9249 Clin Pathol 1994;47:924-927 Pancreatic enzymes in the epithelium of intrahepatic large bile ducts and in hepatic bile in J Clin Pathol: first published as 10.1136/jcp.47.10.924 on 1 October 1994. Downloaded from patients with extrahepatic obstruction

T Terada, T Morita, M Hoso, Y Nakanuma

Abstract Methods Aim-To determine whether pancreatic Hepatic bile was collected from 25 patients enzymes are present in hepatic bile and with extrahepatic biliary obstruction (chole- in intrahepatic bile duct epithelium. docholithiasis n = 10; extrahepatic bile duct Methods-The activity and proteins of carcinoma n = 6; pancreatic carcinoma n = 5; pancreatic enzymes (pancreatic a-amy- cholangiocarcinoma of hilar type n = 4). lase, lipase, trypsin/trypsinogen) in These patients had undergone percutaneous hepatic bile were investigated using transhepatic catheter drainage to reduce biochemical and western blot analyses in obstructive jaundice. The 25 bile samples 25 patients with extrahepatic bile duct (each 10 ml) obtained from the catheter were obstruction. Immunolocalisation of immediately frozen and stored at - 80°C enzyme proteins was evaluated by immu- until use. nohistochemistry in 20 necropsy Pancreatic a-amylase activity was measured with extrahepatic bile duct obstruction. using the Amylase Kit EPS (Boehringer- Results-Western blot analysis showed Mannheim Co. Ltd., Germany) which proteins of pancreatic a-amylase, lipase, requires ethilidine-(G7)-1-4-nitrophenyl-(Gl)- and trypsin in 19 of 25 (76%), 10 of 25 a-D-maltohepaoside as the substrate.9 (40%), and 14 of 25 (56%) patients, Pancreatic lipase activity was measured respectively. Pancreatic a-amylase and (Lipase Kit-VE, Nihon Shoji Ltd., Tokyo, lipase activities was present in every bile Japan) according to the method of Imamura specimen. Radioimmunoassay showed and Misaki.'0 Because trypsin inhibitor may that trypsin was present in every bile be present in hepatic bile, trypsin was mea- sample. Immunohistochemically, the sured (ng/ml) using a radioimmunoassay kit http://jcp.bmj.com/ immunoreactivity of the three enzymes (Ria-Gnost Trypsin II; Behring-Werke Co. was present in epithelia and in the lumina Ltd., Marburg, Germany). of intrahepatic large bile ducts, septal The protein concentration of bile samples bile ducts, and peribiliary glands in all was measured according to the method of cases. Lowry et al.'I The bile samples containing 100 Conclusions-These results strongly ,ug protein were solubilised at 100°C for five suggest that biliary epithelia of larger minutes in 50 ,l sample buffer (50 mM on September 27, 2021 by guest. Protected copyright. intrahepatic ducts produce pancreatic TRIS-HCI (pH 6 8), 2% sodium dodecyl sul- a-amylase, lipase, and trypsin, and that phate (SDS), 10% 2-mercaptoethanol, 01% these enzymes are secreted into the bromophenol blue, and 10% glycerol). Bile lumina ofintrahepatic bile ducts. proteins were separated on 10% SDS poly- acrylamide electrophoresis gels at 60 mnA for (7 Clin Pathol 1994;47:924-927) two hours at room temperature. The gels were then equilibrated for 30 minutes in transfer buffer (50 mM TRIS, 40 mM glycine, 0 04% It has become increasingly evident that intra- SDS, and 30% methanol). Proteins were elec- hepatic bile ducts not only drain canalicular trophoretically transferred on to nylon mem- bile but also have a variety of functions branes (Immobiron PVDF Transfer including secretion and absorption of water Membrane; Daiichi Pure Chemicals, Ltd., and electrolytes,' secretion of mucous,2 secre- Tokyo, Japan) at 120 mA for 60 minutes. The tion of IgA,3 secretion of lactoferrin and membranes were incubated in blocking buffer lysozyme,4 and presence of neuroendocrine (phosphate buffered saline (PBS) containing Second Department of Pathology, Kanazawa hormones.5 The degree of these functions 3% bovine serum albumin) for 12 hours at University School of varies with the anatomical segments and the 4°C. For detection of pancreatic a-amylase, Medicine, Kanazawa type of bile duct. Recently, it has become evi- lipase, and trypsin, the membranes were incu- 920, Japan T Terada dent that intrahepatic large bile ducts, septal bated for 60 minutes at room temperature in T Morita bile duct, and peribiliary glands express mouse monoclonal antibody (IgG class) M Hoso immunoreactive pancreatic a-amylase, pan- against human pancreatic a-amylase Y Nakanuma creatic lipase, and trypsin in normal human (Chemicon Inc., Tomecula, California, USA; Correspondence to: and in various hepatobiliary diseases.8 dilution 1 in 100), mouse monoclonal anti- Dr Tadashi Terada unknown these human Accepted for publication However, it is whether body (IgG class) against pancreatic 19 April 1994 pancreatic enzymes occur in hepatic bile. lipase (Chemicon Inc.; dilution 1 in Pancreatic enzymes in hepatic bile 925

100), or mouse monoclonal antibody (IgG Activity Concentration class) against human trypsinogen-trypsin (IU/I) (ng/ml)

(Chemicon Inc.; dilution 1 in 100). After two J Clin Pathol: first published as 10.1136/jcp.47.10.924 on 1 October 1994. Downloaded from 300 000 washes in T-PBS (0 03% Tween 20 and PBS * (pH7-2), the membranes were incubated for 60 minutes at room temperature with peroxi- r dase labelled goat anti-mouse IgG (Dako Corporation, Santa Barbara, California, USA; 150 000 15 000 dilution 1 in 1500) in T-PBS. After three washes in T-PBS the membranes were incu- bated at room temperature with development buffer (50 mM TRIS-HCl (pH7-2), 0-025% 100 000 F 10 000 diaminobenzidine, and 0 075% hydrogen peroxide). As positive controls the same procedures were performed using human pan- creatic a-amylase (Athens Research and 50 000 Technology Inc., Athens, Georgia, USA), 40 000 human pancreatic lipase (Elastin Products 30 000 20000 Co. Ltd., Owensville, Michigan, USA), and *$ -r ***** ********* ********* human trypsin (Athens Research and ********* -I-- ********* u Technology Inc.). - II A total of 20 recent necropsy livers with Pancreatic Pancreatic Trypsin a amylase lipase extrahepatic biliary obstruction were obtained 19 411 ±62 524 6690±11 905 6432±26327 from patients aged 47-86 years (12 men and (IU/I) (lU/l) (ng/mI) eight women). Five tissue specimens contain- ing large bile ducts were obtained from each Figure 1 Pancreatic a-amylase and lipase activities and trypsin concentrations in the hepatic bile ofpatients with liver, fixed in 4% formaldehyde, and embed- extrahepatic bile duct obstruction. Each dot represents a ded in paraffin wax. As positive controls single bile sample. specimens of the normal were obtained from our surgical files, fixed in 4% formaldehyde, and embedded in paraffin wax. staining procedure. The absorbents included Several serial 5 pm sections were obtained human pancreatic a-amylase (Athens Research from each paraffin wax block, and one of them and Technology Inc.), human pancreatic was stained with haematoxylin and eosin. lipase (Elastin Products Co. Ltd.), and Three serial sections from every block were human trypsin (Athens Research and immunostained for pancreatic a-amylase, Technology Inc.). pancreatic lipase, and trypsin by the three-

step indirect immunoperoxidase method http://jcp.bmj.com/ (avidin biotin peroxidase method) of Hsu et Results al.12 The primary antibodies used were the Figure 1 shows the pancreatic a-amylase, same as those used in the western blot assay. pancreatic lipase, and trypsin activities. The sections were treated with the optimally Pancreatic a-amylase and lipase were present diluted primary antibodies at 4°C overnight. in all 25 bile samples (pancreatic a-amylase, The sections were next incubated with the mean 19 411 IU/l; pancreatic lipase mean =

secondary biotinylated antibody (anti-mouse 6690 IU/1), although the values varied greatly on September 27, 2021 by guest. Protected copyright. IgG; Vector Laboratories, Burlingame, according to the bile samples. Trypsin was California, USA) for 40 minutes and subse- present in all 25 bile samples (mean 6432 quently treated with avidin biotin peroxidase ng/ml), although this varied greatly with the complex (Vectastain ABC Kit, Vector bile samples. Laboratories) for 40 minutes. Reaction Western blotting of pancreatic a-amylase products were developed by immersing the revealed a signal at 54 kilodaltons in 19 of 25 sections in a 3,3'-diaminobenzidine tetra- (76%) of the bile samples (fig 2). Signals for hydrochloride solution (Sigma Chemicals, trypsin and pancreatic lipase were noted at 23 St Louis, Missouri, USA) containing and 52 kilodaltons in 10 of 25 (40%) and 14 of hydrogen peroxide. Positive staining was 25 (56%), respectively (fig 2). The locations abolished when non-immune sera or PBS ofthe signals were consistent with the molecu- were substituted for the primary antibodies. lar weight of the enzymes and corresponded In five cases the immunostaining was per- to the signals of positive controls (fig 2). The formed with Vector Red (Vector Labora- activities of these enzymes were much higher tories) instead of diaminobenzidine. Vector in bile samples with signals (pancreatic a- red produces red fluorescence under a fluo- amylase, mean 22 872 IU/l; trypsin mean rescence microscope with a Thodamine filter 9831 ng/ml; pancreatic lipase mean 18 060 system. The specimens were examined under a IU/1) than in those without signals (pancreatic confocal laser scanning microscope (LSM- a-amylase, mean 126 IU/l; trypsin, mean 351 GB200, Olympus, Tokyo, Japan). An argon ng/ml; pancreatic lipase, mean 101 IU/l). laser with a 514-4 nm wavelength and a 615 The acinar cells of the normal pancreas nm long-pass barrier filter made confocal fluo- were positive for pancreatic a-amylase, lipase, rescence images. and trypsin, and were negative by the absorp- Absorption tests were also performed in tion test, suggesting that each immunostain selected specimens during each immuno- is specific. Immunoreactivity to pancreatic 926 Terada, Morita, Hoso, Nakanuma

Figure 2 Western blot analysis ofhepatic bile. A signal (arrow) is seen at

54 kilodaltons in J Clin Pathol: first published as 10.1136/jcp.47.10.924 on 1 October 1994. Downloaded from pancreatic a-amylase immunoblots. A signal (arrow) is seen at 23 ..: ..:. .::.:i .;. kilodaltons in trypsin .... i:.^i. .:. immunoblots. A signal :j'.,W, ::: (arrow) is seen at 52 :: i. :... ::: .. kilodaltons in pancreatic .. .i O ....F.-a .s lipase immunoblots. The .....:. .:>R:i 9 locations ofthe signals of .,: samples (S) are consistent ..: .. ..: .. with the molecular weight ...... ofthe respective enzymes, :_ and correspond to the .:...... y .:;:' °j signals ofpositive controls *. (C). M = molecular :... *: ..a.o. ,:_ .:: weight marker. ft:. : .... i. ::.i .g ...... s , l

Figure 3 Confocal laser and scanning images of a-amylase, pancreatic lipase, trypsin- intrahepatic large bile ducts trypsinogen was present in the epithelial cells immunostainedfor of the intrahepatic large bile ducts, septal bile pancreatic a-amylase (A), ducts, and peribiliary glands in all cases (figs lipase (B), and trypsin (C). The biliary epithelial 3A-C). The lumina of the bile ducts and peri- cels are positivefor the biliary glands were also positive for the three three enzymes. The lumina enzymes, and the luminal enzymes seemed to are also positivefor the enzymes, and the enzymes be secreted from the biliary epithelia (figs 3A- seem to be secretedffrom the C). The immunoreactivities became negative biliary epithelia with the absorption test. These enzymes were (immunostain). absent in interlobular bile ducts, bile ductules, and .

Discussion In this study, the activities of the three enzymes were present in all bile samples from http://jcp.bmj.com/ patients with extrahepatic bile duct obstruc- tion, indicating that pancreatic a-amylase, pancreatic lipase, and trypsin are present in hepatic bile. The reason why the enzyme activities varied greatly according to the bile

samples remains to be clarified. on September 27, 2021 by guest. Protected copyright. Western blot analysis confirmed the pres- ence of pancreatic a-amylase, pancreatic lipase, and trypsin proteins in the hepatic bile. However, there was a discrepancy between enzyme activity and results of western blot analysis: enzyme activities were present in all bile samples while proteins were detected in only 40-76% of samples by western blot analysis. The reason for this discrepancy may have been that the western blot technique is less sensitive than the biochemical techniques measuring enzyme activity because enzyme activities in bile samples evaluated with west- ern blot signals were high whereas those in bile samples with no western blot signals were low. This study has shown that immunoreactive pancreatic a-amylase, lipase, and trypsin- trypsinogen occur in the epithelium of intrahepatic large bile ducts, septal bile ducts, and peribiliary glands in necropsy livers from patients with extrahepatic bile duct obstruction. The immunoreactivity became negative in the absorption test, suggesting that each immunostain is specific. This finding strongly suggests that epithelial cells in intra- Pancreatic enzymes in hepatic bile 927

hepatic large bile ducts, septal bile ducts, and absorption in man. Lab Invest 1967;16:84-95. 2 Terada T, Nakanuma Y. Development of human intra- peribiliary glands contain pancreatic a-amy- hepatic peribiliary glands: histological, keratin immuno- lase, pancreatic lipase, and trypsinogen- histochemical and mucus histochemical analyses. Lab J Clin Pathol: first published as 10.1136/jcp.47.10.924 on 1 October 1994. Downloaded from Invest 1993;68:261-9. trypsin. Of particular interest was that enzyme 3 Sugiura H, Nakanuma Y. Secretory components and immunoreactivity seemed to be secreted from immunoglobulins in the intrahepatic biliary tree and peribiliary glands in normal livers and hepatolithiasis. the biliary epithelium into bile duct lumina. GastroenterolJpn 1989;24:308-14. Because these enzymes were detected in 4 Saito K, Nakanuma Y. Lactoferrin and lysozyme in the intrahepatic bile duct of normal livers and hepatolithia- hepatic bile, these biliary epithelial cells are sis. JHepatol 1992;15:147-53. very likely to be the cellular source of the 5 Roskam T, Van der Oord JJ, De Vos R, Desmet VJ. Neuroendocrine features of reactive bile ductules in enzymes. cholestatic liver disease. Am J Pathol 1990;137:1019-25. Finally, our experiment was confined to 6 Terada T, Nakanuma Y. Expression of alpha-amylase isoenzymes and trypsin by the proliferating epithelium of hepatic bile in patients with extrahepatic bile large intrahepatic bile ducts and intrahepatic peribiliary duct obstruction. We did not examine normal glands in hepatolithiasis. Histopathology 1993;22:467-73. 7 Terada T, Nakanuma Y. Pancreatic lipase is a useful phe- human hepatic bile, because it is very difficult notypic marker of intrahepatic large and septal bile or impossible to obtain normal human hepatic ducts, peribiliary glands, and their malignant counter- parts. Mod Pathol 1993;6:419-26. bile. It remains to be elucidated whether 8 Terada T, Kida T, Nakanuma Y. Extrahepatic peribiliary normal human hepatic bile contains pancre- glands express alpha-amylase isozymes, trypsin and pan- creatic lipase: an immunohistochemical analysis. atic enzymes. Hepatology 1993;18:803-8. In conclusion, our data strongly suggest 9 Rauscher E, Bulow S, Hagele EO, Neumann U, Schaich E. Ethylidine protected substrate for the assay of human that the biliary epithelia of larger intrahepatic alpha-amylase. Fresenius ZAnalyt Chem 1986;324:304-5. ducts pancreatic a-amylase, lipase, 10 Imanura S, Misaki H. A sensitive method for assay of produce lipase activity by coupling with beta-oxidation enzymes and trypsin, and that these enzymes are of fatty acid. Sel Topics Clin Enzymol 1984;2:73-7. secreted into the lumina of intrahepatic bile 11 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem ducts. 1951;193:265-75. 12 Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxi- dase complex (ABC) in immunoperoxidase techniques: 1 Sasaki H, Schaffner F, Popper H. Bile ductules in a comparison between ABC and unlabelled (PAP) cholestasis: morphologic evidence for secretion and procedures. J Histochem Cytochem 1981;29:557-80. http://jcp.bmj.com/ on September 27, 2021 by guest. Protected copyright.