Practical assessment of the NBT-PABA pancreatic function test using high performance liquid chromatography determination of p-aminobenzoic acid

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Ann Clin Biochem 198; 22: 86-90 Practical assessment of the NBT-PABA pancreatic function test using high performance liquid chromatography determination of p-aminobenzoic acid.

1 Ann Clin Biochem 198; 22: Practical assessment of the NBT-PABA pancreatic function test using high performance liquid chromatography determination of p-aminobenzoic acid.. In unne J D BE R G,* I C H E S N E R t and N LAW SON * From the *Departments of Clinical Chemistry and i Gastroenterology, East Birmingham Hospital, Bordesley Green East, Birmingham B9 ST, UK SUMMARY. A practical method for determining p-aminobenzoic acid in urine by high-performance liquid chromatography has been assessed. The technique is quick, requires no extraction steps and has good precision. Using this method, healthy individuals had a p-aminobenzoic acid excretion index of 94%±18 (mean±l SD). Patients with proven, severe chronic exocrine pancreatic dysfunction had index values of <-2%. Unlike chemical methods available, there were no interferences in any of the urines tested, nor did 12 compounds tested interfere with the analysis. An assessment of exocrine pancreatic function may be made by the oral administration of N-Benzoyl-L-tyrosyl-p-amino benzoic acid (NBT-PABA).l Pancreatic chymotrypsin cleaves this synthetic peptide, releasing p aminobenzoic acid (PABA) which can be absorbed from the small intestine, conjugated by the liver and excreted in to urine. An estimate of exocrine pancreatic function can be made by the measurement of PABA in a timed urine collection after NBT-PABA administration. The test can be improved by administering 14C-PABA at the same time as NBT-PABA and accounting for individual variability in intestinal, hepatic and renal handling of PABA, expressing the result as a PABA excretion index.i: 3 At the present time, the usefulness of this test is difficult to assess, with conflicting reports on the specificity and sensitivity of the test. 4--<l A true appraisal of the test can be made only if a reliable analytical technique is used for urinary PABA measurement. Quantification of urine PABA is often made using colorimetric methods.": 10 A number of reports suggest that, Correspondence: J D Berg, Department of Clinical Biochemistry, Sandwell District General Hospital, Wes' Bromwhich, West Midlands B71 4HJ, UK. 86 when applied to urinary PABA estimation, these colorimetric techniques will give misleading results due to interference from other urine constituents, including drugs. H. 11 A HPLC method for urine PABA measurement has been described 12 and this technique may provide a much more specific assay, but this depends on the retention times of PABA and the internal standard being different from those of other urine constituents. This work presents a practical assessment of using HPLC in determining PABA in urine as part of the NBT-PABA test, and includes studies of 12 compounds which may interfere with the technique. Materials and methods NBT-PABA was obtained from Roche Products Limited, Welwyn Garden City, Hertfordshire, England. et 4 C-carboyxl)-p-amino benzoic acid was purchased from Amersham International Limited, Bucks, UK. Healthy individuals and patients were fasted overnight and any pancreatic supplements stopped 48 h before the day of the test. At 9.00 am the subjects were asked to empty their bladder and then given 3x333 mg tablets of NBT-PABA, IlCi of et 4 C-carboxyl)-PABA in 100 ml of water and 2 g of casein in 400 ml of a

2 NBT-PABA pancreatic function test 87 chocolate flavoured drink (0 g/l of Cadbury's drinking chocolate). The subjects then drank 1 L of water within 2 h of commencing the test and were requested not to eat for 4 h. Urine was collected for 6 h after giving the NBT PABA. Urine aliquots (20 ml) were stored at - 20 C and analysed within 1 week of performing the test. To determine the amount of 14C-PABA excreted into the urine, 1 ml of the 6-h test urine was added to HI ml scintillant (Fisofluor, Fisons, Loughborough, UK) and counted. In 19 healthy individuals (11 men; eight women; age range years) the mean excretion of 14C_ PABA into the urine over 6 h expressed as a percentage of the dose of radioactivity given. was 82%±6 (±1 SD; range 70-94%). To determine the quantity of PABA in the urine, samples had to be hydrolysed first. One ml of urine was added in a glass screwtopped tube to 1 ml of 8 molll NaOH containing the internal standard; 7 0 mmolll m-hydroxybenzoic acid and heated to 120 Cfor 1 h. After cooling, 40 ul, of the hydrolysate was added to 960 ul, of 0 2 mmol/l phosphoric acid, thoroughly mixed and centrifuged at 6000 g for min. The clear supernatant (20 /ll) was analysed using a 2 cm x-t-e mm C18 reverse phase column (Spherosorb ODS, HPLC Ltd, Macclesfield, UK) with a mobile phase of 0 2 molll phosphate buffer, ph 3 :acetonitrile (7:1 v/v), at room temperature with a flow rate of 1 mumin. Applied Chromatography Systems 70/03 pump and a 24 nm UV detector (ACS 70/11) were used (Applied Chromatography Systems Ltd, Luton, England). The chromatographs were integrated using a Hewlett Packard 3390A integrator (Hewlett Packard, Altrincham, Cheshire, England) using peak height quantification. Results were expressed in the form of a PABA excretion index (PEl): PEI= Total amount of PABA excreted in urine (mmol) Total amount of 14C-PABAexcreted in urine (/lci) x--xloo 2 14 where the number refers to the dose of radioactivity administered to each subject ( /lci) and 2 14 is the amount in mmol of PABA administered to the subject in the form of NBT-PABA. Results The conjugates of PABA cannot be identified, separated and quantified by this HPLC system, therefore hydrolysis of the various conjugates to PABA has to be complete. To ensure this a mmol/l p-aminohippuric acid aqueous standard was included in each batch. This is one A B [ a o Po p [ [ [ 10 Retention time (min) FIG. 1. HPLC chromatograms of (A) 2 62 mmol/l PABA aqueous standard; (B) 6-h urine collection from a healthy individual given I g NBT-PABA; (C) 6-h urine collection from a patient with severe, chronic exocrine pancreatic dysfunction given I g NBT-PABA; (D) urine from a patient on salazopyrin treatment, and not given NBT-PABA. All samples were prepared as described in the methods section. 1

3 88 Berg, Chesner and Lawson of the major metabolites of PABA and is the most difficult to hydroiyse.v In this study, employing hydrolysis conditions of 4 mol/l NaOH at 120 C for 1 h, 9-10% of the p amino hippuric acid was converted to PABA. Use of a lower temperature (100 C), lower concentration of NaOH (2 mol/l), and shorter hydrolysis time (30 min) resulted in considerably less p-aminohippuric acid being converted to PABA. A typical chromatograph of a 2 62 mmol/l aqueous standard of PABA, and a chromatograph of a 6-h urine collection from a healthy individual are shown in Fig. la and b. Quantification was accomplished using peak-height ratios, the method being linear from 0 10 to 10 mmol/l of PABA. Inter-assay precision was estimated using two control materials. A control urine sample was prepared by adding a known concentration of PABA to a urine specimen and produced a CV of 3 9% (mean=2'81 mmol/l; n=19). A urine control sample 'prepared from a 6-h urine collection of a healthy individual who had been given 1 g of NBT PABA produced a CV of 3 1% (mean=i 30 mmol/l; n=9). A chromatograph of a urine sample from a patient given 1 g of NBT-PABA who had severe, chronic exocrine pancreatic dysfunction is shown in Fig. lc. There is only a very small peak at the elution time corresponding to PABA. Table 1 shows the mean PEL of 19 healthy individuals, and the range of PEls obtained from patients having severe, chronic exocrine pancreatic dysfunction, diagnosed by having very small rises in duodenal enzymes after a secretin-pancreozymin challenge. There is a clear separation between the control group and the patients having severe exocrine pancreatic dysfunction. Patients who had proven exocrine TABLE 1. PABA excretion index in healthy individuals and patients with severe, chronic pancreatic exocrine dysfunction 19 healthy individuals" Nine patients with severe chronic exocrine pancreatic dysfunction'? PEl (%) 94±18 Range <--2 Results expressed as mean± 1 SD 11 males, eight females; mean age 31 years, range Seven males, two females; mean age 3 years, range pancreatic disease (Xvray, ultrasound), but with equivocal pancreozymin-secretin test results, displayed PEls between 2 and 80%. Fig. ld shows the chromotograph of a patient's urine before NBT PABA was administered. There is no peak with an elution time corresponding to PABA. In 40 different urine samples analysed there were no peaks which coeluted with either the internal standard (m P 4 6 p !L o 10 1 Retention time (min) FIG. 2. HPLC chromatograms of PABA (P) and the internal standard (), m-hydroxybenzoic acid, together with the following: (I) -aminosalicylic acid; (2) m-aminobenzoic acid; (3) p-hydroxybenzoic acid; (4) a-hydroxybenzoic acid; () sulphanilic acid; (6) paracetamol"; (7) sulphadiazine; (8) caffeine"; (9) sulphosalicylic acid; (10) sulphanilamide; (11) theophylline"; (12) a-acetylsalicylic acid. ( Peak lost after alkaline hydrolysis.)

NBT-PABA pancreatic function test 89 hydroxy benzoic acid) or with PABA, although in Fig. ld there are several peaks which are clearly discernible.

4 NBT-PABA pancreatic function test 89 hydroxy benzoic acid) or with PABA, although in Fig. ld there are several peaks which are clearly discernible. This particular patient was undergoing treatment for Crohn's disease with salazopyrin, and the metabolites of this drug are the probable cause of the peaks. Figure 2 shows chromatographs of compounds which may have co-eluted with PABA, and some compounds which can interfere with the chemical determination of PABA. Several compounds, -aminosalicylic acid, m aminobenzoic acid, p-hydroxybenzoic acid, 0 hydroxy benzoic acid (salicylate) and a-acetylsalicylic acid (peaks 1, 2, 3, 4 and 12 respectively in Fig. 2), which have similar structures to PABA, all had elution times different from either PABA or the internal standard, m hydroxybenzoic acid. It is probable that aminosalicylic acid and m-aminobenzoic acid would interfere with the chemical determination of PABA. Fig. 2 also shows the elution times of a number of compounds which are known to interfere with the chemical determination of PABA. Sulphanilic acid, paracetamol, sulphadiazine, sulphosalicylic acid and sulphanilamide (peaks, 6, 7, 9 and 10 respectively in Fig. 2) all had elution times different from either PABA or the internal standard. Two further drugs were separated on this HPLC system: caffeine and theophylline (peaks 8 and 11 respectively in Fig. 2) and neither co-eluted with PABA and m-hydroxybenzoic acid. All of the chromatograms illustrated in Fig. 2 were produced from aqueous mixtures of the compounds which had been treated the same as all other specimens, except that the heating at 120 C for 1 h was omitted. After heating at 120 C the peaks corresponding to caffeine, paracetamol and theophylline disappeared, indicating the instability of these compounds to alkaline hydrolysis. Discussion This work clearly demonstrates the improved specificity that HPLC analysis of PABA can give over colorimetric methods. None of the drugs tested co-eluted with PABA or the internal standard in this HPLC system. Several of the drugs that have been studied are known to interfere with the Bratton and Marshall assay." Furthermore, none of the urine samples analysed in this study produced peaks on the HPLC chromatograms which co-eluted with PABA or the internal standard. It is possible that other drugs or drug metabolites could interfere with the HPLC assay, but by analysing a pre-test urine any such interference should be noticed. In this study, 17% of the patients and controls were on drugs which could have given interference in the colorimetric assay of Bratton Marshall. In particular, aspirin derivatives are often encountered in patients undergoing exocrine pancreatic function tests, but these have been shown not to interfere with the HPLC determination of PABA. Interference by procainamide-type local anaesthetics and the food additives E210 to E219 could occur, as these substances may be metabolised to PABA. The results obtained for the PABA excretion index in healthy individuals and patients with severe exocrine pancreatic dysfunction are very encouraging, but further study is required to define the specificity and sensitivity of the test when employing HPLC determination of PABA. The use of HPLC estimation of PABA provides the possibility of an alternative means of correcting for variation in intestinal absorption together with hepatic and renal handling of PABA. A drug with similar pharmokinetics to PABA could be used to correct for such variability and such a regimen would remove the requirement for 14C-PABA administration. If such a drug were found, it should be possible to measure it using the same HPLC system as that used for PABA estimation. Several such markers of absorption have been tried but at the present time a suitable drug has not been found. Salicylate is absorbed primarily in the stomach and is unsuitable. Enteric-coated salicylate has been tested but shows insufficient absorption in healthy individuals. Results for paracetamol are more encouraging, although, due to its instability in the alkaline hydrolysis step, an alternative deconjugation step needs to be evaluated. The commonly available sulphonamides show very different pharmacokinetics to PABA exhibiting slow absorption and extended half-lives compared to PABA. It is possible that m-aminobenzoic acid could be an ideal alternative to 14C-PABA, but this compound is not available for use in humans at the present time. An alternative approach is to combine the NBT-PABA test with a test of malabsorption such as the xylose absorption test. 13 It may not be possible to do both these tests simultaneously and, if so, there would be no advantage over administering PABA itself on a separate day to NBT-PABA, to determine a PABA excretion index.

5 90 Berg, Chesner and Lawson Serum PABA levels can be used as an alternative to urine estimation. 14. Serum measurement removes the problem of variation due to hepatic and renal handling of PABA. However, variation in intestinal absorption still has to be overcome, and it is not practical to estimate 14C-PABA in serum. The use of an alternative marker of variation in intestinal absorption would also be of benefit for a test based on estimation of PABA in serum. Employing HPLC analysis of PABA removes major problems of non-specificity and should enable a more comprehensive evaluation of the use of the NBT-PABA test in clinical practice. The technique is straightforward and, although an investment in HPLC equipment is required, the running costs are low. HPLC analysis of PABA should be seriously considered by anyone wishing to introduce the NBT-PABA test. Acknowledgements Dr P Asquith and Dr H I Pandov are thanked for their guidance and Dr K Cleur of Roche for his support. References Lankisch PG, Lembecke B. Indirect pancreatic function tests: chemical and radioisotope methods. Clin Gastroenterol 1984; 13: Mitchell Cl, Field HP, Simpson FG et al. Preliminary evaluation of a single day tubeless test of pancreatic function. Br Med J 1981; 282: Tetlow VA, Labley RW, Herman K et al. A one day oral pancreatic function test using a chymotrypsin labile peptide and a radioactive marker. Clin Trials J 1981; 17: Braganza 1. Prospective comparison of 3 noninvasive tests for pancreatic disease. Br Med J 1984; 289: 62. Cobden I, Lendrum R, lames OFW, et al. Prospective comparison of three non-invasive tests for pancreatic disease. Br J Med 1984; 289: Foster PN, Mitchell Cl, Robertson ORC et al. Prospective comparison of three non-invasive tests for pancreatic disease. Br Med J 1984; 289: Faulder GC, Strange RC. Brief study of the urinary PABA test for assessment of pancreatic exocrine function. Clin Chern 1984; 30: Braganza 1M, Kay GH, Tetlow VA, Herman Kl. Observations on the BT PABAl I4C-PABA tubeless test of pancreatic function. Clin Chim Acta 1983; 130: 33~7. 9 Bratton AG, Marshall EK. A new coupling component for sulphanilamide determination. J Bioi Chern 193; 128: Yamato C, Kinoshita K. A simple assay for measurement of urinary p-aminobenzoic acid in the oral pancreatic function test. Anal Biochem 1979; 98: Braganza 1. Prospective comparison of 3 noninvasive tests of pancreatic disease. Br Med J 1984; 289: Ito S, Maruta K, Imai Y et al. Urinary p aminobenzoic acid determined in the pancreatic function test by liquid chromatography with electrochemical detection. Clin Chern 1982; 28: Sherding RG, Stradley RP, Rogers WA, Johnson SE. Bentiromide:Xylose test in healthy cats. Ann J Vet Res 1982; 430: Oelchier JC, Soule JC, BT-PABA test with plasma PABA measurements: evaluation of sensitivity and specificity. Gut 1983; 24: Lang C. Gyr K. Tanko I et al. Value of serum PABA as a pancreatic function test. Gut 1984; 2: Accepted for publication 8 March /98

Pancreatic function testing: serum PABA

Gut, 1988, 29, 1736-1740 Pancreatic function testing: serum PABA measurement is a reliable and accurate measurement of exocrine function A R TANNER AND D P ROBINSON From the Departments of Medicine and