P<0&middot;001 and P<0&middot;05respectively). A good correlation was obtained compar-ing 8-hour cumulative recovery of P.A.B.A. with maximalbicarbonate concentration during secretin cholecystok-inin/pancreozymin (C.C.K./P.Z.) test (P<0&middot;001), mean

intraduodenal concentration of trypsin (P<0&middot;01), and72-hour f&aelig;cal fat determination (p<0&middot;05). These dataindicate that urinary excretion of a tracer marker(P.A.B.A.) of a synthetic peptide (1) correlates well withother parameters of exocrine pancreatic function and (2)may prove a useful, practical, and accurate diagnostictest in chronic pancreatitis with pancreatic insuffi-

ciency.

Introduction

IT is generally agreed that there are no simple andpractical diagnostic tests to assess pancreatic exocrinefunction. The currently available tests require duodenalintubation, stimulation of exocrine pancreatic secretionby hormones or a test meal, and complex analyticalmethods for determination of pancreatic enzymes andbicarbonate concentration. There is great need for a

simple, accurate and reproducible test of pancreaticfunction.The synthetic peptide N-benzoyl-L-tyrosyl-p-amino-

benzoic acid (Bz-ty-P.A.B.A.) is specifically cleaved by thepancreatic endopeptidase chymotrypsin to Bz-ty andP.A.B.A. (fig. 1). Chymotrypsin breaks peptide bonds inwhich the carbonyl group is part of an aromatic amino-acid. When administered orally, Bz-ty-P.A.B.A., ’uponreaching the small intestine, is hydrolysed in the pre-sence of chymotrypsin with the liberation of P.A.B.A.

Fig. 1-Chemical formula of the synthetic peptide N-benzoyl-L-tyrosyl-p-aminobenzoic acid.

The peptide bond is specifically cleaved by chymotrypsin (inter-rupted line) liberating Bz-ty and P.A.B.A.

p-aminobenzoic acid is rapidly absorbed and its metabo-lites are excreted by the kidney. Accordingly, the recov-ery of P.A.B.A. in the urine after administration ofBz-tY-P.A.B.A.’ indirectly reflects intraluminal chymo-trypsin activity and appears to be a reliable index ofexocrine pancreatic function. Previous studies by Im-ondi et al.1 in experimental pancreatic insufficiency, in-duced by ligation of the pancreatic duct in rats, swine,and dogs, demonstrated that urinary recovery of P.A.B.A.after oral administration of Bz-ty-p.A.B.A. accurately ref-lected impaired exocrine pancreatic function. Recently,Gyr et aLl have reported that Bz-ty-P.A.B.A. is of diag-nostic value in protein-deficient monkeys with pancrea-tic insufficiency.We have examined the role of Bz-ty-P.A.B.A. in the

diagnosis of pancreatic disease in man. The dataobtained indicate that the urinary excretion of P.A.B.A.correlates well with other parameters of exocrine pan-creatic function in patients with chronic pancreatitisand pancreatic carcinoma.

Patients and Methods

The following groups were studied: (1) sixteen normal con-trols ; (2) sixteen patients with chronic pancreatitis (calcificpancreatitis and pancreatic steatorrhoea); (3) four patientswith proven pancreatic carcinoma; and (4) one patient withcholedocholithiasis. Informed consent was obtained from allthe subjects prior to study.

Controls

There were ten male and six female normal volunteers, aged19-42 years (mean 27). None of them presented with any clini-cal or laboratory evidence of gastrointestinal, hepatobiliary,pancreatic, or renal disease. None had a history of alcoholism.

Patients

Chronic pancreatitis.-There were thirteen male and threefemale patients, aged 26-60 years (mean 48). Patients admit-ted to the study fulfilled two or more of the following diagnos-tic criteria: (1) history of chronic relapsing pancreatitis; (2)history of pancreatic pseudocyst, drained spontaneously or atsurgery; (3) presence of pancreatic calcifications on plainX-ray of the abdomen; and (4) demonstration of pancreaticsteatorrhoea by determination of 72 h faecal fat (normal < 18

g/72 h). The aetiology of chronic pancreatic desease in all six-teen patients was related to longstanding heavy alcohol intake.One patient had, in addition, a history of symptomatic gall-bladder disease and had undergone cholecystectomy.

Pancreatic carcinoma.-There were four male patients,aged 42-65 years (mean 53) with adenocarcinoma of the pan-creas proven at surgery. One of them had undergone partial