Arch Dis Child: first published as 10.1136/adc.63.7.780 on 1 July 1988. Downloaded from

Archives of Disease in Childhood, 1988, 63, 780-784

Simplified oral pancreatic function test

J W L PUNTIS,* J D BERG,t B M BUCKLEY,t I W BOOTH,* AND A S McNEISH* *Institute of Child Health, University of Birmingham, Birmingham Children's Hospital, and tDepartment of Clinical Biochemistry, Sandwell District General Hospital, West Bromwich, West Midlands

SUMMARY The standard Bentiromide test and a new modified test using p-aminosalicylic acid (PAS) as a pharmacokinetic marker for p-aminobenzoic acid (PABA) have been evaluated in the detection of pancreatic exocrine insufficiency in children. The conventional two day test using a colorimetric assay for urinary PABA discriminated poorly between five children with pancreatic insufficiency and 13 others with normal pancreatic function. Two further groups of patients, comprising 28 with pancreatic exocrine insufficiency and 20 with normal pancreatic function underwent the modified test, and urine samples were analysed by high performance liquid chromatography. The results showed a complete separation between groups. The use of PAS eliminates a number of sources of error inherent in a two day Bentiromide test and provides a simplified and accurate diagnostic test for pancreatic insufficiency. The PABA-PAS modified test enables collection of the urine to be done during a single six hour period.

Exocrine pancreatic insufficiency is often considered ordered gut motility or an enteropathy (which result in the differential diagnosis of failure to thrive, in low PABA absorption) or in those with hepatic or protracted diarrhoea, or steatorrhoea. Though it is renal dysfunction, when either conjugation or urin- most commonly seen in patients with cystic fibrosis, ary excretion of PABA may be reduced.3 In

it may occur in a number of other disorders. ' addition, drugs such as paracetamol and sulphona- http://adc.bmj.com/ Duodenal intubation followed by hormonal stimula- mides, or certain foods, may interfere with the tion of the pancreas with intravenous pancreozymin urinary assay of PABA. and secretin is still considered the most reliable test An attempt to correct for these variables has been for the diagnosis of pancreatic exocrine insuf- reported whereby a dose of free PABA alone is ficiency. This test is, however, unpleasant for the given the day after the Bentiromide test. A PABA patient, subsequent laboratory analysis is both time excretion index is then derived by dividing the consuming and complex, and reliable supplies of amount of PABA recovered on day 1 by the amount secretin are difficult to obtain. recovered on day 2. This makes the test more on September 24, 2021 by guest. Protected copyright. Simple screening tests for pancreatic insufficiency cumbersome but can improve specificity4 by reduc- have therefore been developed and evaluated ing the number of false positives. Subsequent largely in adult patients as alternatives to duodenal refinements have included administration of PABA intubation.2 The Bentiromide test has been most labelled with 14C at the same time as the widely evaluated and relies on hydrolysis of an Bentiromide,s which avoids the need to repeat the orally administered synthetic compound N-benzoyl- test on a second day. Though it is undesirable to give 1-tyrosyl p-aminobenzoic acid (Bentiromide) within the 0I emitter 14C to children in a diagnostic test, the the small bowel. This compound is cleaved selec- single day test with 14C PABA has gained accept- tively by pancreatic chymotrypsin to release p- ance as a screening test for pancreatic disease in aminobenzoic acid (PABA). PABA is absorbed in adults.6 the small intestine, conjugated in the liver, and As an alternative to 14C PABA we have evaluated excreted in the urine. The amount recovered from a the use of a structural analogue of PABA, p- timed urine collection expressed as a proportion of aminosalicylic acid (PAS), and compared the initial the PABA given in the dose of Bentiromide gives an PABA excretion index obtained with 14C PABA indication of duodenal chymotrypsin activity and with that using PAS. The advantage of using PAS is hence of exocrine pancreatic function. False postive that it is structurally and pharmacokinetically results may occur, however, in patients with dis- related to PABA and is safe. Furthermore, after low 780 Arch Dis Child: first published as 10.1136/adc.63.7.780 on 1 July 1988. Downloaded from

Simplified oral pancreatic function test 781 doses, its concentration in urine can be measured performed 48 hours later according to an identical simultaneously with PABA by high performance protocol. liquid chromatography. The comparison of PABA Urine volumes for the six hours were measured excretion index obtained using 14C PABA and then and aliquots of 20 ml frozen at -20 C until analysis. PAS in a group of adult patients with a wide range of Urinary PABA was assayed by the Bratton and pancreatic exocrine function showed a high correla- Marshall method for sulphanilamide as modified by tion (r=0-96) confirming the pharmacokinetic simi- Smith et al.8 larities of PABA and PAS.7 We have therefore assessed the ability of the test MODIFIED ONE STAGE TEST, INCLUDING PAS incorporating PAS to discriminate between normal Twenty eight children with known pancreatic insuf- and abnormal pancreatic function in children, and ficiency were studied; 25 had cystic fibrosis proved have compared our experience with the conven- by sweat testing, and had clinical steatorrhoea that tional two stage Bentiromide and free PABA test. had improved when enzyme supplements were given. The remaining three subjects, two with Subjects and methods Shwachman's syndrome, and one who had had a pancreatectomy, had abnormally low pancreatic UNMODIFIED TWO STAGE TEST enzyme secretion in response to stimulation with Five children with clinically apparent pancreatic pancreozymin and secretin. The median (range) age insufficiency were investigated. Two had greatly was 10 years (1 year 5 months-16 years) and weight decreased duodenal pancreatic enzyme secretion in 23 kg (8.2-54). Pancreatic enzyme replacements response to pancreozymin and secretin stimulation were stopped 48 hours before testing. (one had inflammatory bowel disease and one had Twenty other subjects had normal pancreatic cystic fibrosis), and two had undergone pancreatec- function. Fourteen had been investigated for di- tomy and had almost undetectable faecal chymo- arrhoea with or without associated failure to thrive trypsin. The remaining child had cystic fibrosis and (five had enteropathies on histological examination low faecal chymotrypsin concentrations, and was of jejunal biopsy specimens) and had normal pan- about to start pancreatic enzyme supplementation creozymin and secretin tests. There were two for poor weight gain. Their median (range) age was normal children, and four others who had normal 21/2 years (6 weeks-5 years) and weight 13-2 kg stools but who failed to thrive from a variety of (4-8-17-4). Pancreatic enzyme supplements were causes. Each of these had a negative sweat test that

discontinued 48 hours before the test. excluded cystic fibrosis, and their faecal chymo- http://adc.bmj.com/ Thirteen other subjects had normal pancreatic trypsin concentrations were within the normal function. Nine had duodenal enzyme secretion range. Their median (range) age was 2½/2 years (4 within the reference range after stimulation by months-11 years) and weight 8-5 kg (3-25). intravenous pancreozymin and secretin carried out The test protocol for the second group was similar during investigation of gastrointestinal symptoms. to that for the first group except that 4 5 mg/kg PAS Four others with symptoms of chronic chest disease was given simultaneously with 15 mg/kg Bentir- were under investigation for suspected cystic omide and a second day test was not performed. A fibrosis, but subsequently shown to have normal 20 ml aliquot from the six hour urine collection was on September 24, 2021 by guest. Protected copyright. sweat tests and faecal chymotrypsin concentrations. stored at -20°C until analysis. The median (range) age of the control subjects was 2 After the alkaline hydrolysis of PABA and PAS years (3 weeks to 13 years) and weight 9 kg conjugates, urine samples were assayed by high (2.9-45.2). performance liquid chromatography as previously After an overnight fast a urine specimen was described9; the technique was modified to measure taken before the test to determine the presence of PABA and PAS simultaneously.7 In a few patients interfering substances. Bentiromide 15 mg/kg body already receiving antibiotics, problems of interfer- weight was then given, together with a standard ence with the measurement of PABA and PAS were breakfast of unmodified cows' milk and cornflakes. overcome by using a 30x0-3 cm [t-Bondapak Babies not yet taking solids were given their usual column (Millipore Ltd, Harrow, Middlesex) in place milk feed. Urine was then collected for the next six of the 15xO-5 cm polymer column previously hours. Adhesive collecting bags were used for described.9 children not old enough to cooperate with voiding. Results from patients with pancreatic insuffici- Older children were encouraged to drink fluids of ency were compared with those from subjects with their choice during the six hour collecting period and pancreatic sufficiency by the Mann-Whitney U test. were allowed to eat lunch. The second phase of the Because results for the control group in the six hour test using free PABA alone (4-5 mg/kg) was test were non-parametrically distributed, logarith- Arch Dis Child: first published as 10.1136/adc.63.7.780 on 1 July 1988. Downloaded from

782 Puntis, Berg, Buckley, Booth, and McNeish mic transformation of the data permitted calculation 140- of the mean and two standard deviations as a reference range. Approval for the study was obtained from the 120- research ethical committee of the Central Birming- ham Health Authority. Informed parental consent was obtained before the tests. 100- Mean (2SD) Results 80- Urine [ PABA I I. The results of the two stage unmodified Bentir- Urine [PAS) omide test are shown in fig 1. The median PABA 60- S excretion index for patients with exocrine pancreatic insufficiency was 29 (range 2-65) and for subjects with normal pancreatic function 60 (range 30-118, 40- p<0-05) showing considerable overlap between the groups. The results of the six hour PABA/PAS test are 20- shown in fig 2. The PABA excretion index is derived from the ratio of concentrations of PABA and PAS 0 derivatives in the urine.7 The median PABA excre- Pancreatic Pancreatic insufficiency sufficiency tion index for patients with pancreatic insufficiency (n=28) (n=20) was 19 (range 4-60) and for patients with pancreatic sufficiency 79 (range 66-140, p<0-0001) and there Fig 2 PABA excretion index results after modified one- was complete separation between the two groups. stage Bentiromide test ofexocrine pancreaticfunction, The PABA excretion index of the two patients incorporating p-aminosalicylic acid. with Shwachman's syndrome were 58 and 60, PABA recovery being 47% and 50%, respectively. Discussion http://adc.bmj.com/ 120 Results with the unmodified two day test (fig 1) show a wide spread of PABA excretion index in subjects with and without pancreatic insufficiency, 100- with considerable overlap of the two groups. Though the number of patients studied with this test was small, the practical difficulties of a two day procedure and the wide spread of results suggest 80- that it has little clinical application. In contrast, we on September 24, 2021 by guest. Protected copyright. have found the modified oral pancreatic function test using PAS as a pharmacokinetic marker for Urine PABA recovery 60- day PABA a simple and accurate diagnostic test result- day 2 ing in complete separation between patients with and without pancreatic exocrine insufficiency (fig 2). 40- 0 All patients with pancreatic sufficiency were cor- rectly identified including those children with 0 diarrhoea and failure to thrive secondary to an 20- enteropathy. This makes the test particularly useful for rapid exclusion of pancreatic disease as a cause for altered bowel habit with or without failure of growth. The test also identified 93% of those 0- 0 Pancreatic subjects with pancreatic disease (the patients with insuffkicency Shwachman's syndrome being the two exceptions). (n=5) Results with the modified test are therefore superior Fig 1 PABA excretion index results after unmodified two to recently reported findings using fluorescein stage Bentiromide test ofexocrine pancreatic function. dilaurate which, when given as an alternative to the Arch Dis Child: first published as 10.1136/adc.63.7.780 on 1 July 1988. Downloaded from

Simplified oral pancreatic function test 783 Bentiromide test, produced a false positive result in ences in completeness of urine collections in the two 20% of control subjects.10 day test protocol that can be an important source of Patients with the rare condition of isolated lipase error. A quick and simple test is particularly useful deficiency cannot be identified by the Bentiromide in children who are not old enough to be able to void test because of its chymotrypsin specificity, nor urine on demand. The test procedure itself proved would an early indication of pancreatic exocrine acceptable to patients and no adverse reactions were failure such as decreased bicarbonate secretion-be associated with the administration of Bentiromide detected. In addition, the test cannot supplant and PAS. conventional analysis of enzymes in aspirated Using a colorimetric assay, up to 13% of tests fail duodenal juice for studying the ontogeny of pan- because of interference from drugs and foodstuffs. 18 creatic exocrine function, because it is likely that Analysis by high performance liquid chromatogra- each pancreatic enzyme has a unique maturational phy reduces this problem, though if patients are pattern. 1 receiving antibiotics care must be taken to ensure The two patients with Shwachman's syndrome, that this does not interfere with analysis. Apparatus despite almost undetectable duodenal chymotrypsin for high performance liquid chromatography is secretion following pancreozymin and secretin sti- becoming standard in clinical chemistry laborator- mulation, were still able to hydrolyse Bentiromide. ies, so that this test may be carried out in most Anomalous Bentiromide test results in patients with district general hospitals. Alternatively, PABA and Shwachman's syndrome have been reported PAS conjugates remain stable in urine samples at before,12 13 indicating that Bentiromide hydrolysis ambient temperature for several days, and could be may not be entirely due to pancreatic chymotrypsin. sent by post to a reference centre. Sterchi et al have characterised a small intestinal We recommend that when clinical features sug- brush border enzyme capable of cleaving Bentir- gest pancreatic exocrine insufficiency, consideration omide, and have named it PABA peptide should be given to using the modified PABA/PAS hydrolase.14 There is a proximal to distal gradient in test before resorting to more invasive forms of the activity of this enzyme with the lowest activity in investigation. the proximal small bowel. The activity of PABA peptide hydrolase was measured in jejunal biopsy We thank Dr AJ Shipman of Roche Products for providing specimens from both the children with Shwach- Bentiromide and PAS, Dr M Lentze for undertaking the assays of PABA peptide hydrolase activity, and Mrs D Sule for analysis of man's syndrome. Interestingly, the child with neg- the duodenal enzyme activities and the faecal chymotrypstn ligible exocrine function showed the highest PABA concentrations. Dr JWL Puntis held a Sheldon Research Fel- http://adc.bmj.com/ peptide hydrolase activity (105-19 imol/hour/gram lowship from the West Midlands Regional Health Authority. of protein; normal control mean (SD): 46-39 (6.97) imol/hour/gram of protein) whereas the second References patient, who had some residual pancreatic function, 'Mila PJ. The investigation and management of pancreatic had lower PABA peptide hydrolase activity (40.79 disease in childhood. In: Mitchell CJ, Kelleher J, eds. Pancreatic [imol/hour/gram of protein), within the control disease in clinical practice. London: Pitman, 1981:291-305. range (M Lentze, personal communication). The 2 Lankisch PG, Schreiber A, Otto J. Pancreolauryl test. Evalua-

tion of a tubeless pancreatic function test in comparison with on September 24, 2021 by guest. Protected copyright. importance of this finding remains uncertain. other indirect and direct tests for exocrine pancreatic function. Shwachman's syndrome is a rare condition with a Dig Dis. Sci 1983;28:490-3. number of associated features,15 making it unlikely 3 Niederau C, Grendell JH. Diagnosis of chronic pancreatitis. Gastroenterology 1985;88:1973-95. that a normal result from a Bentiromide test could in 4 Mitchell CJ, Humphrey CS, Bullen AW, Kelieher J, Losowsky itself lead to misdiagnosis. MS. Improved diagnostic accuracy of a modified oral pancreatic Despite these shortcomings the test has a number function test. Scand J Gastroenterol 1979;14:737-41. of clinical applications. It will readily exclude Tetlow VA, Lobley RW, Herman K, Braganza JM. A one-day oral pancreatic function test using a chymotrypsin-labile peptide pancreatic exocrine insufficiency in most children and a radio-active marker. Clinical Trials Journal 1980;17: being investigated for altered bowel habit, and 121-30. would immediately call into question the diagnosis 6 Foster PN, Mitchell CJ, Robertson DRC, et al. Prospective of cystic fibrosis if used in patients with a false comparison of three non-invasive tests for pancreatic disease. Br sweat In Bentiromide is Med J 1984;289:13-16. positive test.16 addition, T Berg JD, Chesner IM, Allen-Narker RAC, Buckley BM, useful in assessing the effectiveness of exogenous Lawson N. Exocrine pancreatic function as determined in a pancreatic enzyme replacement treatment in same-day test with use of Bentiromide and p-aminosalicylic patients with proved pancreatic insufficiency.'7 acid. Clin Chem 1986;32:1010-12. 8 Smith HW, Finkelstein N, Aliminosa L, Crawford B, Graber M. The PABA/PAS modification of the Bentiromide The renal clearances of substituted hippuric acid derivatives and test overcomes a number of problems. A single other aromatic acids in dog and man. J Clin Invest 1945;24:388- collection of urine over six hours removes differ- 404. Arch Dis Child: first published as 10.1136/adc.63.7.780 on 1 July 1988. Downloaded from

784 Puntis, Berg, Buckley, Booth, and McNeish 9Berg JD, Chesner IM, Lawson N. Practical assessment of the N-benzoyl-L-tyrosyl-p-aminobenzoic acid (PABA-peptide) in NBT-PABA pancreatic function test using high performance the human small intestine. Clin Sci 1982;62:557-60. liquid chromatography determination of p-aminobenzoic acid in 15 Aggett PJ, Cavanagh NPC, Matthew DJ, Pincott JR, Sutcliffe J, urine. Ann Clin Biochem 1985;22:586-90. Harries JT. Shwachman's syndrome. Arch Dis Child Cumming JGR, Forsyth JS, Boyd EJS, Frost GJ, Cuschieri A. 1980;55:331-47. Diagnosis of exocrine pancreatic insufficiency in cystic fibrosis 16 Shaw NJ, Littlewood JM. Misdiagnosis of cystic fibrosis. Arch by use of fluorescein dilaurate test. Arch Dis Child 1986;61: Dis Child 1987;62:1271-2. 573-5. 17 Hubbard VS, Wolf RO, Lester LA, Egge AC. Diagnostic and Lebenthal E, Lee PC. Development of functional response in therapeutic applications of Bentiromide screening test for human exocrine pancreas. Pediatrics 1980;66:556-60. exocrine pancreatic insufficiency in patients with cystic fibrosis. 12 Kai H, Nose 0, Harada T, Maki I, Tajiri H, Yabuuchi H. Dig Dis Sci 1984;29:881-9. Failure of p-aminobenzoic acid screening test to diagnose Ward CD, Rowe DJF, Fraser K. Problems of interference in the pancreatic insufficiency in Shwachman's syndrome. J Pediatr PABA test for the assessment of pancreatic exocrine function. Gastroenterol Nutr 1982;1:445-8. Clin Chem 1985;31:661. 13 Weizman Z, Forstner GG, Gaskin KJ, Kopelman H, Wong S, Durie PR. Bentiromide test for assessing pancreatic dysfunction Correspondence to Dr JWL Puntis, Institute of Child Health, using analysis of para-aminobenzoic acid in plasma and urine. Francis Road, Edgbaston, Birmingham B16 8ET. Gastroenterology 1985;89:596-604. 4 Sterchi EE, Green JR, Lentze MJ. Non-pancreatic hydrolysis of Accepted 15 January 1988 http://adc.bmj.com/ on September 24, 2021 by guest. Protected copyright.